In Silico Analysis and Expression Profiling of Expansin A4, BURP Domain protein RD22- like and E6-like Genes Associated with Fiber Quality in Cotton

In Silico Analysis and Expression Profiling of Expansin A4, BURP Domain protein RD22- like and E6-like Genes Associated with Fiber Quality in Cotton | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In Silico Analysis and Expression Profiling of Expansin A4, BURP Domain protein RD22- like and E6-like Genes Associated with Fiber Quality in Cotton Farzana Ashraf, Asif Ali Khan, Nadia Iqbal, Zahid Mahmood, Abdul Ghaffar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1327190/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background Wild Gossypium species and races are rich source of genetic polymorphism due to environmental dispersal and continuous natural selection. These genetic resources hold mass of outclass genes that can be used in cotton improvement breeding programs to exploit possible traits such as fibre quality, abiotic stress tolerance, and disease and insect resistance. Therefore, use of new molecular techniques such as genomics, transcriptomics and bioinformatics is very important to utilize the genetic potential of wild species in cotton improvement programs. Methods Interspecific lines and Gossypium species used in the study were grown at Central Cotton Research Institute (CCRI), Multan. After retrieving DNA sequence of the genes from NCBI, the primers for gene expression and full-length gene sequence were designed. Expression profiling of Expansin A4 , BURP Domain protein RD22-like and E6-like fibre genes was performed through Real Time PCR. BLAST and DNA sequence alignment was conducted for sequence comparison of interspecific lines and Gossypium species. Different in silico analysis were used for characterization of fibre genes and identification of cis acting promoter elements in promoter region. Results Variable expression of genes related to fibre development was observed at different stages. BLAST and DNA sequence alignment exhibited resemblance of interspecific lines with G. hirsutum . In silico analysis on the sequence data also confirmed the role of Expansin A4 , BURP Domain protein RD22-like and E6-like fibre genes in fibre development. Similarly, several stress tolerant and light responsive cis acting elements were identified through promotor analysis, which may contribute for fibre development in the breeding programs. Conclusion Expansin A4 , BURP Domain RD22-like and E6-like have positive role in fibre development with variable expression at fiber length and strength associated stages. DNA sequencing Expression analysis Fibre genes In silico analysis Cotton Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Globally, synthetic fibre consumption is continuously increasing and projected to reach at 130 million tons by 2030. The consumption of synthetic fibre is 62.7% compared to 24.3% cotton fibre consumption [ 1 ]. Competition of cotton fibre with polyester is creating negative influence on the demand of cotton. Genetic improvement of cotton for fibre traits is very crucial to meet the challenges of the textile industry. So, there is need to devise clear-cut policies for cotton breeding program to enhance the quality cotton production. In a breeding program, germplasm collection, its conservation and utilization, trait specific screening programs and modern genomics have key role in variety development [ 2 ]. Cotton genetic resources have been extensively studied over the last many decades to introduce valuable traits in cotton [ 3 – 5 ]. These genetic resources include wild Gossypium germplasm, innovative cytogenetic stocks with specific chromosomes additions or deletions in different species, large mapping families, recombinant inbred lines, near isogenic lines and interspecific lines. While there are some queries about narrow genetic base of these cultivars and most breeders would admit that in breeding programs maximum utilization of genetic diversity within their material should be ensured. Breeders will have to utilize wild cotton relatives, as well as advance lines or cultivars to develop cotton varieties with superior traits. At the cellular level, cotton fibre development is supported by several genes which facilitates the elongation process, for example, Expansins are involved in fibre elongation at various development stages [ 6 ]. High transcript abundance of GhEXP1 was observed in cotton fibre during the elongation phase of fibre development, which steadily decreased from 16 to 20 DPA [ 7 , 8 ]. In cotton, GhEXPA1 along with GhRDL1 showed an increase in fiber length and an enlargement of endopleura cells of ovules [ 9 ]. The BURP Domain is a plant-specific protein characterized by repetitive units of amino acid [ 10 ]. This protein is mainly involved in promoting the fibre cells elongation when over-expressed. Because GhRDL1 directly interacts with cotton α- Expansin fibre gene therefore, Expansin s mediate GhRDL1’s effect on overall fibre cell enlargement [ 9 , 11 ]. It was suggested that E6 protein is involved in fibre development, but no support was present to justify this hypothesis as no conclusive evidence was presented [ 12 ]. When E6 antisense suppression construct was used, there was knockdown to uncover a phenotype E6-like . E6 proteins play a comprehensive role in cell wall, and are deposited during fibre elongation, which give high transcripts in fibre cell during transcriptomic analysis [ 13 ]. Transcriptional profiling is a unique tool to gain knowledge about gene mechanisms, regulatory pathways, and gene expression [ 14 , 15 ]. Number of techniques are used for specific gene expression studies but Real Time PCR is the most reliable technology for absolute and comparative quantification of the gene transcription [ 16 ]. This comprehensive wide-ranging gene expression study is supportive to sightsee the role of genes, which are up regulated, entirely expressed, or down regulated during different cotton fibre development stages. Through transcriptomic data, one can explain the fibre expansion process and can discover highly expressed genes for the development of transgenic cotton varieties with superior fibre traits. Profiling of fibre genes in interspecific lines will enable us to unravel variable expression pattern of selected fibre genes. Application of in silico methods along with expression profiling is important for characterization of fibre genes. DNA sequence of interspecific lines and Gossypium species were aligned to have information about differences and similarities. Diploid and tetraploid genomes of various Gossypium species have repeatedly sequences making their entire genome sequences. These valuable repeatedly sequenced data revealed the evolutionary history of the cotton with polyploidization and decaploidization leading to the of the formation of genus Gossypium [ 17 ]. Multiple sequence alignment approaches envisage algorithmic explanation about evolutionarily sequences alignments. Fibre genes were subjected to BLAST analysis for expression validation and multiple DNA sequence alignment for similarities and differences of interspecific lines and parent species. Genomics combines recombinant DNA technology, DNA sequencing and bioinformatics sequence to analyze the structure and function of genes [ 18 ]. Bioinformatics is a systematic field that utilizes advance approaches for computational analysis of biological data [ 18 ]. Bioinformatics also aids to recognize different promoters involve in fibre yield and quality, abiotic stress tolerance and disease resistance. Strength and specificity related character of promoter sequence can be exhibited through expression profiling. Strong promoters predict high expression and vice versa. Fibre genes protein E6 , Expansin A4 and BURP Domain RD22-like also have strong promoters, which can be used in future breeding program. Cotton breeders have extensively carried out interspecific hybridization for utilization of desirable genes from wild species to cultivated cotton and developed interspecific cotton varieties. Among them, a lot of upland cotton lines with improved traits including fibre quality and insect pest resistance have been developed [ 19 – 22 ]. All these upland cotton lines are designated as introgression lines of interspecific hybridization. These interspecific lines with their practical value in cotton breeding program have changed genetic basis from narrow line to a wide broad base in the present upland cotton germplasm and have broken the bottlenecks of breeding. However, the full potential of interspecific lines have not yet been obtained for beneficial traits exploitation in traditional and advanced breeding programs [ 23 ]. Therefore, this study was designed to evaluate the expression of fibre genes in diverse interspecific lines and Gossypium species and their role in different fibre development stages. Results of this study will be directive for development of high-quality cotton varieties. Materials And Methods DNA Sequence retrieval and primer designing DNA sequences of selected fibre genes ( Expansin A4, BURP Domain protein RD22- like and E6-like ) were retrieved from NCBI website https://www.ncbi.nlm.nih.gov/ . RT-PCR Primers were designed using PRIMER 3.0 software (Table 1). Collection of fibre tissues Three interspecific lines (SL-19, SL-79 and SL-369) of varying fibre length categorized as long fibre (34.7mm), medium fibre (28.5 mm) and short fibre (24.0 mm) along with three parent species ( G. arboreum , G. anomalum and G. hirsutum ) were used for fibre tissue collection. Cotton bolls were collected at different stages (0, 05, 10, 15 and 20 days after anthesis). Collected bolls were rinsed with diethyl pyro carbonate (DEPC) treated water and were stored in liquid nitrogen. These frozen bolls were further used for RNA extraction. Plant RNA extraction and cDNA synthesis RNA was extracted following Gynidium isothiocynate method [ 24 , 25 ]. RNA quality was observed by electrophoresis and monitored under UV light. RNA samples were quantified through nanodrop (Thermo Scientific ND 2000) and concentrations was optimized prior to cDNA synthesis. Extracted RNA from fibre tissues was used for cDNA synthesis. Real Time PCR analysis To certify the sequence for specific gene, BLAST short (http://www.ncbi.nlm.nih.gov) was used. For expression analysis, Real Time PCR was performed by with SYBR Green Super Mix (Bio-Rad, USA) and 10 ng/μl of both set of primers. 18S rRNA constitutive gene primers were used as data normalizer in this assay. Table 1 Primers used for Real Time qPCR Assay Gene annotation Primer pair Primer sequence (5'-3') Primer length Product length (bp) Accession No. 18S rRNA RT18S -F AAACGGCTACCACATCCAAG 20 153 U42827.1 RT18S R CCTCCAATGGATCCTCGTTA 20 E6-like RTE6-F ATGGCTTCCTCACCAAAACTCTTCT 25 211 DQ023519 RTE6-R TTTCAGGGATGAACCTTGGCTCTT 24 Expansin A4 RT EXPF ATGGCAACCAAAACGATGATGT 22 220 DQ204495 RT EXPR AAGCTGCTGTGCTCGTTCCAT 21 BURP Domain RD22-like-like RTRD22-F ATGAAGGTTCTCTCCCCAATTCT 23 198 XM_016894801 RTRD22-R GACGTTTACACCACCACCTCCT 22 Full length gene specific primer designing Full length primers (Table 2) were retrieved from phytozome https://phytozome.jgi.doe.gov/pz/portal.html . Table 2 Detail of full-length fibre genes Gene Accession No Size 5'F 5'R E6-like DQ023519 726 ATGGCTTCCTCACCAAAACTCTTCT TCAGGGTTCGAACTCTTCCTCGCTT Expansin A4 DQ204495 777 ATGGCAACCAAAACGATGATGT TTAAAACTGGCCTCCTTCAAAAGT RD-22 XM_016894801 1008 ATGAAGGTTCTCTCCCCAATTCT TTACTTAGGGACCCAAACAATGT Sequencing of PCR product PCR products of full-length primers were sent to Macrogen Korea for Sanger sequencing. Sequencing PCR was performed using gene specific forward primers. Sequencing comparison of interspecific lines and species Multiple alignment of predicted DNA sequences and phylogenetic tree analysis was performed at https://www.ebi.ac.uk/Tools/msa/clustalo [ 26 ]. In silico analysis of fibre genes Sequence of Sus gene was taken from NCBI database ( https://www.ncbi.nlm.nih.gov/ ) by searching accession number in all data bases. Coding sequences were identified with amino acid residues. Translation of gene sequence into amino acid sequences was done through EXPASY ( https://web.expasy.org/translate/ ) into six reading frames. Theoretical computation of physicochemical properties Basic physiochemical properties and hydropathy index of protein sequences were computed through Expasy’s ProtParam Proteomic server ( http://web.expasy.org/protparam/ ). Functional annotation of protein For Subcellular Location DeepLoc-1.0 ( http://www.cbs.dtu.dk/services/DeepLoc ) databases was used. Moreover, SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/) was used to check existence of signal peptide. Promoter sequence analysis Promoter analysis was carried out at http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ . Results Expression profiling of Expansin A4 , BURP Domain protein RD22-like and E6-like Overall expression of Expansin A4 gene was remarkably high in rapid elongating fibre during 10 DPA in all interspecific lines and Gossypium species. Maximum transcripts were found in SL-19 (Fig. 1). Expression of BURP Domain protein RD22-like was almost remained constant from 10-20 DPA fibre in all genotypes except in Gossypium anomalum . Transcripts of BURP Domain protein RD22-like gene were maximum in 10 DAP fibre as compared to 5 DPA. In all three interspecific lines, highest expression was detected at 15 and 20 DPA fibre stages in SL-19, SL-79 and SL-369 respectively (Fig. 2). Expression pattern of E6-like showed that high expression was detected at 10 and 15 DPA fibre stages predicting its main role in fibre elongation. In interspecific lines, transcripts of E6-like gene were varied from 0 DPA till 20 DPA. In SL-19, expression of fibre gene starts to increase from 0 DPA and reached at maximum level at 15 DPA and after that slightly decreases at 20 DPA (Fig. 3). To validate expression results, the target gene transcriptomic profiles (E 6-like , Expansin A4 & BURP Domain protein RD22-like) were validated by using existing RNA-seq data on Cotton FGD. The results of available fibre specific genes were generally similar with our expression analysis results. Heat map was created on the basis of RNA-seq data of related expressed in transcript per Million (TPM) during different fibre development stages. E6-like , Expansin A4 and BURP Domain RD22-like showed similarity with gene Gh-D05G160200, Gh_A10G149600 and Gh_D05G052400 respectively. (Fig. 4). An expression trend of gradual increasing from 5 DPA to 10 DPA were identified, while similar tendencies were also observed in our experiment. values of log2, day post anthesis and fragments per kilobase of transcript per million mapped reads . Sequence comparison of interspecific lines and species In E6-like DNA sequencing, all interspecific lines exhibited sequences more similar to G. hirsutum as depicted at nucleotide positions 213, 217 and 221-226. In Expansin A4 , interspecific lines were also more closely related to Gossypium hirsutum predicted at 390, 393, 507, 519 & 657bp which also confirm its breeding history. In B BURP Domain RD22-like , it was also predicted that almost all dissimilar nucleotide (241-300, 301-360, 361-420) were observed in G. anomalum as compared to other species of cotton (Fig. 5). In silico analysis of E6-like , Expansin A4 and BURP Domain RD-22 Physicochemical properties Expasy’s Protpam analysis of predicted protein showed that Protein E6-like and RD-22 was characterized as unstable as value of instability index was 47.75 and 44.72 respectively (Table 3). Expansin A4 was characterized as a stable protein with value of instability index of 29.01. Table 3 Physicochemical properties of fibre genes Physicochemical properties E6-like Expansin A4 BURP Domain RD-22 Number of amino acids 241 258 335 Total negatively amino acid charged residues (Asp + Glu) 37 13 35 Total positively amino acid charged residues (Arg + Lys) 25 16 34 Molecular weight 28223.37 27936.46 36595.05 Theoretical pI 5.00 8.36 6.89 Aliphatic index 32.37 62.83 75.64 Grand average of hydropathicity (GRAVY) -1.356 -0.090 -0.266 Instability index (II) 47.75 29.01 44.72 Subcellular Localization DeepLoc analysis designated that protein. Proteins E6-like , Expansin A4 and BURP Domain RD22-like were a membrane soluble protein family. Location in different organelles with the approximate values (Table 4) predicted the probability of protein location in different organelles. Highest Extracellular values of Proteins E6-like , Expansin A4 and BURP Domain RD22-like (0.819, 0.729 and 0.843 respectively) showed that these proteins are extracellular. Table 4 Predicted subcellular localization of E6-like , Expansin A4 and BURP Domain RD-22 Fibre gene Extracellular Lysosome Endoplasmic reticulum Cell membrane Golgi apparatus Cytoplasm E6-like 0.8195 0.1706 0.0083 0.0013 0.0002 0.0002 Expansin A4 0.7293 0.2373 0.0329 0.0005 0 0 BURP Domain RD-22 0.8435 0.1316 0.0237 0.0008 0 0.0003 Signal peptide analysis In E6-like , Expansin-A4 and BURP Domain RD22 were characterizes as extracellular membrane that’s why signal peptide was present in protein coding sequence. Score values of C, S, 3Y is more than 0.45 (Table 5) that shows that peptide signal is present. Table 5 Signal peptide Analysis of E6-like , Expansin A4 and BURP Domain RD22-like Fibre gene Measure Position Value Cut Off Signal Peptide E6-like max.C 26 0.792 max.Y 26 0.840 max.S 15 0.941 Mean S 1-25 0.891 D 1-25 0.868 0.450 Yes Expansin A4 max.C 30 0.427 max.Y 30 0.586 max.S 9 0.950 Mean S 1-29 0.821 D 1-29 0.713 0.45 yes BURP Domain RD22-like max.C 30 0.427 max.Y 30 0.586 max.S 9 0.950 Mean S 1-29 0.821 D 1-29 0.713 0.450 Yes Promoter sequence Analysis Sequence analysis of cotton E6-like , Expansin A4 and BURP Domain protein RD22-like promoter using PlantCARE predicted many vital motifs in this region related to gene expression (Fig. 4). There are few transcriptions activation related motifs along with core promoter elements like TATA and CAAT boxes. These motifs are light responsive, hormone and stress regulated cis elements. These motifs are involved in the light, stress and hormones responsiveness. There were other vital core promoter elements required for promoter activity including TATA box and CAAT box (Tale-6). Cis-acting essential element for the abscisic acid reaction ( Hordeum vulgare ), light response elements ( Arabidopsis thaliana ), gibberellin-enhancer element ( Brassica oleracea ) and element for variation of the palisade mesophyll cells ( Arabidopsis thaliana ) were present in E6-like promoter region. Similarly, in Expansin A4 various cis acting premotor elements were identified. Abscisic acid responsiveness elements were identified in Arabidopsis thaliana , light responsiveness in Zea mays , element responsive for transcription start in Brassica oleracea and MeJA-responsiveness in Hordeum vulgare . In BURP Domain RD22-like , elements essential for light responsiveness were present in Petroselinum crispum while promoter and enhancer regions were identified in Arabidopsis thaliana . MYBHv1 binding site, MeJA and anaerobic induction responsive elements were present in Hordeum vulgare and Zea mays respectively. Table 6 Cis acting promoter elements in promoter region E6-like Site Name Organism Position Strand Score. Sequence Function ABRE Hordeum vulgare 425 - 9 GCAACGTGTC cis-acting element involved in the abscisic acid responsiveness AE-box Arabidopsis thaliana 748 + 8 AGAAACAA part of a module for light response CAAT-box Arabidopsis thaliana 638 + 5 CCAAT common cis-acting element in promoter and enhancer regions CAAT-box Pisum sativum 852 - 5 CAAAT common cis-acting element in promoter and enhancer regions GARE motif Brassica oleracea 615 - 7 TCTGTTG gibberellin-responsive element HD-Zip 1 Arabidopsis thaliana 564 - 8 CAAT(A/T) ATTG element involved in differentiation of the palisade mesophyll cells TATA-box Arabidopsis thaliana 575 - 4 TATA core promoter element around -30 of transcription start TC-richrepeats Nicotiana tabacum 380 + 9 GTTTTCTTAC cis-acting element involved in defense and stress responsiveness TCT-motif Arabidopsis thaliana 384 + 6 TCTTAC part of a light responsive element Expansin A-4 G-Box Pisum sativum 507 - 6 CACGTT cis-acting regulatory element involved in light responsiveness ABRE Arabidopsis thaliana 508 + 5 ACGTG cis-acting element involved in the abscisic acid responsiveness ABRE Arabidopsis thaliana 508 + 5 ACGTG cis-acting element involved in the abscisic acid responsiveness ATC-motif Zea mays 384 - 9 TGCTATCCG part of a conserved DNA module involved in light responsiveness CAAT-box Pisum sativum 361 - 5 CAAAT common cis-acting element in promoter and enhancer regions CAAT-box Arabidopsis thaliana 581 - 8 CCCAATTT common cis-acting element in promoter and enhancer regions CAAT-box Petunia hybrida 694 - 7 TGCCAAC common cis-acting element in promoter and enhancer regions TATA-box Arabidopsis thaliana 527 - 4 TATA core promoter element around -30 of transcription start TGACG-motif Hordeum vulgare 532 - 5 TGACG cis-acting regulatory element involved in the MeJA-responsiveness BURP Domain RD22-like ABRE Triticum aestivum 181 - 9 GACACGTGGC cis-acting element involved in the abscisic acid responsiveness ARE Zea mays 542 + 6 AAACCA cis-acting regulatory element essential for the anaerobic induction Box 4 Petroselinum crispum 450 - 6 ATTAAT part of a conserved DNA module involved in light responsiveness CAAT-box Arabidopsis thaliana 55 + 5 CCAAT common cis-acting element in promoter and enhancer regions CCAAT-box Hordeum vulgare 440 + 6 CAACGG MYBHv1 binding site CGTCA-motif Hordeum vulgare 515 + 5 CGTCA cis-acting regulatory element involved in the MeJA-responsiveness TATA-box Arabidopsis thaliana 291 + 4 TATA core promoter element around -30 of transcription start TGACG-motif Hordeum vulgare 512 + 5 TGACG cis-acting regulatory element involved in the MeJA-responsiveness TGACG-motif Hordeum vulgare 515 - 5 TGACG cis-acting regulatory element involved in the MeJA-responsiveness Discussion Realistic genetic resources are accessible for innovative cotton breeders to make more perfection in crop improvement. Transcriptomic analysis of interspecific lines and Gossypium species for fibre traits identified in this study will improve our understanding of fibre genes that have key role in fibre development. Transcriptomic analysis simplifies the breeding through expression profiling of highly expressed genes. Transcriptomic analysis was performed for the identification of differentially expressed genes at different fibre growth stages in interspecific lines and three Gossypium species. Our study predicts expression analysis of selected fibre genes during 0, 5, 10, 15 and 20 DPA fibre stages. High level variable regulation of genes encoding for fibre development was observed at different stages. Transcriptomic profiling has been effectively used for gene identification in cotton crop [ 27 – 31 ]. Here, we describe transcriptome profiling of genes in cotton fibre through quantitative Real Time PCR. This is the initial comprehensive expression profiling that identified the differentially expressed genes with different stages contributing to fibre development in contrasting interspecific lines of cotton. Real Time PCR results predicted high expression levels specifically in the interspecific lines SL-19 (long staple line) as compared to parent species (Fig. 1 - 3 ) envisaging that when genome of two different species merge with each other, its progenitors possess more DNA content, which can be associated with fibre elongation and amplified size of single-celled fibres. It was also concluded that transgressive segregates are possible with hybrid vigor because of different genome groups of Gossypium , which make it possible to get interspecific lines with good fibre length, fibre strength and fibre fineness [ 32 – 35 ]. Expression profiling was compared with RNA sequence data submitted in different bio projects on FGD (Fig. 5 ). In Expansin A 4 , our results were according to PRJNA490626 project in which transcripts were detected in 5 experiments including fibre development at various stages (0-25 DPA). Maximum expression was at 10 DPA which was similar to our results. GhEXPA4a and GhEXPA4b are specific fibre related genes that exhibited high expression during the fibre initiation and elongation stages (0 to 15 DPA). Over-expression of GhEXPA8 predicted that these genes have ability to improve the fibre length and fineness in cotton crop [ 6 ]. Expansin proteins indorse the spillage between different microfibrils by Hemicellulose and cellulose cleavage [ 36 ]. Moreover, our data also suggested that Expansin protein has essential role in cotton fibre development by enlargement of fibre cells through sliding apart cellulose micro fibrils. Expression levels for E6-like genes was also compared. E6-like gene has similarity with genes Gh-D05G160200 for fibre related gene. It also plays its role fibre development. E6 gene was firstly recognized as fibre gene with high expression during cotton fibre development and similar E6 -like was predicted in Angiosperms [ 13 ]. BURP Domain proteins are known as important proteins that has significant roles in plant growth and stress responses [ 37 , 38 ]. Number of BURP proteins have been recognized and characterized on the basis of sequences features. However, different members from different subfamilies predicted variable expression patterns. In our findings, BURP Domain RD22-like genes actually execute main function in fibre elongation and maturation. Although low copy number of TPM of BURP Domain RD22-like gene were observed but this has a role in fibre development. The cotton fibre related gene (AtRD-22-Like) with over expression in elongating fibre cells, translates a BURP Domain-containing protein [ 9 ]. Cotton plants with high expression of GhRDL1 and GhEXPA1 give more number of bolls, resulting up to 40% more lint yield plant −1 without disturbing fibre quality and non-reproductive growth. [ 9 ]. It is further concluded from the study that there is a direct association between Expansin A4 , E6-like , BURP Domain protein RD22-like and fibre quality traits. Thus, these are key target for improving the fibre characteristics. Transformation of these highly expressed genes in local cotton varieties can fulfill the mechanized textile industry requirements. Moreover, genetically modified cotton produced by over expression of these genes will be the best source for use as a long staple variety or use as a parent in breeding program. Biological sequences comparison in molecular biology and bioinformatics has been an imperative approach to supports analysis, such as prediction of protein sub-cellular localization [ 39 ], Physio chemical properties [ 40 ] and the field of taxonomy [ 41 ]. E6-like was characterized as unstable as value of instability index was 47.75. A protein whose instability index is less than 40 is expected as stable while a value greater than 40 indicates that the protein may be unstable. Similarly, Expansin A4 was characterized as a stable protein with value of instability index of 29.01. An imperative step on this mode is prediction of subcellular localization of each protein. E6-like , Expansin A4 and BURP Domain RD22-like were characterized as a membrane soluble protein family. In silico analysis also confirm the role of genes in fibre elongation, Expansin -A4, BURP Domain protein RD22-like-like and E6-like play its main role in rapid elongation and also with predominantly effect in transition stage of elongation supporting to secondary cell wall synthesis. DNA sequence alignment is a criterion for almost all comparative genomic analyses, including documentation of well-preserved sequence motifs and investigation of genes and species historical relationships [ 42 ]. E6-like , Expansin A4 and BRUP Domain RD22-like PCR amplified full length gene was sequenced and subjected to BLAST analysis followed by multiple sequence alignment of DNA sequence and protein sequence for similarities and differences of interspecific lines and parent species (Fig. 5 ). It was concluded from the sequence comparison of interspecific lines and species of cotton that tri-species introgression lines are more closely related to Gossypium hirsutum as compared to Gossypium arboreum and Gossypium anomalum depicted. This confirms its back crossing with Gossypium hirsutum for yield improvement. These interspecific lines were also originate from BC 4 S 5 population { G. hirsutum × 2( G. arboreum × G. anomalum ) developed at Cytogenetics Section, CCRI, Multan [ 22 ]. In interspecific hybrids of Gossypium , a greater proportion of female gametes than male gametes is generally useful with few exceptions [ 43 ], hence backcross breeding should be subjugated. Review of backcrossing with distinct reference to cotton traits improvement exhibited that during repeated backcrossing one set of chromosomes retained with genes balanced. This technique has been used successfully in crosses of different Gossypium species [ 44 – 46 ]. In silico analysis tries to find proteins with consistent annotations about their interaction and functions in the cellular machinery. An imperative step on this mode is prediction of subcellular localization of each protein. E6 -like, Expansin A4 and BURP Domain RD22-like were characterized as a membrane soluble protein family. In E6-like , Expansin-A4 and BURP Domain RD22-like were characterizes as extracellular membrane that’s why signal peptide was present in protein coding. As validation of specific genes for crop improvement programs is also becoming popular engendering novel properties [ 47 – 49 ].Promoter regions In silico analysis of fibre related gene could be used to predict gene expression profiles in cotton plant. Many stresses resistant, light responsive which can contribute for fibre development were present in E6-like , Expansin A4 and Burp Domain RD22-like (Fig. 6 and Table 6 ). To explore the molecular mechanisms regulating cotton fibre development, promoters of several cotton fibre genes have been identified. E6 was the first of such genes to be reported, and the E6 promoter has been used for engineering cotton fibre quality [ 50 ]. GhRDL1 , a gene highly expressed in cotton fibre cells at the elongation stage, encodes a BURP domain-containing protein [ 51 ], and the GaRDL1 promoter exhibited a trichome-specific activity in transgenic Arabidopsis plants [ 52 ]. The aim of our analysis was to predict promoter and regulatory elements of genes encoding useful stress responsive leading to fibre production. In cotton, basic information related to different cis acting elements was generated to support the effort of improving cotton plant for a stress resistant with more fibre production. Conclusion The SL-19 appeared to be a promising source for cotton quality improvement with maximum expression for all fibre genes. To address the negative correlation between yield and fibre quality, use of genetic engineering is recommended to break this linkage by transferring E6-like , Expansin A4 and BURP Domain RD22-like genes in local cotton cultivars. Declarations Acknowledgements Authors are the whole cotton group working at MNS University of Agriculture, Multan and Central Cotton Research Institute, Multan, for providing technical support and germplasm for this study. Author contributions AAK, NI and ZK designed the research plan. FA carried out the experiments and drafted the manuscript. ZM and AG supported in experimentation and manuscript review and improvement. ZK and NI helped in data analysis. AAK, NI and ZK reviewed the final manuscript. Funding Funding for this study was provided by Pakistan Science Foundation (PSF), Islamabad under PSF-NSFC-IV/Agr/P-MNSUAM (30). The authors are thankful to PSF for supporting this research work. Declarations Conflict of Interests The authors declare that they have no conflict of interests. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. References Sandin G, Peters GM (2018) Environmental impact of textile reuse and recycling–A review. 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Afr J Agric Res 10:1612–1618 Pang C, Du X, Ma Z (2005) The progress of enhancement and utilization of upland cotton elite germplasm with wild cotton genes. Cotton Sci (in Chinese) 17:171–177 Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163:16–20 Dolferus R, Jacobs M, Peacock WJ, Dennis ES (1994) Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene. Plant Physiol 105:1075–1087 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA et al (2007) Clustal W and Clustal X version 2.0. bioinformatics 23: 2947-2948 Arpat A, Waugh M, Sullivan JP, Gonzales M, Frisch D et al (2004) Functional genomics of cell elongation in developing cotton fibers. Plant Mol Biol 54:911–929 Wilkins TA, Arpat AB (2005) The cotton fiber transcriptome. 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Adv Agron 78:139 Senchina DS, Alvarez I, Cronn RC, Liu B, Rong J et al (2003) Rate variation among nuclear genes and the age of polyploidy in Gossypium. Mol Biol Evol 20:633–643 Soltis PS, Marchant DB, Van de Peer Y, Soltis DE (2015) Polyploidy and genome evolution in plants. Curr Opin Genet Dev 35:119–125 McCann MC, Knox JP (2018) Plant cell wall biology: polysaccharides in architectural and developmental contexts.Annual Plant Reviews online:343–366 Yamaguchi-Shinozaki K, Shinozaki K (1993) The plant hormone abscisic acid mediates the drought-induced expression but not the seed-specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis thaliana. Mol Gen Genet MGG 238:17–25 Phillips K, Ludidi N (2017) Drought and exogenous abscisic acid alter hydrogen peroxide accumulation and differentially regulate the expression of two maize RD22-like genes. Sci Rep 7:1–12 Zhao Y, Li X, Qi Z (2014) Novel 2D graphic representation of protein sequence and its application. J Fiber Bioeng Inf 7:23–33 Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD et al (2005) Protein identification and analysis tools on the ExPASy server. The proteomics protocols handbook: 571-607 Huang D-S, Yu H-J (2013) Normalized feature vectors: a novel alignment-free sequence comparison method based on the numbers of adjacent amino acids. IEEE/ACM Trans Comput Biol Bioinf 10:457–467 Kumar S, Filipski A (2007) Multiple sequence alignment: in pursuit of homologous DNA positions. Genome Res 17:127–135 Harland SC, Atteck OM (1941) The genetics of cotton. XVIII. Transference of genes from diploid North American wild cottons (Gossypium thurberi Tod., G. armourianum Kearney, G. aridum comb. nov. Skovsted) to tetraploid New World cottons (G. barbadense L. and G. hirsutum L.). J Genet 42:1–19 Deodikar G (1949) Cytogenetic studies on crosses of G. anomalum with cultivated cottons. I (G. hirsutum× G. anomalum) doubled× G. hirsutum. Indian J Agric Sci 19:389–399 Marappan P, Santhanam V (1962) Breeding behaviour of some arboreum-anomalum backcrosses. Indian Cot Gr Rev 16:24–30 Mehetre SS (2010) Wild Gossypium anomalum: a unique source of fibre fineness and strength.Current Science:58–71 Lata C, Prasad M (2011) Role of DREBs in regulation of abiotic stress responses in plants. J Exp Bot 62:4731–4748 Puranik S, Sahu PP, Srivastava PS, Prasad M (2012) NAC proteins: regulation and role in stress tolerance. Trends Plant Sci 17:369–381 Singh RK, Deshmukh R, Muthamilarasan M, Rani R, Prasad M (2020) Versatile roles of aquaporin in physiological processes and stress tolerance in plants. Plant Physiol Biochem 149:178–189 John M (1996) Metabolic pathway engineering in cotton: Biosynthesis of polyester in fiber; Li C-H, Zhu Y-Q, Meng Y-L, Wang J-W, Xu K-X et al (2002) Isolation of genes preferentially expressed in cotton fibers by cDNA filter arrays and RT-PCR. Plant Sci 163:1113–1120 Wang E, Hall JT, Wagner GJ (2004) Transgenic Nicotiana tabacum L. with enhanced trichome exudate cembratrieneols has reduced aphid infestation in the field. Mol Breeding 13:49–57 Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 10 Feb, 2022 Reviewers invited by journal 10 Feb, 2022 Editor assigned by journal 07 Feb, 2022 First submitted to journal 04 Feb, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1327190","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":83048075,"identity":"69431019-8cf1-4b0e-8c1f-87ca215053d7","order_by":0,"name":"Farzana Ashraf","email":"","orcid":"","institution":"IPBB: Institute of Plant Biology and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Farzana","middleName":"","lastName":"Ashraf","suffix":""},{"id":83048076,"identity":"128e65f8-4c38-473c-b528-2ef9604f87c8","order_by":1,"name":"Asif Ali Khan","email":"","orcid":"","institution":"IPBB: Institute of Plant Biology and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Asif","middleName":"Ali","lastName":"Khan","suffix":""},{"id":83048077,"identity":"3142beb1-09d4-43bb-a6b0-4dd8b80fd25f","order_by":2,"name":"Nadia Iqbal","email":"","orcid":"","institution":"IPBB: Institute of Plant Biology and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Iqbal","suffix":""},{"id":83048078,"identity":"512badbe-d7a3-420b-893e-c2943a4539ac","order_by":3,"name":"Zahid Mahmood","email":"","orcid":"","institution":"Central Cotton Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Zahid","middleName":"","lastName":"Mahmood","suffix":""},{"id":83048079,"identity":"31aad8b5-1c6b-4c3a-a39e-d29be836995a","order_by":4,"name":"Abdul Ghaffar","email":"","orcid":"","institution":"MNSUA: Muhammad Nawaz Shareef University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Abdul","middleName":"","lastName":"Ghaffar","suffix":""},{"id":83048080,"identity":"efc07ae7-93f4-4790-86a1-dc1835635e6b","order_by":5,"name":"Zulqurnain Khan","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-6910-7389","institution":"Muhammad Nawaz Shareef University of Agriculture","correspondingAuthor":true,"prefix":"","firstName":"Zulqurnain","middleName":"","lastName":"Khan","suffix":""}],"badges":[],"createdAt":"2022-02-04 12:14:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1327190/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1327190/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18243626,"identity":"9d27b211-b5c5-46d3-afe3-a0515260331a","added_by":"auto","created_at":"2022-02-15 15:32:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":72910,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiling of \u003cem\u003eExpansin A4\u003c/em\u003e in \u003cem\u003eGosssypium\u003c/em\u003e species and interspecific lines: \u003cstrong\u003eA\u003c/strong\u003e (Expression of \u003cem\u003eExpansin A4\u003c/em\u003e in \u003cem\u003eG. arboreium\u003c/em\u003e), \u003cstrong\u003eB\u003c/strong\u003e (Expression of \u003cem\u003eExpansin A4\u003c/em\u003e in \u003cem\u003eG. hirsutum\u003c/em\u003e), \u003cstrong\u003eC\u003c/strong\u003e (Expression of \u003cem\u003eExpansin A4\u003c/em\u003e in \u003cem\u003eG. anomalum\u003c/em\u003e), \u003cstrong\u003eD\u003c/strong\u003e (Expression of \u003cem\u003eExpansin A4\u003c/em\u003e in SL-19) \u003cstrong\u003eE\u003c/strong\u003e (Expression of \u003cem\u003eExpansin A4\u003c/em\u003e in SL-79), \u003cstrong\u003eF\u003c/strong\u003e (Expression of \u003cem\u003eExpansin A4\u003c/em\u003e in SL-369).\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig01.png","url":"https://assets-eu.researchsquare.com/files/rs-1327190/v1/c545a9e4802d03f3088fb551.png"},{"id":18243629,"identity":"b3c8e93f-2913-42fb-a77c-65e74e1a6ef4","added_by":"auto","created_at":"2022-02-15 15:32:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75516,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiling of \u003cem\u003eBURP Domain RD-22 \u003c/em\u003ein \u003cem\u003eGosssypium\u003c/em\u003e species and interspecific lines: \u003cstrong\u003eA\u003c/strong\u003e (Expression of \u003cem\u003eRD-22\u003c/em\u003e in \u003cem\u003eG. arboreium\u003c/em\u003e), \u003cstrong\u003eB\u003c/strong\u003e (Expression of \u003cem\u003eRD-22\u003c/em\u003e in \u003cem\u003eG. hirsutum\u003c/em\u003e), \u003cstrong\u003eC\u003c/strong\u003e (Expression of \u003cem\u003eRD-22\u003c/em\u003e in \u003cem\u003eG. anomalum\u003c/em\u003e), \u003cstrong\u003eD\u003c/strong\u003e (Expression of \u003cem\u003eRD-22\u003c/em\u003e in SL-19) \u003cstrong\u003eE\u003c/strong\u003e (Expression of \u003cem\u003eRD-22\u003c/em\u003e in SL-79), \u003cstrong\u003eF\u003c/strong\u003e (Expression of \u003cem\u003eRD-22\u003c/em\u003e in SL-369).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig02.png","url":"https://assets-eu.researchsquare.com/files/rs-1327190/v1/6fb307575661549069431f27.png"},{"id":18244405,"identity":"f994c5f2-ddcc-4e56-a2d3-b67774e803d6","added_by":"auto","created_at":"2022-02-15 15:35:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":78054,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiling of \u003cem\u003eE6-like \u003c/em\u003ein \u003cem\u003eGosssypium\u003c/em\u003e species and interspecific lines: \u003cstrong\u003eA\u003c/strong\u003e (Expression of \u003cem\u003eE6-like \u003c/em\u003ein \u003cem\u003eG. arboreium\u003c/em\u003e), \u003cstrong\u003eB\u003c/strong\u003e (Expression of \u003cem\u003eE6-like \u003c/em\u003ein \u003cem\u003eG. hirsutum\u003c/em\u003e), \u003cstrong\u003eC\u003c/strong\u003e (Expression of \u003cem\u003eE6-like \u003c/em\u003ein \u003cem\u003eG. anomalum\u003c/em\u003e), \u003cstrong\u003eD\u003c/strong\u003e (Expression of \u003cem\u003eE6-like \u003c/em\u003ein SL-19) \u003cstrong\u003eE\u003c/strong\u003e (Expression of \u003cem\u003eE6-like \u003c/em\u003ein SL-79), \u003cstrong\u003eF\u003c/strong\u003e (Expression of \u003cem\u003eE6-like \u003c/em\u003ein SL-369).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig03.png","url":"https://assets-eu.researchsquare.com/files/rs-1327190/v1/a5c0079dbd7e25dfdcd0f036.png"},{"id":18244399,"identity":"9f874f9c-a22e-45bd-9ed6-34605e499c0b","added_by":"auto","created_at":"2022-02-15 15:35:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20628,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of expression levels (log-transformed transcript per kilobase million (TPM) values). Figure was generated based on available RNA-seq data of \u003cem\u003eBURP Domain RD2-like-2\u003c/em\u003e, \u003cem\u003eExpansin\u003c/em\u003e and \u003cem\u003eE6-like\u003c/em\u003e submitted bio projects from cotton FGD data base. Red indicates high expression, yellow indicates intermediate expression and green indicates no expression. It is straightforward to identify highly expressed genes in specific tissues from this figure. Tissues are labeled with Days After post anthesis (DPA).\u0026nbsp;Rows indicates the fibre gens and column show the fibre stages (250DPA ovule -25 DPA fibre). The data denotes the logarithm-transformed values of log2, day post anthesis and fragments per kilobase of transcript per million mapped reads\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig04.png","url":"https://assets-eu.researchsquare.com/files/rs-1327190/v1/90291f53d1a12ff8d45f5396.png"},{"id":18244401,"identity":"e5548930-2b2a-4f14-8063-d23ff9762202","added_by":"auto","created_at":"2022-02-15 15:35:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":234485,"visible":true,"origin":"","legend":"\u003cp\u003eDNA sequence alignments of fibre genes. A (\u003cem\u003eE6-like\u003c/em\u003e), B (\u003cem\u003eExpansin A4\u003c/em\u003e), C (\u003cem\u003eBURP Domain\u003c/em\u003e \u003cem\u003eRD22-like\u003c/em\u003e). White shadings indicate the polymorphic nucleotides. Interspecific lines and \u003cem\u003eGossypium\u003c/em\u003e species names are indicated in the left and number of bases depicted in each line is marked by the number shown at the top right of each section.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig05.png","url":"https://assets-eu.researchsquare.com/files/rs-1327190/v1/03d873298b5552252b0376fa.png"},{"id":18245002,"identity":"cd45251e-e009-44f5-83db-eda9af10c506","added_by":"auto","created_at":"2022-02-15 15:38:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":587161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e analysis of promoter sequences of fibre genes. \u003cstrong\u003eA\u003c/strong\u003e (cis-acting regulatory elements in \u003cem\u003eE6-like\u003c/em\u003e)-like, \u003cstrong\u003eB\u003c/strong\u003e (cis-acting regulatory elements in \u003cem\u003eExpansin A4\u003c/em\u003e), \u003cstrong\u003eC\u003c/strong\u003e (cis-acting regulatory elements in \u003cem\u003eBURP Domain RD-22\u003c/em\u003e) Highlighted regions show cis regulatory motifs present in the promoter regions with specific function.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig06.png","url":"https://assets-eu.researchsquare.com/files/rs-1327190/v1/d62f5241234fd0de3274e9fa.png"},{"id":18245025,"identity":"f3084539-36a7-4ae1-9970-88c5c07fae7d","added_by":"auto","created_at":"2022-02-15 15:38:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1202236,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1327190/v1/45d2d8f4-d861-43b2-87b9-3b62df003782.pdf"}],"financialInterests":"","formattedTitle":"In Silico Analysis and Expression Profiling of Expansin A4, BURP Domain protein RD22- like and E6-like Genes Associated with Fiber Quality in Cotton","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobally, synthetic fibre consumption is continuously increasing and projected to reach at 130 million tons by 2030. The consumption of synthetic fibre is 62.7% compared to 24.3% cotton fibre consumption [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Competition of cotton fibre with polyester is creating negative influence on the demand of cotton. Genetic improvement of cotton for fibre traits is very crucial to meet the challenges of the textile industry. So, there is need to devise clear-cut policies for cotton breeding program to enhance the quality cotton production. In a breeding program, germplasm collection, its conservation and utilization, trait specific screening programs and modern genomics have key role in variety development [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Cotton genetic resources have been extensively studied over the last many decades to introduce valuable traits in cotton [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These genetic resources include wild \u003cem\u003eGossypium\u003c/em\u003e germplasm, innovative cytogenetic stocks with specific chromosomes additions or deletions in different species, large mapping families, recombinant inbred lines, near isogenic lines and interspecific lines. While there are some queries about narrow genetic base of these cultivars and most breeders would admit that in breeding programs maximum utilization of genetic diversity within their material should be ensured. Breeders will have to utilize wild cotton relatives, as well as advance lines or cultivars to develop cotton varieties with superior traits.\u003c/p\u003e \u003cp\u003eAt the cellular level, cotton fibre development is supported by several genes which facilitates the elongation process, for example, \u003cem\u003eExpansins\u003c/em\u003e are involved in fibre elongation at various development stages [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. High transcript abundance of \u003cem\u003eGhEXP1\u003c/em\u003e was observed in cotton fibre during the elongation phase of fibre development, which steadily decreased from 16 to 20 DPA [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In cotton, \u003cem\u003eGhEXPA1\u003c/em\u003e along with \u003cem\u003eGhRDL1\u003c/em\u003e showed an increase in fiber length and an enlargement of endopleura cells of ovules [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The BURP Domain is a plant-specific protein characterized by repetitive units of amino acid [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This protein is mainly involved in promoting the fibre cells elongation when over-expressed. Because \u003cem\u003eGhRDL1\u003c/em\u003e directly interacts with cotton α-\u003cem\u003eExpansin\u003c/em\u003e fibre gene therefore, \u003cem\u003eExpansin\u003c/em\u003es mediate \u003cem\u003eGhRDL1\u0026rsquo;s\u003c/em\u003e effect on overall fibre cell enlargement [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It was suggested that E6 protein is involved in fibre development, but no support was present to justify this hypothesis as no conclusive evidence was presented [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. When E6 antisense suppression construct was used, there was knockdown to uncover a phenotype \u003cem\u003eE6-like\u003c/em\u003e. E6 proteins play a comprehensive role in cell wall, and are deposited during fibre elongation, which give high transcripts in fibre cell during transcriptomic analysis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTranscriptional profiling is a unique tool to gain knowledge about gene mechanisms, regulatory pathways, and gene expression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Number of techniques are used for specific gene expression studies but Real Time PCR is the most reliable technology for absolute and comparative quantification of the gene transcription [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This comprehensive wide-ranging gene expression study is supportive to sightsee the role of genes, which are up regulated, entirely expressed, or down regulated during different cotton fibre development stages. Through transcriptomic data, one can explain the fibre expansion process and can discover highly expressed genes for the development of transgenic cotton varieties with superior fibre traits. Profiling of fibre genes in interspecific lines will enable us to unravel variable expression pattern of selected fibre genes.\u003c/p\u003e \u003cp\u003eApplication of \u003cem\u003ein silico\u003c/em\u003e methods along with expression profiling is important for characterization of fibre genes. DNA sequence of interspecific lines and \u003cem\u003eGossypium\u003c/em\u003e species were aligned to have information about differences and similarities. Diploid and tetraploid genomes of various \u003cem\u003eGossypium\u003c/em\u003e species have repeatedly sequences making their entire genome sequences. These valuable repeatedly sequenced data revealed the evolutionary history of the cotton with polyploidization and decaploidization leading to the of the formation of genus \u003cem\u003eGossypium\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Multiple sequence alignment approaches envisage algorithmic explanation about evolutionarily sequences alignments. Fibre genes were subjected to BLAST analysis for expression validation and multiple DNA sequence alignment for similarities and differences of interspecific lines and parent species. Genomics combines recombinant DNA technology, DNA sequencing and bioinformatics sequence to analyze the structure and function of genes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Bioinformatics is a systematic field that utilizes advance approaches for computational analysis of biological data [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Bioinformatics also aids to recognize different promoters involve in fibre yield and quality, abiotic stress tolerance and disease resistance. Strength and specificity related character of promoter sequence can be exhibited through expression profiling. Strong promoters predict high expression and vice versa. Fibre genes protein \u003cem\u003eE6\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and BURP \u003cem\u003eDomain RD22-like\u003c/em\u003e also have strong promoters, which can be used in future breeding program.\u003c/p\u003e \u003cp\u003eCotton breeders have extensively carried out interspecific hybridization for utilization of desirable genes from wild species to cultivated cotton and developed interspecific cotton varieties. Among them, a lot of upland cotton lines with improved traits including fibre quality and insect pest resistance have been developed [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. All these upland cotton lines are designated as introgression lines of interspecific hybridization. These interspecific lines with their practical value in cotton breeding program have changed genetic basis from narrow line to a wide broad base in the present upland cotton germplasm and have broken the bottlenecks of breeding. However, the full potential of interspecific lines have not yet been obtained for beneficial traits exploitation in traditional and advanced breeding programs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, this study was designed to evaluate the expression of fibre genes in diverse interspecific lines and \u003cem\u003eGossypium\u003c/em\u003e species and their role in different fibre development stages. Results of this study will be directive for development of high-quality cotton varieties.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eDNA Sequence retrieval and primer designing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA sequences of selected fibre genes (\u003cem\u003eExpansin A4, BURP Domain protein RD22- like and E6-like\u003c/em\u003e) were retrieved from NCBI website\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/a\u003e. RT-PCR Primers were designed using PRIMER 3.0 software (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of fibre tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree interspecific lines (SL-19, SL-79 and SL-369) of varying fibre length categorized as long fibre (34.7mm), medium fibre (28.5 mm) and short fibre (24.0 mm) along with three parent species (\u003cem\u003eG. arboreum\u003c/em\u003e, \u003cem\u003eG. anomalum\u003c/em\u003e and \u003cem\u003eG. hirsutum\u003c/em\u003e) were used for fibre tissue collection. Cotton bolls were collected at different stages (0, 05, 10, 15 and 20 days after anthesis). Collected bolls were rinsed with diethyl pyro carbonate (DEPC) treated water and were stored in liquid nitrogen. These frozen bolls were further used for RNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant RNA extraction and cDNA synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted following Gynidium isothiocynate method\u0026nbsp;[\u003ca href=\"#_ENREF_24\" title=\"Logemann, 1987 #678\"\u003e24\u003c/a\u003e,\u003ca href=\"#_ENREF_25\" title=\"Dolferus, 1994 #679\"\u003e25\u003c/a\u003e]. RNA quality was observed by electrophoresis and monitored under UV light. RNA samples were quantified through nanodrop (Thermo Scientific ND 2000) and concentrations was optimized prior to cDNA synthesis. Extracted RNA from fibre tissues was used for cDNA synthesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal Time PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo certify the sequence for specific gene, BLAST short (http://www.ncbi.nlm.nih.gov) was used. For expression analysis, Real Time PCR was performed by with SYBR Green Super Mix (Bio-Rad, USA) and 10 ng/\u0026mu;l of both set of primers. 18S rRNA constitutive gene primers were used as data normalizer in this assay.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u0026nbsp;\u003c/strong\u003ePrimers used for Real Time qPCR Assay\u003c/p\u003e\n\u003ctable align=\"left\" border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"16.49484536082474%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene annotation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer pair\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.927835051546392%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer sequence \u0026nbsp; \u0026nbsp;(5\u0026apos;-3\u0026apos;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.34020618556701%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer length\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProduct length (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.371134020618557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"16.49484536082474%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e18S rRNA\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003eRT18S -F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.927835051546392%\"\u003e\n \u003cp\u003eAAACGGCTACCACATCCAAG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.34020618556701%\"\u003e\n \u003cp\u003e20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"12.371134020618557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eU42827.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.454545454545453%\"\u003e\n \u003cp\u003eRT18S R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"54.54545454545455%\"\u003e\n \u003cp\u003eCCTCCAATGGATCCTCGTTA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"16.49484536082474%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE6-like\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003eRTE6-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.927835051546392%\"\u003e\n \u003cp\u003eATGGCTTCCTCACCAAAACTCTTCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.34020618556701%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"12.371134020618557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDQ023519\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.454545454545453%\"\u003e\n \u003cp\u003eRTE6-R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"54.54545454545455%\"\u003e\n \u003cp\u003eTTTCAGGGATGAACCTTGGCTCTT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"16.49484536082474%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eExpansin A4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003eRT EXPF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.927835051546392%\"\u003e\n \u003cp\u003eATGGCAACCAAAACGATGATGT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.34020618556701%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"12.371134020618557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDQ204495\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.454545454545453%\"\u003e\n \u003cp\u003eRT EXPR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"54.54545454545455%\"\u003e\n \u003cp\u003eAAGCTGCTGTGCTCGTTCCAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"16.49484536082474%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBURP Domain RD22-like-like\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003eRTRD22-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.927835051546392%\"\u003e\n \u003cp\u003eATGAAGGTTCTCTCCCCAATTCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.34020618556701%\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"12.371134020618557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eXM_016894801\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.454545454545453%\"\u003e\n \u003cp\u003eRTRD22-R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"54.54545454545455%\"\u003e\n \u003cp\u003eGACGTTTACACCACCACCTCCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eFull length gene specific primer designing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFull length primers (Table 2) were retrieved from phytozome\u0026nbsp;\u003ca href=\"https://phytozome.jgi.doe.gov/pz/portal.html\"\u003ehttps://phytozome.jgi.doe.gov/pz/portal.html\u003c/a\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003e Detail of full-length fibre genes\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccession No\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.185567010309279%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSize\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.927835051546392%\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u0026apos;F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"31.95876288659794%\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u0026apos;R\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE6-like\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003eDQ023519\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.185567010309279%\"\u003e\n \u003cp\u003e726\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.927835051546392%\"\u003e\n \u003cp\u003eATGGCTTCCTCACCAAAACTCTTCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"31.95876288659794%\"\u003e\n \u003cp\u003eTCAGGGTTCGAACTCTTCCTCGCTT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eExpansin A4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003eDQ204495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.185567010309279%\"\u003e\n \u003cp\u003e777\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.927835051546392%\"\u003e\n \u003cp\u003eATGGCAACCAAAACGATGATGT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"31.95876288659794%\"\u003e\n \u003cp\u003eTTAAAACTGGCCTCCTTCAAAAGT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eRD-22\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003eXM_016894801\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.185567010309279%\"\u003e\n \u003cp\u003e1008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"30.927835051546392%\"\u003e\n \u003cp\u003eATGAAGGTTCTCTCCCCAATTCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"31.95876288659794%\"\u003e\n \u003cp\u003eTTACTTAGGGACCCAAACAATGT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing of PCR product\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePCR products of full-length primers were sent to Macrogen Korea for Sanger sequencing. Sequencing PCR was performed using gene specific forward primers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing comparison of interspecific lines and species\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultiple alignment of predicted DNA sequences and phylogenetic tree analysis was \u0026nbsp;performed at\u0026nbsp;\u003ca href=\"https://www.ebi.ac.uk/Tools/msa/clustalo\"\u003ehttps://www.ebi.ac.uk/Tools/msa/clustalo\u003c/a\u003e [\u003ca href=\"#_ENREF_26\" title=\"Larkin, 2007 #858\"\u003e26\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn silico\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eanalysis of fibre genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence of Sus gene was taken from NCBI database \u003cstrong\u003e(\u003c/strong\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/a\u003e) by searching accession number in all data bases. Coding sequences were identified with amino acid residues. Translation of gene sequence into amino acid sequences was done through EXPASY (\u003ca href=\"https://web.expasy.org/translate/\"\u003ehttps://web.expasy.org/translate/\u003c/a\u003e) into six reading frames.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTheoretical computation of physicochemical properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBasic physiochemical properties and hydropathy index of protein sequences were computed through Expasy\u0026rsquo;s ProtParam Proteomic server (\u003ca href=\"http://web.expasy.org/protparam/\"\u003ehttp://web.expasy.org/protparam/\u003c/a\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional annotation of protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor Subcellular Location DeepLoc-1.0 (\u003ca href=\"http://www.cbs.dtu.dk/services/DeepLoc\"\u003ehttp://www.cbs.dtu.dk/services/DeepLoc\u003c/a\u003e) databases was used. Moreover, SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/) was used to check existence of signal peptide.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePromoter sequence analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePromoter analysis was carried out at\u0026nbsp;\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/a\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExpression profiling of \u003cem\u003eExpansin A4\u003c/em\u003e, \u003cem\u003eBURP Domain protein RD22-like\u0026nbsp;\u003c/em\u003eand \u003cem\u003eE6-like\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall expression of \u003cem\u003eExpansin A4\u003c/em\u003e gene was remarkably high in rapid elongating fibre during 10 DPA in all interspecific lines and \u003cem\u003eGossypium\u0026nbsp;\u003c/em\u003especies. Maximum transcripts were found in SL-19 (Fig. 1). Expression of \u003cem\u003eBURP Domain protein RD22-like\u003c/em\u003e was almost remained constant from 10-20 DPA fibre in all genotypes except in \u003cem\u003eGossypium anomalum\u003c/em\u003e. Transcripts of \u003cem\u003eBURP Domain protein RD22-like\u0026nbsp;\u003c/em\u003egene were maximum in 10 DAP fibre as compared to 5 DPA. In all three interspecific lines, highest expression was detected at 15 and 20 DPA fibre stages in SL-19, SL-79 and SL-369 respectively (Fig. 2). Expression pattern of \u003cem\u003eE6-like\u003c/em\u003e showed that high expression was detected at 10 and 15 DPA fibre stages predicting its main role in fibre elongation. In interspecific lines, transcripts of \u003cem\u003eE6-like\u003c/em\u003e gene were varied from 0 DPA till 20 DPA. In SL-19, expression of fibre gene starts to increase from 0 DPA and reached at maximum level at 15 DPA and after that slightly decreases at 20 DPA (Fig. 3).\u003c/p\u003e\n\u003cp\u003eTo validate expression results, the target gene transcriptomic profiles (E\u003cem\u003e6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e \u0026amp; \u003cem\u003eBURP Domain protein\u0026nbsp;\u003c/em\u003eRD22-like) were validated by using existing RNA-seq data on Cotton FGD. The results of available fibre specific genes were generally similar with our expression analysis results. Heat map was created on the basis of RNA-seq data of related expressed in transcript per Million (TPM) during different fibre development stages. \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBURP Domain RD22-like\u003c/em\u003e showed similarity with gene Gh-D05G160200, \u003ca href=\"https://cottonfgd.org/profiles/gene/Gh_A10G149600/\" target=\"_blank\"\u003eGh_A10G149600\u003c/a\u003e and \u003ca href=\"https://cottonfgd.org/profiles/gene/Gh_D05G052400/\" target=\"_blank\"\u003eGh_D05G052400\u003c/a\u003e respectively. (Fig. 4). An expression trend of gradual increasing from 5 DPA to 10 DPA were identified, while similar tendencies were also observed in our experiment.\u003c/p\u003e\n\u003cp\u003evalues of log2, day post anthesis and fragments per kilobase of transcript per million mapped reads\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence comparison of interspecific lines and species\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eE6-like\u003c/em\u003e DNA sequencing, all interspecific lines exhibited sequences more similar to \u003cem\u003eG. hirsutum\u003c/em\u003e as depicted at nucleotide positions 213, 217 and 221-226. In \u003cem\u003eExpansin A4\u003c/em\u003e, interspecific lines were also more closely related to \u003cem\u003eGossypium hirsutum\u003c/em\u003e predicted at 390, 393, 507, 519 \u0026amp; 657bp which also confirm its breeding history. In B\u003cem\u003eBURP Domain RD22-like\u003c/em\u003e, it was also predicted that almost all dissimilar nucleotide (241-300, 301-360, 361-420) were observed in \u003cem\u003eG. anomalum\u003c/em\u003e as compared to other species of cotton (Fig. 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn silico\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eanalysis of \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBURP Domain RD-22\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical properties\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpasy\u0026rsquo;s Protpam analysis of predicted protein showed that Protein \u003cem\u003eE6-like\u003c/em\u003e and \u003cem\u003eRD-22\u0026nbsp;\u003c/em\u003ewas characterized as unstable as value of instability index was 47.75 and 44.72 respectively (Table 3). \u003cem\u003eExpansin A4\u0026nbsp;\u003c/em\u003ewas characterized as a stable protein with value of instability index of 29.01.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003ePhysicochemical properties of fibre genes\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.484848484848484%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhysicochemical properties\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE6-like\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eExpansin A4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBURP Domain RD-22\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.484848484848484%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of amino acids\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e258\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e335\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.484848484848484%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal negatively amino acid charged residues (Asp + Glu)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.484848484848484%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal positively amino acid charged residues (Arg + Lys)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.484848484848484%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMolecular weight\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e28223.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e27936.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e36595.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.484848484848484%\"\u003e\n \u003cp\u003e\u003cstrong\u003eTheoretical pI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e5.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e8.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e6.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.484848484848484%\"\u003e\n \u003cp\u003e\u003cstrong\u003eAliphatic index\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e32.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e62.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e75.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.484848484848484%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGrand average of hydropathicity (GRAVY)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e-1.356\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e-0.090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e-0.266\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"48.484848484848484%\"\u003e\n \u003cp\u003e\u003cstrong\u003eInstability index (II)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e47.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e29.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.171717171717173%\"\u003e\n \u003cp\u003e44.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eSubcellular Localization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeepLoc analysis designated that protein. Proteins \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBURP Domain RD22-like\u0026nbsp;\u003c/em\u003ewere a membrane soluble protein family. Location in different organelles with the approximate values (Table 4) predicted the probability of protein location in different organelles. Highest Extracellular values of Proteins \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBURP Domain RD22-like\u0026nbsp;\u003c/em\u003e(0.819, 0.729 and 0.843 respectively) showed that these proteins are extracellular.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003ePredicted subcellular localization of \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBURP Domain\u003c/em\u003e \u003cem\u003eRD-22\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"17.52577319587629%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFibre gene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e\u003cstrong\u003eExtracellular\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.371134020618557%\"\u003e\n \u003cp\u003e\u003cstrong\u003eLysosome\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e\u003cstrong\u003eEndoplasmic reticulum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.402061855670103%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell membrane\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.34020618556701%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGolgi apparatus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCytoplasm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"17.52577319587629%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE6-like\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e0.8195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.371134020618557%\"\u003e\n \u003cp\u003e0.1706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.0083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.402061855670103%\"\u003e\n \u003cp\u003e0.0013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.34020618556701%\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"17.52577319587629%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eExpansin A4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e0.7293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.371134020618557%\"\u003e\n \u003cp\u003e0.2373\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.0329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.402061855670103%\"\u003e\n \u003cp\u003e0.0005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.34020618556701%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"17.52577319587629%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBURP Domain\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eRD-22\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e0.8435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.371134020618557%\"\u003e\n \u003cp\u003e0.1316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.0237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.402061855670103%\"\u003e\n \u003cp\u003e0.0008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.34020618556701%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.463917525773196%\"\u003e\n \u003cp\u003e0.0003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eSignal peptide analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin-A4\u003c/em\u003e and BURP Domain RD22 were characterizes as extracellular membrane that\u0026rsquo;s why signal peptide was present in protein coding sequence. Score values of C, S, 3Y is more than 0.45 (Table 5) that shows that peptide signal is present.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e\u0026nbsp; \u0026nbsp;Signal peptide Analysis of \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBURP Domain RD22-like\u003c/em\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25.510204081632654%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFibre gene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.306122448979592%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeasure\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.26530612244898%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.306122448979592%\"\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.244897959183673%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCut Off\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.367346938775512%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSignal Peptide\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"25.510204081632654%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE6-like\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.306122448979592%\"\u003e\n \u003cp\u003emax.C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.26530612244898%\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.306122448979592%\"\u003e\n \u003cp\u003e0.792\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.244897959183673%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.367346938775512%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003emax.Y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.840\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003emax.S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003eMean S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e1-25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.891\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e1-25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.868\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\n \u003cp\u003e0.450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"25.510204081632654%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eExpansin A4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.306122448979592%\"\u003e\n \u003cp\u003emax.C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.26530612244898%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.306122448979592%\"\u003e\n \u003cp\u003e0.427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.244897959183673%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.367346938775512%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003emax.Y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.586\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003emax.S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.950\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003eMean S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e1-29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.821\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e1-29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.713\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\n \u003cp\u003eyes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"25.510204081632654%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBURP Domain RD22-like\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.306122448979592%\"\u003e\n \u003cp\u003emax.C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.26530612244898%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.306122448979592%\"\u003e\n \u003cp\u003e0.427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.244897959183673%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"18.367346938775512%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003emax.Y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.586\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003emax.S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.950\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003eMean S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e1-29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.821\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"17.80821917808219%\"\u003e\n \u003cp\u003e1-29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"20.54794520547945%\"\u003e\n \u003cp\u003e0.713\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"16.438356164383563%\"\u003e\n \u003cp\u003e0.450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.65753424657534%\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003ePromoter sequence Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence analysis of cotton \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBURP Domain protein RD22-like\u003c/em\u003epromoter using PlantCARE predicted many vital motifs in this region related to gene expression (Fig. 4). There are few transcriptions activation related motifs along with core promoter elements like TATA and CAAT boxes. These motifs are light responsive, hormone and stress regulated cis elements. These motifs are involved in the light, stress and hormones responsiveness. There were other vital core promoter elements required for promoter activity including TATA box and CAAT box (Tale-6).\u0026nbsp;Cis-acting essential element for the abscisic acid reaction (\u003cem\u003eHordeum vulgare\u003c/em\u003e), light response elements (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e),\u0026nbsp;gibberellin-enhancer element (\u003cem\u003eBrassica oleracea\u003c/em\u003e) and element for variation of the palisade mesophyll cells (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e) were present in \u003cem\u003eE6-like\u003c/em\u003e promoter region. Similarly, in \u003cem\u003eExpansin A4\u003c/em\u003e various cis acting premotor elements were identified. Abscisic acid responsiveness elements were identified in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, light responsiveness in \u003cem\u003eZea mays\u003c/em\u003e, element responsive for transcription start in \u003cem\u003eBrassica oleracea\u003c/em\u003e and MeJA-responsiveness in \u003cem\u003eHordeum vulgare\u003c/em\u003e. In \u003cem\u003eBURP Domain RD22-like\u003c/em\u003e, elements essential for light responsiveness were present in \u003cem\u003ePetroselinum crispum\u003c/em\u003e while promoter and enhancer regions were identified in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. MYBHv1 binding site, MeJA and anaerobic induction responsive elements were present in \u003cem\u003eHordeum vulgare\u0026nbsp;\u003c/em\u003eand \u003cem\u003eZea mays\u003c/em\u003e respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6\u0026nbsp;\u003c/strong\u003eCis acting promoter elements in promoter region\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"98%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE6-like\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSite Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrganism\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrand\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e\u003cstrong\u003eScore.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFunction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27GCAACGTGTC%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eABRE\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eHordeum vulgare\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eGCAACGTGTC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting element involved in the abscisic acid responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27AGAAACAA%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eAE-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eAGAAACAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003epart of a module for light response\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27CCAAT%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eCAAT-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCCAAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecommon cis-acting element in promoter and enhancer regions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27CAAAT%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eCAAT-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003ePisum sativum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e852\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCAAAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecommon cis-acting element in promoter and enhancer regions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TCTGTTG%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eGARE motif\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eBrassica oleracea\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTCTGTTG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003egibberellin-responsive element\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27CAAT%28A%2FT%29ATTG%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eHD-Zip 1\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCAAT(A/T) ATTG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003eelement involved in differentiation of the palisade mesophyll cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TATA%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eTATA-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTATA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecore promoter element around -30 of transcription start\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27GTTTTCTTAC%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eTC-richrepeats\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eNicotiana tabacum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e380\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eGTTTTCTTAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting element involved in defense and stress responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TCTTAC%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eTCT-motif\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e384\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTCTTAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003epart of a light responsive element\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eExpansin A-4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27CACGTT%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eG-Box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003ePisum sativum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCACGTT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting regulatory element involved in light responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27ACGTG%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eABRE\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e508\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eACGTG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting element involved in the abscisic acid responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27ACGTG%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eABRE\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e508\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eACGTG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting element involved in the abscisic acid responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TGCTATCCG%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eATC-motif\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eZea mays\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e384\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTGCTATCCG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003epart of a conserved DNA module involved in light responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27CAAAT%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eCAAT-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003ePisum sativum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCAAAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecommon cis-acting element in promoter and enhancer regions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27CCCAATTT%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eCAAT-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e581\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCCCAATTT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecommon cis-acting element in promoter and enhancer regions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TGCCAAC%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eCAAT-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003ePetunia hybrida\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e694\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTGCCAAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecommon cis-acting element in promoter and enhancer regions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TATA%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eTATA-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e527\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTATA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecore promoter element around -30 of transcription start\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TGACG%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eTGACG-motif\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eHordeum vulgare\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e532\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTGACG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting regulatory element involved in the MeJA-responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBURP Domain RD22-like\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27GACACGTGGC%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eABRE\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eTriticum aestivum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e181\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eGACACGTGGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting element involved in the abscisic acid responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27AAACCA%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eARE\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eZea mays\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e542\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eAAACCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting regulatory element essential for the anaerobic induction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27ATTAAT%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eBox 4\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003ePetroselinum crispum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eATTAAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003epart of a conserved DNA module involved in light responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27CCAAT%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eCAAT-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCCAAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecommon cis-acting element in promoter and enhancer regions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27CAACGG%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eCCAAT-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eHordeum vulgare\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCAACGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003eMYBHv1 binding site\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27CGTCA%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eCGTCA-motif\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eHordeum vulgare\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e515\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eCGTCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting regulatory element involved in the MeJA-responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TATA%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eTATA-box\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e291\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTATA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecore promoter element around -30 of transcription start\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TGACG%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eTGACG-motif\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eHordeum vulgare\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTGACG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting regulatory element involved in the MeJA-responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"9.375%\"\u003e\n \u003cp\u003e\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/cgi-bin/show_site_info.htpl?QWhere=ID_of_Site%20like%20%27TGACG%27\u0026StartAt=0\u0026NbRecs=10\"\u003e\u003cstrong\u003eTGACG-motif\u003c/strong\u003e\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eHordeum vulgare\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"8.333333333333334%\"\u003e\n \u003cp\u003e515\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.291666666666667%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.583333333333334%\"\u003e\n \u003cp\u003eTGACG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"38.541666666666664%\"\u003e\n \u003cp\u003ecis-acting regulatory element involved in the MeJA-responsiveness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eRealistic genetic resources are accessible for innovative cotton breeders to make more perfection in crop improvement. Transcriptomic analysis of interspecific lines and \u003cem\u003eGossypium\u003c/em\u003e species for fibre traits identified in this study will improve our understanding of fibre genes that have key role in fibre development. Transcriptomic analysis simplifies the breeding through expression profiling of highly expressed genes. Transcriptomic analysis was performed for the identification of differentially expressed genes at different fibre growth stages in interspecific lines and three \u003cem\u003eGossypium\u003c/em\u003e species. Our study predicts expression analysis of selected fibre genes during 0, 5, 10, 15 and 20 DPA fibre stages. High level variable regulation of genes encoding for fibre development was observed at different stages. Transcriptomic profiling has been effectively used for gene identification in cotton crop [\u003cspan additionalcitationids=\"CR28 CR29 CR30\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Here, we describe transcriptome profiling of genes in cotton fibre through quantitative Real Time PCR.\u003c/p\u003e \u003cp\u003eThis is the initial comprehensive expression profiling that identified the differentially expressed genes with different stages contributing to fibre development in contrasting interspecific lines of cotton. Real Time PCR results predicted high expression levels specifically in the interspecific lines SL-19 (long staple line) as compared to parent species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) envisaging that when genome of two different species merge with each other, its progenitors possess more DNA content, which can be associated with fibre elongation and amplified size of single-celled fibres. It was also concluded that transgressive segregates are possible with hybrid vigor because of different genome groups of \u003cem\u003eGossypium\u003c/em\u003e, which make it possible to get interspecific lines with good fibre length, fibre strength and fibre fineness [\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExpression profiling was compared with RNA sequence data submitted in different bio projects on FGD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In \u003cem\u003eExpansin A 4\u003c/em\u003e, our results were according to PRJNA490626 project in which transcripts were detected in 5 experiments including fibre development at various stages (0-25 DPA). Maximum expression was at 10 DPA which was similar to our results. GhEXPA4a and GhEXPA4b are specific fibre related genes that exhibited high expression during the fibre initiation and elongation stages (0 to 15 DPA). Over-expression of \u003cem\u003eGhEXPA8\u003c/em\u003e predicted that these genes have ability to improve the fibre length and fineness in cotton crop [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Expansin proteins indorse the spillage between different microfibrils by Hemicellulose and cellulose cleavage [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, our data also suggested that Expansin protein has essential role in cotton fibre development by enlargement of fibre cells through sliding apart cellulose micro fibrils. Expression levels for \u003cem\u003eE6-like\u003c/em\u003e genes was also compared. \u003cem\u003eE6-like\u003c/em\u003e gene has similarity with genes Gh-D05G160200 for fibre related gene. It also plays its role fibre development. \u003cem\u003eE6\u003c/em\u003e gene was firstly recognized as fibre gene with high expression during cotton fibre development and similar \u003cem\u003eE6\u003c/em\u003e-like was predicted in Angiosperms [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBURP Domain proteins are known as important proteins that has significant roles in plant growth and stress responses [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Number of BURP proteins have been recognized and characterized on the basis of sequences features. However, different members from different subfamilies predicted variable expression patterns. In our findings, \u003cem\u003eBURP Domain RD22-like\u003c/em\u003e genes actually execute main function in fibre elongation and maturation. Although low copy number of TPM of \u003cem\u003eBURP Domain RD22-like\u003c/em\u003egene were observed but this has a role in fibre development. The cotton fibre related gene (AtRD-22-Like) with over expression in elongating fibre cells, translates a BURP Domain-containing protein [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Cotton plants with high expression of GhRDL1 and \u003cem\u003eGhEXPA1\u003c/em\u003e give more number of bolls, resulting up to 40% more lint yield plant\u003csup\u003e\u0026minus;1\u003c/sup\u003e without disturbing fibre quality and non-reproductive growth. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is further concluded from the study that there is a direct association between \u003cem\u003eExpansin A4\u003c/em\u003e, \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eBURP Domain protein RD22-like\u003c/em\u003e and fibre quality traits. Thus, these are key target for improving the fibre characteristics. Transformation of these highly expressed genes in local cotton varieties can fulfill the mechanized textile industry requirements. Moreover, genetically modified cotton produced by over expression of these genes will be the best source for use as a long staple variety or use as a parent in breeding program.\u003c/p\u003e \u003cp\u003eBiological sequences comparison in molecular biology and bioinformatics has been an imperative approach to supports analysis, such as prediction of protein sub-cellular localization [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], Physio chemical properties [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and the field of taxonomy [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. \u003cem\u003eE6-like\u003c/em\u003e was characterized as unstable as value of instability index was 47.75. A protein whose instability index is less than 40 is expected as stable while a value greater than 40 indicates that the protein may be unstable. Similarly, \u003cem\u003eExpansin A4\u003c/em\u003e was characterized as a stable protein with value of instability index of 29.01. An imperative step on this mode is prediction of subcellular localization of each protein. \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBURP Domain RD22-like\u003c/em\u003e were characterized as a membrane soluble protein family. \u003cem\u003eIn silico\u003c/em\u003e analysis also confirm the role of genes in fibre elongation, \u003cem\u003eExpansin\u003c/em\u003e-A4, \u003cem\u003eBURP Domain protein RD22-like-like\u003c/em\u003eand \u003cem\u003eE6-like\u003c/em\u003e play its main role in rapid elongation and also with predominantly effect in transition stage of elongation supporting to secondary cell wall synthesis.\u003c/p\u003e \u003cp\u003eDNA sequence alignment is a criterion for almost all comparative genomic analyses, including documentation of well-preserved sequence motifs and investigation of genes and species historical relationships [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBRUP Domain RD22-like\u003c/em\u003e PCR amplified full length gene was sequenced and subjected to BLAST analysis followed by multiple sequence alignment of DNA sequence and protein sequence for similarities and differences of interspecific lines and parent species (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It was concluded from the sequence comparison of interspecific lines and species of cotton that tri-species introgression lines are more closely related to \u003cem\u003eGossypium hirsutum\u003c/em\u003e as compared to \u003cem\u003eGossypium arboreum\u003c/em\u003e and \u003cem\u003eGossypium anomalum\u003c/em\u003e depicted. This confirms its back crossing with \u003cem\u003eGossypium hirsutum\u003c/em\u003e for yield improvement. These interspecific lines were also originate from BC\u003csub\u003e4\u003c/sub\u003eS\u003csub\u003e5\u003c/sub\u003e population {\u003cem\u003eG. hirsutum\u003c/em\u003e \u0026times; 2(\u003cem\u003eG. arboreum\u003c/em\u003e \u0026times; \u003cem\u003eG. anomalum\u003c/em\u003e) developed at Cytogenetics Section, CCRI, Multan [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In interspecific hybrids of \u003cem\u003eGossypium\u003c/em\u003e, a greater proportion of female gametes than male gametes is generally useful with few exceptions [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], hence backcross breeding should be subjugated. Review of backcrossing with distinct reference to cotton traits improvement exhibited that during repeated backcrossing one set of chromosomes retained with genes balanced. This technique has been used successfully in crosses of different \u003cem\u003eGossypium\u003c/em\u003e species [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn silico\u003c/em\u003e analysis tries to find proteins with consistent annotations about their interaction and functions in the cellular machinery. An imperative step on this mode is prediction of subcellular localization of each protein. \u003cem\u003eE6\u003c/em\u003e-like, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBURP Domain RD22-like\u003c/em\u003e were characterized as a membrane soluble protein family. In \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin-A4\u003c/em\u003e and \u003cem\u003eBURP Domain RD22-like\u003c/em\u003e were characterizes as extracellular membrane that\u0026rsquo;s why signal peptide was present in protein coding. As validation of specific genes for crop improvement programs is also becoming popular engendering novel properties [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].Promoter regions \u003cem\u003eIn silico\u003c/em\u003e analysis of fibre related gene could be used to predict gene expression profiles in cotton plant. Many stresses resistant, light responsive which can contribute for fibre development were present in \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBurp Domain RD22-like\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Table \u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). To explore the molecular mechanisms regulating cotton fibre development, promoters of several cotton fibre genes have been identified. \u003cem\u003eE6\u003c/em\u003e was the first of such genes to be reported, and the \u003cem\u003eE6\u003c/em\u003e promoter has been used for engineering cotton fibre quality [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. \u003cem\u003eGhRDL1\u003c/em\u003e, a gene highly expressed in cotton fibre cells at the elongation stage, encodes a BURP domain-containing protein [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], and the \u003cem\u003eGaRDL1\u003c/em\u003e promoter exhibited a trichome-specific activity in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The aim of our analysis was to predict promoter and regulatory elements of genes encoding useful stress responsive leading to fibre production. In cotton, basic information related to different cis acting elements was generated to support the effort of improving cotton plant for a stress resistant with more fibre production.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe SL-19 appeared to be a promising source for cotton quality improvement with maximum expression for all fibre genes. To address the negative correlation between yield and fibre quality, use of genetic engineering is recommended to break this linkage by transferring \u003cem\u003eE6-like\u003c/em\u003e, \u003cem\u003eExpansin A4\u003c/em\u003e and \u003cem\u003eBURP Domain RD22-like\u003c/em\u003e genes in local cotton cultivars.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are the whole cotton group working at MNS University of Agriculture, Multan and Central Cotton Research Institute, Multan, for providing technical support and germplasm for this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAAK, NI and ZK designed the research plan. FA carried out the experiments and drafted the manuscript. ZM and AG supported in experimentation and manuscript review and improvement. ZK and NI helped in data analysis. AAK, NI and ZK reviewed the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this study was provided by Pakistan Science Foundation (PSF), Islamabad under PSF-NSFC-IV/Agr/P-MNSUAM (30). The authors are thankful to PSF for supporting this research work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSandin G, Peters GM (2018) Environmental impact of textile reuse and recycling\u0026ndash;A review. 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Mol Breeding 13:49\u0026ndash;57\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"DNA sequencing, Expression analysis, Fibre genes, In silico analysis, Cotton ","lastPublishedDoi":"10.21203/rs.3.rs-1327190/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1327190/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eWild \u003cem\u003eGossypium\u003c/em\u003e species and races are rich source of genetic polymorphism due to environmental dispersal and continuous natural selection. These genetic resources hold mass of outclass genes that can be used in cotton improvement breeding programs to exploit possible traits such as fibre quality, abiotic stress tolerance, and disease and insect resistance. Therefore, use of new molecular techniques such as genomics, transcriptomics and bioinformatics is very important to utilize the genetic potential of wild species in cotton improvement programs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eInterspecific lines and \u003cem\u003eGossypium\u003c/em\u003e species used in the study were grown at Central Cotton Research Institute (CCRI), Multan. After retrieving DNA sequence of the genes from NCBI, the primers for gene expression and full-length gene sequence were designed. Expression profiling of \u003cem\u003eExpansin A4\u003c/em\u003e, \u003cem\u003eBURP Domain protein RD22-like \u003c/em\u003eand \u003cem\u003eE6-like\u003c/em\u003e fibre genes was performed through Real Time PCR. BLAST and DNA sequence alignment was conducted for sequence comparison of interspecific lines and \u003cem\u003eGossypium \u003c/em\u003especies. Different \u003cem\u003ein silico\u003c/em\u003e analysis were used for characterization of fibre genes and identification of cis acting promoter elements in promoter region.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eVariable expression of genes related to fibre development was observed at different stages. BLAST and DNA sequence alignment exhibited resemblance of interspecific lines with \u003cem\u003eG. hirsutum\u003c/em\u003e. \u003cem\u003eIn silico\u003c/em\u003e analysis on the sequence data also confirmed the role of \u003cem\u003eExpansin A4\u003c/em\u003e, \u003cem\u003eBURP Domain protein RD22-like \u003c/em\u003eand \u003cem\u003eE6-like\u003c/em\u003e fibre genes in fibre development. Similarly, several stress tolerant and light responsive cis acting elements were identified through promotor analysis, which may contribute for fibre development in the breeding programs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eExpansin A4\u003c/em\u003e, \u003cem\u003eBURP Domain RD22-like \u003c/em\u003eand \u003cem\u003eE6-like\u003c/em\u003e have positive role in fibre development with variable expression at fiber length and strength associated stages.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"In Silico Analysis and Expression Profiling of Expansin A4, BURP Domain protein RD22- like and E6-like Genes Associated with Fiber Quality in Cotton","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-15 15:32:36","doi":"10.21203/rs.3.rs-1327190/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2022-02-11T04:56:08+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-02-11T03:51:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-02-07T16:01:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2022-02-04T07:14:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"eee46cdd-ed1f-4b9a-b1fa-80dfca8b0c13","owner":[],"postedDate":"February 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-03-25T14:46:52+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-15 15:32:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1327190","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1327190","identity":"rs-1327190","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}