Genetic Diversity Assessment of Bixa orellana L., a Natural Seed Dye Species of the Tropics, using ISSR Markers

Genetic Diversity Assessment of Bixa orellana L., a Natural Seed Dye Species of the Tropics, using ISSR Markers | 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 Genetic Diversity Assessment of Bixa orellana L., a Natural Seed Dye Species of the Tropics, using ISSR Markers Manasa Mogilicharla, Reeja S, VBS Lalitha T, Arjun Ramachandran, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6660535/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bixa orellana .L is an important dye yielding tree. The bixin content in the seeds yields a red colour pigment in varying colours which is used as dye. The variation in bixin content could be attributed to both environmental and genetic changes. In this study a total of 24 genotypes of Bixa orellana .L were raised from FC&RI, Annatto gene bank of Mettupalayam, India and their genetic diversity were analysed using ISSR markers. Out of 33 primers used, 23 primers were effectively screened for amplification and their polymorphism ranged from 50–100% indicating high genetic diversity among the genotypes. The values of polymorphic information content (PIC) ranged from 0.50–0.60. The other marker traits viz., effective marker ratio, discriminating power and heterozygosity are in higher values revealing the effectiveness of the ISSR markers. The total number of bands varied from 6 to 13. Among all the bands generated, 22 unique bands were detected in eight genotypes, out of which TSMLGB18 produced maximum number of unique bands. The UPGMA clustering pattern revealed that 24 genotypes were divided into seven distinct clusters. The genetic relationship obtained through Jaccard’s similarity co-efficient ranged between 0.57 to 0.88. The result provided valid guidelines for collection, conservation and characterization of Bixa orellana genetic resources. Bixa orellana PIC unique bands genetic diversity UPGMA ISSR markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Bixa orellana is a medicinal dye source plant. It is normally admitted as “annatto”, “sindhur”, “lipstick tree”. Originally, it is a native of South American region and has its place in their culinary as a food ingredient and the most important ingredient of beauty products (Alcazar-Alay et al. , 2017). The seed coat extract gives out red orange pigment dye known as bixin that industrially competes well in the food and textile industries as a natural colorant and dye. It is the second most commercial non- toxic food colorant as compared to all other natural food colorants (Lopa pattanaik et al., 2019 ). Other than the fact that it is used in textile industries, it has medicinal values that escalates its demands. It is a small tree or shrub that is perennially foliated and belongs to Bixaceae family. It is the only one in the Bixaceae family that has well-known uses. It possesses wide adaptability and is well distributed all over India in the following states Tamilnadu, Kerala, Karnataka, Chhattisgarh, Maharashtra, Madhya Pradesh, Gujarat and Andhra Pradesh (Kumaran K,2014). It may flourish in the temperature between 24℃ – 38℃ with various soils. A mean annual rainfall demand was 800-1500mm. It can even reach the height of 6 meters, if it will be provided with good irrigation (Kumaran K,2014). The leaf is simple, smooth when mature, long petioles and are arranged alternating the branches (Franco et al., 2008 ; Leal and Clavijo, 2012 ). The flowers are vibrant and big, color ranges from white through to shades of pink and violet. They are bisexual and possess five sepals and they will usually see them flower right at the tips of the branches. The fruit is dehiscent, ovoid capsule made up of two/three carpels; covered by soft spines when young. It can be reddish, greenish or brownish and about 3–5 cm in length; it contains dark seeds covered in a reddish waxy Aril (bixin) with an unsettling smell. The fruits are borne in clusters as every fruit can hold up to 17 pods (Dequigiovani et al., 2017 ). The seeds in a given fruit may vary from 50–60 seeds. It is capable of adjusting itself to numerous ecological and edaphic conditions (Kumaran K et al. , 2014), means that, there is enough genetic variability available which can be exploited for its appropriate repercussions. Environmental factors have a dominant effect on the presence of bixin in seeds (Smith et al. , 2006). Due to environmental impact amount of bixin varies from place to place thereby suggesting variation is on population basis and not on individual variation (Vilares et al., 1992 ). The study of variation by the help of morphometric techniques was the dominating one earlier, and now, it is refined by the help of molecular techniques to evaluate the genetic variation which is less made in Bixa orellana. Environmental influences have a minimal impact on molecular markers and one can carry out analysis from DNA at any stage of development of the organism. PCR-assisted molecular markers – like random amplified polymorphic DNA (RAPD; Methods, such as, inter-simple sequence repeat (ISSR; Zietkiewicz et al. , 1994), amplified fragment length polymorphism (AFLP; Vos et al., 1995 ), sequence-related amplified polymorphism (SRAP; Li & Quiros 2001 )—have emerged However, little work is known of the diversity and genetic structure of this species (Diquigiovanni et al. ,2017). Until recently the genetic diversity analysis on that of DNA markers viz. microsatellites, SRAP and RAPD (Sequence Related Amplified product and Randomly Amplified Polymorphic DNA) were used to evaluate genetic diversity in bixa. The current research attempts to evaluate the reliability of ISSR markers in the task of studying genetic variability of Bixa orellana which wasn’t realized earlier. ISSR comes to the dominant marker class; they are emerged as an alternative system, with reliability and benefits of SSR markers and therefore it is reliable, fast and very informative system for studying the diversity (Zhang et al., 2017 ). The present study made an attempt to study genetic diversity in the studied genotypes of Bixa orellana. Materials and Methodology Plant material; To assess the genetic variation, seeds of Bixa were brought from field germplasm bank of Forest college & Research Institute(FCRI), (latitude 11 9’N and longitude 76 56’E) TNAU, Mettupalayam. The seeds were the individual collections from the progenies which are initially raised from the seeds of plus trees obtained from different parts of Southern India. For the purpose of this study, seeds from 24 such progenies (genotypes) were used to raise the seedlings & assembled at FCRI, Mulugu (17 º .728544 N to 78 º .63296 E), Telangana and were given accession codes (Table 1 ). Genomic DNA was extracted from young leaves following the standard method of CTAB, based on the procedure given by Doyle and Doyle ( 1991 ) with some minor changes. Table 1 Details of accession number and genotypes of Bixa orellana .L Accession No. Genotypes TSMLGB1 G1 TSMLGB2 G2 TSMLGB3 G3 TSMLGB4 G4 TSMLGB5 G5 TSMLGB6 G6 TSMLGB7 G7 TSMLGB8 G8 TSMLGB9 G9 TSMLGB10 G10 TSMLGB11 G11 TSMLGB12 G12 TSMLGB13 G13 TSMLGB14 G14 TSMLGB15 G15 TSMLGB16 G16 TSMLGB17 G17 TSMLGB18 G18 TSMLGB19 G19 TSMLGB20 G20 TSMLGB21 G21 TSMLGB22 G22 TSMLGB23 G23 TSMLGB24 G24 The leaf tissues of Bixa orellana (400mg) were grinded into to fine powder in liquid nitrogen and homogenized it with extraction buffer by adding 3ml of CTAB buffer,1%PVP, 250 µl of beta-mercapto-ethanol and incubated for 30min at By using the ratio of 24:1 v/v of chloroform and isoamyl-alcohol solution supernant was extracted twice. 25 µl of RNase A was introduced in the extract which was then kept for incubation at room temperature. With the chilled iso-propanol, the DNA was pelleted and washed 2 times with 70% ethanol. Then air dried pellet added with 200 µl of nuclease free grade water and stored at -20°C. The quality of DNA was tested in nanodrop (ND1000, Thermo Scientific, US) spectrophotometer at 260 and 280 nm and quantity was measured in ng/µl. Portions that had between 200 µl to 600 µl DNA were best suited for PCR process. ISSR amplification Total of 33 random UBC primers were screened initially to assess genetic diversity among 24 genotypes of B.orellana. The 10 µl volume of ISSR PCR reaction mixture composed of 5 µl PCR emerald master mix, 2 µl water, 1.5 µl selected primer, 1.5 µl DNA sample. PCR reactions were performed as, i) denaturation was done at 94º for 4 minutes followed by ii) Annealing of primer at which temperature was specific to each primer and run for 1 minute then iii) Extension was done at 72ºC for 2minutes.The reaction was repeated for 30 cycles. The amplified product was run for 1 hour at 100 V in 1.2% agarose gel (Himedia) immersed in 1X TBE buffer by placing the 1kb DNA ladder as reference. After running, the gel was scanned under UV Gel scanner and photographed in the system (Biorad). Data analysis DNA fingerprint patterns received were transformed into binary data matrices consisting of arrays of 0 and 1 s. The bands were awarded points based on their presence (1) or lack (0) of bands of different molecular weight sizes. Polymorphic and reproducible bands were the only ones taken into account for the analysis. Data were analysed with SIMQUAL route for creating Jaccard’s similarity coefficient in NTSYS-pc version 2.02i (Numerical Taxonomy System) (Rohlf 1998). Dendrograms were done with using similarity matrices UPGMA (Unweighted Pair Group Method with Arithmetic Average) algorithm. The percentage polymorphism was done in Microsoft-excel. Some of the important measures which states the informativeness of marker was calculated using an online tool i.e.,marker efficiency calculator ( Amiryousefi et al ,2018; https://irscope.shinyapps.io/iMEC/ ). Polymorphic Information Content (PIC) and Heterozygosity(H) were calculated using Botstein et al. ( 1980 ) and Liu ( 1998 ). PIC = 1-(∑ pi2 – Σ pi2 pj2) When pi and pj are population frequency of the ith and jth allele. H = 1 – Σ pi2, in this pi is the frequency of the allele for the ith allele. Effective Multiplex Ratio (EMR) and Marker Index (MI) was calculated according to Powell et al. (1996). EMR = n β, β = np / (np + nnp), p and np represent polymorphic and nonpolymorphic fraction of the markers, hence np and nnp refer to the counting numbers of the same. MI = EMR*Havp, Havp refers to an average heterozygosity for the polymorphic makers. It is equal to Σ Hn/np, where, Hn represents the markers’ polymorphic fraction’s heterozygosity, and the summation is done over all the polymorphic loci np. The discriminating power of a marker was determined by D = 1-C (Tessier et al., 1999 ). Where, C = Σ pi (Npi-1)/(N-1), N – Individuals, C – Totality of loci for all the patterns formed by the primer. Results Among the 33 ISSR primers used, 23 primers that yielded distinct,reproducible and polymorphic banding profiles were screened for subsequent molecular charachterization (Table 2 ). The total number of bands generated is 249 and per primer it is 10.8. Maximum number of bands were generated by UBC 826(9) while minimum was reported by ISSR-O4(6) with an average band size varied from 100bp to 3500bp. The polymorphism ranged from 50–100% among all genotypes. Of the 23 primers used in the study, 15 primers have shown 100% polymorphism viz., UBC841, UBC857, UBC826, UBC815, UBC888, UBC891, UBC810, UBC845, UBC847, UBC864, UBC880, ISSR-O3, ISSR-O4, ISSR-O6, ISSR-F2 (Fig. 1 ). Minimum polymorphism was reported in UBC823 (50%) followed by UBC 864(62.5%) (Fig. 2 ). The extent of polymorphism by all primers was presented in Table 3 . Among all the bands generated, twenty-two bands were detected as unique among eight genotypes (Table 4 ), out of which TSMLGB18 was recorded for maximum number of unique bands (Fig. 3 &Figure 4 ). Molecular markers particular to 24 genotypes were identified; this can be transformed into SCAR primers, to increase accuracy and reliability of the marker assisted selection. Table 5 were presented to show the molecular marker characters. The highest PIC was showed by UBC 809(0.60) followed by ISSR O6(0.59) and the minimum was reported for the UBC 857(0.505). All the 23 markers had > 0.50 PIC reporting the informative nature of the marker. The heterozygosity ranged from 0.2 to 0.6. In UBC 864 the highest values for PIC was 0.60 and that of heterozygosity was 0.66. The estimates of marker properties for ISSRs had wide range. The effective MP ratio is 1 for all the primers. Marker index (MI) was 0.66(UBC 864) to 0.24(ISSR O6). The maximum discriminating value was found in the case of UBC811 (0.86) and minimal in UBC 809(0.31). Table 2 Primers selected for amplification and their specific annealing temperatures S .NO Primer Sequence Annealing Temperature 1 UBC 811 GAGAGAGAGAGAGAGC 43⁰C 2 UBC 841 GAGAGAGAGAGAGAYC 40⁰C 3 UBC 857 ACACACACACACACACG 59⁰C 4 UBC 826 ACACACACACACACACC 54⁰C 5 UBC 815 CTCTCTCTTCTCTCTG 41⁰C 6 UBC 888 BDBCACACACACACACA 44⁰C 7 UBC 889 DBDACACACACACACAC 39⁰C 8 UBC 891 HVHTGTGTGTGTGTGTG 52⁰C 9 UBC 813 CTCTCTCTCTCTCTCTT 41⁰C 10 ISSR M5 (GA)9-C 43⁰C 11 ISSR-03 (CT)8-GC 51⁰C 12 ISSR-04 (CA)6-AC 34⁰C 13 ISSR-06 (CT)6-AG 34⁰C 14 ISLA(CT)8TG (CT)8-TG 43⁰C 15 ISSR F2 (AG)8-CG 54⁰C 16 UBC-809 AGAGAGAGAGAGAGAG 40⁰C 17 UBC-810 GAGAGAGAGAGAGAGT 44⁰C 18 UBC-823 TCTCTCTCTCTCTCTCC 43⁰C 19 UBC-840 GAGAGAGAGAGAGAGT 51⁰C 20 UBC-845 CTCTCTCTCTCTCTCTRG 43⁰C 21 UBC-847 CACACACACACACACC 44⁰C 22 UBC-864 ATGATGATGATGATGTG 49⁰C 23 UBC-880 GGAGAGGAGAGGAGA 49⁰C Table 3 Percentage polymorphism of selected primers S .NO Primer Sequence 5’-3’ Per cent polymorphism 1 UBC 811 GAGAGAGAGAGAGAGC 87.5% 2 UBC 841 GAGAGAGAGAGAGAYC 100% 3 UBC 857 ACACACACACACACACG 100% 4 UBC 826 ACACACACACACACACC 100% 5 UBC 815 CTCTCTCTTCTCTCTG 100% 6 UBC 888 BDBCACACACACACACA 100% 7 UBC 889 DBDACACACACACACAC 66.6% 8 UBC 891 HVHTGTGTGTGTGTGTG 100% 9 UBC 813 CTCTCTCTCTCTCTCTT 77.7% 10 ISSR M5 (GA)9-C 75% 11 ISSR-03 (CT)8-GC 100% 12 ISSR-04 (CA)6-AC 100% 13 ISSR-06 (CT)6-AG 100% 14 ISLA(CT)8TG (CT)8-TG 83.3% 15 ISSR F2 (AG)8-CG 100% 16 UBC-809 AGAGAGAGAGAGAGAG 66.6% 17 UBC-810 GAGAGAGAGAGAGAGT 100% 18 UBC-823 TCTCTCTCTCTCTCTCC 50% 19 UBC-840 GAGAGAGAGAGAGAGT 62.5% 20 UBC-845 CTCTCTCTCTCTCTCTRG 100% 21 UBC-847 CACACACACACACACC 100% 22 UBC-864 ATGATGATGATGATGTG 87.5% 23 UBC-880 GGAGAGGAGAGGAGA 100% Table 4 Details of unique bands displayed by different primers SL.NO PRIMER GENOTYPE UNIQUE BAND (base pairs) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 ISSR-03 ISSR-04 UBC-810 UBC-823 ISLA(CT) 8 TG UBC-864 ISSR-06 UBC-811 UBC-841 UBC-889 UBC-826 UBC-880 ISSR M5 UBC-813 UBC-891 UBC-840 UBC-809 UBC-815 UBC-847 TSMLGB18 TSMLGB19 TSMLGB18 TSMLGB18 TSMLGB19 TSMLGB18 TSMLGB24 TSMLGB17 TSMLGB18 TSMLGB1 TSMLGB18 TSMLGB18 TSMLGB17 TSMLGB18 TSMLGB18 TSMLGB24 TSMLGB18 TSMLGB1 TSMLGB2 TSMLGB17 TSMLGB18 TSMLGB6 TSMLGB18 TSMLGB16 TSMLGB18 TSMLGB18 TSMLGB2 TSMLGB13 TSMLGB2 TSMLGB18 TSMLGB8 TSMLGB18 1000 2500 800,750,600 1200,800 2100 1200,900,800,500 1300 1100,800 1400 1500 400 1300,1250,1000,900 650,300 1500,900 2000,1000,100 300 1250,800,550,400 250 1100 350 2300,1600,1250,180 1400 350 1400,900 1750,500 1500,1400,1000,750 800 1750 650 1200 3100,500 2000,500,300 Table 5 List of ISSR primers and their molecular characters Primer H PIC EMR MI D UBC 811 0.46 0.51 1 0.46 0.86 UBC 841 0.48 0.51 1 0.48 0.67 UBC 857 0.49 0.50 1 0.49 0.70 UBC 826 0.42 0.54 1 0.42 0.51 UBC 815 0.42 0.53 1 0.42 0.52 UBC 888 0.45 0.52 1 0.44 0.56 UBC 889 0.32 0.57 1 0.31 0.35 UBC 891 0.34 0.57 1 0.34 0.38 UBC 813 0.38 0.55 1 0.37 0.43 ISSR M5 0.29 0.58 1 0.29 0.32 ISSR-03 0.34 0.56 1 0.34 0.39 ISSR-04 0.33 0.57 1 0.33 0.38 ISSR-06 0.24 0.59 1 0.24 0.26 ISLA(CT)8TG 0.34 0.57 1 0.34 0.34 ISSR F2 0.37 0.58 1 0.37 0.38 UBC-809 0.30 0.60 1 0.30 0.31 UBC-810 0.53 0.55 1 0.53 0.53 UBC-823 0.53 0.57 1 0.53 0.54 UBC-840 0.56 0.56 1 0.56 0.57 UBC-845 0.60 0.55 1 0.60 0.61 UBC-847 0.60 0.55 1 0.60 0.62 UBC-864 0.66 0.55 1 0.66 0.66 UBC-880 0.60 0.54 1 0.60 0.62 The Jaccard’s co-efficient of similarity varied from 0.57(between TSMLGB18 and TSMLGB1) to 0.88 (between TSMLGB10 and TSMLGB11) (Fig. 5 ). A dendrogram based UPGMA analysis with ISSR data was shown in Fig. 5 .The dendrogram divided the 24 genotypes into 7 clusters based on Jaccard’s coefficient similarity matrix at 0.75 similarity. Cluster I consisted of TSMLGB1, TSMLGB13 and cluster II includes maximum number (15) of genotypes viz., TSMLGB3, TSMLGB4, TSMLGB6, TSMLGB8, TSMLGB5, TSMLGB7, TSMLGB9, TSMLGB10, TSMLGB11, TSMLGB12, TSMLGB14, TSMLGB15, TSMLGB17, TSMLGB19 and TSMLGB20.Cluster III includes TSMLGB16, cluster IV has TSMLGB21, TSMLGB22, TSMLGB24.Cluster V, VI, VII consisted of single genotype each namely TSMLGB23, TSMLGB2, TSMLGB18 respectively. Discussion In the present investigation, ISSR markers were successfully employed in molecular diversity analysis uto assess the phylogenetic relationships at the intraspecific level amongst 24 genotypes of B.orellana collected from the germplasm of TNAU, Mettupalayam, India. The 23 ISSR primers generated 249 bands among 24 genotypes, the molecular band size covered from 100 bp to 3500bp.The amplified fragment ranged from 6(ISSR-O4) to 13(UBC 826) with an average of 10.8 bands per primer. Similar results were reported in Elaeocarpus serratus (Raji R et al ., 2021), Ricinus communis (Gajera et al., 2010 ), Pistacia khinjuk (Karimi et al., 2012 ) and Citrullus colocynthis (Verma et al., 2017 ) for the number of bands in ISSR markers. The primers have recorded high to moderate polymorphism indicating wider genetic diversity in the species. Similar results for ISSR markers have been reported in various woody species viz., J atropha curcas (Singh et al .2010), Pinus roxburghii (Rawat ,2014), Pongamia pinnata (Pradeep et al. , 2010), Simarouba glauca (Sekar et al. , 2003), Albizia lebbeck (Radhakrishnan et al,. 2001), Tectona grandis (Parthibhan 2001). The presence of unique bands facilitates the discrimination of the unknown genotypes, indicating that TSMLGB18 can be reliably identified based on its unique bands. The size of the amplified products varied from 100bp-3000bp and the PIC ranged between 0.6 − 0.5 (Table 3 ), signifying ISSR as an informative molecular marker. The high heterozygosity, multiplex ratio and discriminating power of markers revealed by the study exhibited the proficiency of ISSR markers for genetic diversity studies of Bixa orellana . The results were in line with the other species for marker values viz., Elaeocarpus serratus (Raji R et al. ,2021), Acacia senegal (Josiah et al. 2008),and Populus cathayana (Z lu et al. ,2006). The Jaccard’s co-efficient of similarity varied from 0.57(between TSMLGB18 and TSMLGB1) to 0.88 (between TSMLGB10 and TSMLGB11) which indicated moderate level of genetic variation among the genotypes in the study. The highest JC value between TSMLGB10 and TSMLGB11 indicate their genetic similarity, whereas low JC between TSMLGB1 and TSMLGB18 denotes significant genetic differences. UPGMA cluster placed TSMLGB18 distantly from all other genotypes in the study possibly due to its distinctiveness whereas TSMLGB10 and TSMLGB11 placed closely at 0.88 similarity could rendered to its similarity in genetic structure. Generally, the individuals from different geographic origins have different genetic constitution due to its natural selection for different traits (Raji et al. , 2021) and fall in same cluster based on their origin. The present study is a novel attempt to evaluate genetic diversity using ISSR markers in Bixa orellana . The molecular analysis grouped the genotypes into 7 clusters. The wide divergence and a broad genetic base allow for identification and selection of divergent parents for any hybridization or improvement plans and contribute significantly to the future breeding and conservation initiatives in the species. The study also revealed considerable variation in banding patterns, high heterozygosity and wide range of polymorphism among the genotypes, which demonstrated the suitability of ISSR markers as powerful tools for genetic characterization, diversity assessment and population structure analysis of Bixa orellana genetic resources. Previous studies on genetic diversity analysis using ISSR markers were reported on many species with similar results including Santalum album (Sandeep et al., 2020 ), Eucalyptus cladocalyx (Ballesta et al .,2015), Elaeocarpus serratus (Raji et al .2021), Morus spp (Tikadar et al. , 2003), Tectona grandis (Ansari et al., 2012 ), Bamboo spp (Amom et al., 2020 ) elucidating the effectiveness of ISSR markers in analysing the diversity. Conclusion This study presents a novel application of ISSR markers for assessing genetic diversity in Bixa orellana . The analysis revealed high levels of polymorphism, demonstrating the reliability and informativeness of ISSR markers for genetic characterization in this species. The substantial genetic variability observed highlights the heterogeneous nature of B. orellana , indicating considerable potential for heterosis in future breeding efforts. A well-designed breeding strategy could facilitate the development of superior progenies with enhanced bixin content and associated agronomic traits. These findings provide a valuable genetic framework for the selection of diverse and promising genotypes for future genetic advancement programs targeting bixin yield improvement. Abbreviations ISSR : Inter Simple Sequence Repeats SSR : Simple Sequence Repeats UPGMA : Unweighted pair group mean arthematics DNA : De-oxy ribo Nucleic Acid CTAB : Cetyl trimethyl ammonium bromide UBC : University of British Columbia PCR : Polymerase Chain Reaction ISSR : Inter simple sequence repeats UV : Ultraviolet SIMQUAL : Similarity for Qualitative data SCAR : Sequence Characterized Amplified Region TNAU : Tamilnadu Agriculture University TBE : Tris/Borate/EDTA kb : Kilobytes ml : Milli-litres µl : Micro-litres nm : Nano-metres min : Minutes % : Percentage °C : Degree-centigrade Declarations Acknowledgment; The infrastructure support provided by Forest College and Research Institute, Mulugu, is greatly appreciated by the authors. We are very grateful to FC&RI, Mettupalayam, Tamilnadu for supplying the seed material on schedule. Conflict of interest: Authors do not have any conflict of interests to declare. Ethical issues: None. Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution Manasa conducted the entire research as part of her postgraduate studies. Reeja framed the research objectives, design and methodology and provided guidance. Lalitha supported the research execution, in field and lab, while Arjun contributed to the selection of primers and supported statistical interpretations. Shalini participated in design and methodology. Kumaran provided the seed material from germplasm bank, Tamil Nadu. Sreedhar assisted in statistical analysis and Chiranjeeva provided technical guidance during the seedling nursery stage. All authors have read and approved the final manuscript. References Alcázar-Alay S. C., Osorio-Tobón J. F., Forster-Carneiro T., and Meireles M. A. A. (2017) Obtaining bixin from semi-defatted annatto seeds by a mechanical method and solvent extraction: process integration and economic evaluation. Food research international , 99 : 393–402. Amiryousefi A, Hyvönen J, Poczai P. (2018) iMEC: Online Marker Efficiency Calculator. Appl Plant Sci. Jun 24;6(6):e01159. doi: 10.1002/aps3.1159 . PMID: 30131901; PMCID: PMC6025818. Amom T., Tikendra L., Apana N., Goutam M., Sonia P., Koijam A.S. and Nongdam P. (2020) Efficiency of RAPD, ISSR, iPBS, SCoT and phytochemical markers in the genetic relationship study of five native and economical important bamboos of North-East India. Phytochemistry , 174 : 112–330. Ansari S. A., Narayanan C., Wali S. A., Kumar R., Shukla N. and Rahangdale S. K. (2012) ISSR markers for analysis of molecular diversity and genetic structure of Indian teak ( Tectona grandis Lf) populations. Annals of Forest Research , 55 (1): 11–23. Ballesta P., Mora F., Contreras-Soto R. I., Ruiz E. and Perret S. (2015)Analysis of the genetic diversity of Eucalyptus cladocalyx (sugar gum) using ISSR markers. Acta Scientiarum. Agronomy , 37 :133–140. Botstein, D., White R. L., Skolnick M., and Davis R. W. (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics 32: 314–331. Dequigiovani G., Ramos S. L. F., Alves-Pereira A., Fabri E. G., Carvalho P. R. N., da Silva M. G. and Veasey E. A. (2017) Genetic diversity and structure in a major Brazilian annatto ( Bixa orellana ) germplasm bank revealed by microsatellites and phytochemical compounds. Genetic Resources and Crop Evolution , 64 (7): 1775–1788. Doyle, J. (1991) DNA protocols for plants. In Molecular techniques in taxonomy (pp. 283–293). Berlin, Heidelberg: Springer Berlin Heidelberg. Franco C.F.O., Fabri E.G., Barreiro Neto M. (2008). Urucum: sistemas de produc¸a˜o para o Brasil. EMEPA, Joa˜o Pessoa Gajera BB, Kumar N, Singh AS et al (2010) Assessment of genetic diversity in castor ( Ricinus communis L.) using RAPD and ISSR markers. Ind Crops Prod 32(3):491–498. https://irscope.shinyapps.io/iMEC/ Josiah C. C., George D. O., Eleazar O. M. and Nyamu W. F. (2008) Genetic diversity in Kenyan populations of Acacia senegal (L.) willd revealed by combined RAPD and ISSR markers. African Journal of Biotechnology , 7 (14). Karimi HR, Abadi MHH, Kohbanani AM (2012) Genetic diversity of Pistacia khinjuk Stocks. using RAPD markers and leaf morphological characters. Plant Syst Evol 298(5):963–968 Kumaran, K. (2014) Production potential of annatto ( Bixa orellana L.) as a source of natural edible dye. In the proceedings of the International workshop on natural dyes, Hyderabad, India . Leal F., de Clavijo CM.(2012).Annatto: botany and horticulture. In: Janick J (ed) Horticultural Reviews. Wiley, Hoboken. 389–419. Li G., and Quiros C. F. (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theoretical and applied genetics , 103 (2), 455–461. Liu, B. H. (1998) Statistical genomics: Linkage, mapping and QTL analysis. CRC Press, Boca Raton, Florida, USA. Lu Z., Wang Y., Peng Y., Korpelainen H. and Li C. (2006) Genetic diversity of Populus cathayana Rehd populations in southwestern China revealed by ISSR markers. Plant Science , 170 (2): 407–412. Parthiban K. T. (2001) Seed source variations, molecular characterization and clonal propagation in teak ( Tectona grandis Linn. F.). PhD, Tamil Nadu Agricultural University, Coimbatore, India . Pattanaik L., Naik S. N. and Hariprasad P. (2019) Valorization of waste Indigofera tinctoria L. biomass generated from indigo dye extraction process—potential towards biofuels and compost. Biomass Conversion and Biorefinery , 9 (2): 445–457. Pradeep kumar T., Hegde V., Sujatha R. and George T.(2010)Assessment of Pongamia pinnata (L.)–a biodiesel producing tree species using ISSR markers. Current Science , 99 (10):13–27. Radhakrishnan S. (2001) Genetic divergence and DNA based molecular characterization in Albizia lebbeck (L.) Benth (Doctoral dissertation, Ph. D. (For.) Thesis, Tamil Nadu Agricultural University, Coimbatore). Raji R. and Siril E. A.(2021) Genetic diversity analysis of promising Ceylon olive ( Elaeocarpus serratus L.) genotypes using morphological traits and ISSR markers. Current Plant Biology , 26 : 100–201. Rawat A., Barthwal S., and Ginwal, H. S. (2014) Comparative assessment of SSR, ISSR and AFLP markers for characterization of selected genotypes of Himalayan Chir pine ( Pinus roxburghii Sarg.) based on resin yield. Silvae Genetica , 63 ;1–6:94–108. Sandeep C., Kumar A., Rodrigues V., Viswanath S., Shukla A. K. and Sundaresan V. (2020) Morpho-genetic divergence and population structure in Indian Santalum album L. Trees , 34 (5): 1113–1129. Sekar I. (2003) Seed storage, seed source variations, molecular characterization and in vitro propagation in Simarouba glauca DC (Doctoral dissertation, Ph. D. Thesis, Tamil Nadu Agricultural University, Coimbatore, India). Singh P., Singh S., Mishra S. P. and Bhatia S. K. (2010) Molecular characterization of genetic diversity in Jatropha curcas L. Genes Genomes Genomics , 4 :1–8. Smith J., and Wallin H. (2006) Annatto extracts. Chemical and technical assessment Tessier, C., David J., This P., Boursiquot J. M., and Charrier A. (1999) Optimization of the choice of molecular markers for varietal identification in Vitis vinifera L. Theoretical and Applied Genetics 98: 171–177. Tikader A., Rao A. A. and Thangavelu K. (2003) Genetic divergence in exotic mulberry (Morus spp.) germplasm. Sericologia , 43 (4): 495–501. Verma, K. S., ul Haq, S., Kachhwaha, S., & Kothari, S. L. (2017) RAPD and ISSR marker assessment of genetic diversity in Citrullus colocynthis (L.) Schrad: a unique source of germplasm highly adapted to drought and high-temperature stress. 3 Biotech, 7, 1–24. Vilares A.S., Sa˜o Jose´ A.R., Rebouc T.N.H., Souza I.V.B. (1992) Estudo da biologia floral de urucuzeiro ( Bixa orellana L.). Rev Bras Corantes Nat, 1:101–105 Vos P., Hogers R., Bleeker M., Reijans M., Lee T. V. D., Hornes M., and Zabeau M. (1995) AFLP: a new technique for DNA fingerprinting. Nucleic acids research , 23 (21):4407–4414. Williams J.G., Kubelik A.R., Livak K.J., Rafalski J.A.,Tingey S.V. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic acids research , 18 (22): 6531–6535. Zhang, Y., Zhang, X., Wang, Y. H., & Shen, S. K. (2017) De novo assembly of transcriptome and development of novel EST-SSR markers in Rhododendron rex Lévl . through Illumina sequencing. Frontiers in Plant Science, 8, 1664. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6660535","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":465747609,"identity":"a78aa960-119a-4d4e-a0dc-32e3edfb0dd7","order_by":0,"name":"Manasa Mogilicharla","email":"","orcid":"","institution":"SKLTSHU/Forest College and Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Manasa","middleName":"","lastName":"Mogilicharla","suffix":""},{"id":465747610,"identity":"69667064-8522-47c8-8b4f-1cbab9c167ba","order_by":1,"name":"Reeja S","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYJACAwYbBgY2BgbGB0AODx9xWtKAWtgYmA1AWtiIsweoBWgNmwQDmCYAzNubDxT8SLDL55Nvf1b5NcdOBmjbw0c38GiROXMswbAnIdmyjY3H7LbstmSgw9iMjXPwaJGQyDEw4P1xwICNjYfttuQ2ZqAWHjZpvFrk338w/JMA0sL+rFhyWz0RWiR4GIx5wFoYzBg/bjtMhBaeNANjmYRkoJYcY2nGbcd52JgJ+YX98DPDNwl2BvLNxx9+/Lmt2p6fvfnhY3xagIDNAMZi5gGT+JWDlTyAsRh/EFY9CkbBKBgFIxAAAKz0O1CA0rbCAAAAAElFTkSuQmCC","orcid":"","institution":"SKLTSHU/Forest College and Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Reeja","middleName":"","lastName":"S","suffix":""},{"id":465747611,"identity":"7d93ac78-9272-4260-bb5b-bab74d008c2c","order_by":2,"name":"VBS Lalitha T","email":"","orcid":"","institution":"SKLTSHU/Forest College and Research Institute","correspondingAuthor":false,"prefix":"","firstName":"VBS","middleName":"Lalitha","lastName":"T","suffix":""},{"id":465747612,"identity":"06e67326-9db5-4727-8ed7-3499ab4b137e","order_by":3,"name":"Arjun Ramachandran","email":"","orcid":"","institution":"SKLTSHU/Forest College and Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Arjun","middleName":"","lastName":"Ramachandran","suffix":""},{"id":465747613,"identity":"690acec3-dc1d-484b-8a8a-7a670a687f6f","order_by":4,"name":"Shalini Mudalkar","email":"","orcid":"","institution":"SKLTSHU/Forest College and Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Shalini","middleName":"","lastName":"Mudalkar","suffix":""},{"id":465747614,"identity":"372a1f25-567d-4aac-ac21-7cb60a431358","order_by":5,"name":"Kumaran K","email":"","orcid":"","institution":"Anbil Dharmalingam Agricultural College and Research Institute, TamilNadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kumaran","middleName":"","lastName":"K","suffix":""},{"id":465747615,"identity":"59e43c07-2585-4a30-9e1f-3b7facbd66ba","order_by":6,"name":"Sreedhar Bodiga","email":"","orcid":"","institution":"SKLTSHU/Forest College and Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sreedhar","middleName":"","lastName":"Bodiga","suffix":""},{"id":465747616,"identity":"a63ad89c-5965-4218-94d3-10aabfa12bb1","order_by":7,"name":"Chiranjeeva Milkuri","email":"","orcid":"","institution":"SKLTSHU/Forest College and Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Chiranjeeva","middleName":"","lastName":"Milkuri","suffix":""}],"badges":[],"createdAt":"2025-05-14 05:23:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6660535/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6660535/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84015937,"identity":"eb228bb8-2864-4e0d-bf16-fb0e4c381dbb","added_by":"auto","created_at":"2025-06-05 17:59:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":203609,"visible":true,"origin":"","legend":"\u003cp\u003eISSR profile deciphering 100% polymorphism for primers a) ISSR F2 and b)UBC 845; Lane L is the 1kb DNA ladder; Lane 1 to 24 indicates the DNA of genotypes under study.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6660535/v1/08b881f552772e862b9ff986.jpg"},{"id":84015941,"identity":"dda2e640-8aa1-4e03-9b73-bcad90beafd1","added_by":"auto","created_at":"2025-06-05 17:59:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":186776,"visible":true,"origin":"","legend":"\u003cp\u003eISSR profile deciphering minimum polymorphism for primers a) UBC 823 and b) UBC 864 with 50%\u0026amp;62.5% respectively; Lane L is the 1kb DNA ladder; Lane 1 to 24 displayed the DNA of genotypes under study.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6660535/v1/502871857bcbe786f85281d8.jpg"},{"id":84015944,"identity":"542e6b86-5f99-4198-8b44-04d0cf4c62d2","added_by":"auto","created_at":"2025-06-05 17:59:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":314806,"visible":true,"origin":"","legend":"\u003cp\u003eISSR profile produced with unique bands in TSMLGB18 for a)1750bps for UBC 813 and b) ISSR-O4 at 800 bps; where lane L is 1kb ladder; numbers from 1 to 24 are genotypes DNA under study\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6660535/v1/8167ec4ea50c56184e996f09.jpg"},{"id":84016234,"identity":"52af51be-e244-4a5f-832a-e0b70e1bca9c","added_by":"auto","created_at":"2025-06-05 18:07:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":129131,"visible":true,"origin":"","legend":"\u003cp\u003eISSR profile produced with unique bands in TSMLGB2 for a) 1100 bps for UBC 826 and b) UBC 840 at 800 bps; where lane L is 1kb ladder; numbers from 1 to 24 are genotypes DNA under study\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6660535/v1/1a5c61c1b2dbe8354c3b31b5.jpg"},{"id":84015939,"identity":"3d7c210b-50ad-4a72-a36b-0b41876be980","added_by":"auto","created_at":"2025-06-05 17:59:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":52956,"visible":true,"origin":"","legend":"\u003cp\u003eUPGMA dendrogram computed using genetic distance matrix based on ISSR data\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6660535/v1/568f51cc31a5fe5d9425d1aa.jpg"},{"id":94598793,"identity":"6fff95c3-4c62-4729-97b1-386a1a85f278","added_by":"auto","created_at":"2025-10-28 18:56:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1751683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6660535/v1/03ded73e-e84c-439b-a705-840430e12b77.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic Diversity Assessment of Bixa orellana L., a Natural Seed Dye Species of the Tropics, using ISSR Markers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBixa orellana is a medicinal dye source plant. It is normally admitted as \u0026ldquo;annatto\u0026rdquo;, \u0026ldquo;sindhur\u0026rdquo;, \u0026ldquo;lipstick tree\u0026rdquo;. Originally, it is a native of South American region and has its place in their culinary as a food ingredient and the most important ingredient of beauty products (Alcazar-Alay \u003cem\u003eet al.\u003c/em\u003e, 2017). The seed coat extract gives out red orange pigment dye known as bixin that industrially competes well in the food and textile industries as a natural colorant and dye. It is the second most commercial non- toxic food colorant as compared to all other natural food colorants (Lopa pattanaik et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Other than the fact that it is used in textile industries, it has medicinal values that escalates its demands. It is a small tree or shrub that is perennially foliated and belongs to Bixaceae family. It is the only one in the Bixaceae family that has well-known uses. It possesses wide adaptability and is well distributed all over India in the following states Tamilnadu, Kerala, Karnataka, Chhattisgarh, Maharashtra, Madhya Pradesh, Gujarat and Andhra Pradesh (Kumaran K,2014). It may flourish in the temperature between 24℃ \u0026ndash; 38℃ with various soils. A mean annual rainfall demand was 800-1500mm. It can even reach the height of 6 meters, if it will be provided with good irrigation (Kumaran K,2014).\u003c/p\u003e \u003cp\u003eThe leaf is simple, smooth when mature, long petioles and are arranged alternating the branches (Franco et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Leal and Clavijo, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe flowers are vibrant and big, color ranges from white through to shades of pink and violet. They are bisexual and possess five sepals and they will usually see them flower right at the tips of the branches. The fruit is dehiscent, ovoid capsule made up of two/three carpels; covered by soft spines when young. It can be reddish, greenish or brownish and about 3\u0026ndash;5 cm in length; it contains dark seeds covered in a reddish waxy Aril (bixin) with an unsettling smell. The fruits are borne in clusters as every fruit can hold up to 17 pods (Dequigiovani et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The seeds in a given fruit may vary from 50\u0026ndash;60 seeds. It is capable of adjusting itself to numerous ecological and edaphic conditions (Kumaran K \u003cem\u003eet al.\u003c/em\u003e, 2014), means that, there is enough genetic variability available which can be exploited for its appropriate repercussions. Environmental factors have a dominant effect on the presence of bixin in seeds (Smith \u003cem\u003eet al.\u003c/em\u003e, 2006). Due to environmental impact amount of bixin varies from place to place thereby suggesting variation is on population basis and not on individual variation (Vilares et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe study of variation by the help of morphometric techniques was the dominating one earlier, and now, it is refined by the help of molecular techniques to evaluate the genetic variation which is less made in Bixa orellana. Environmental influences have a minimal impact on molecular markers and one can carry out analysis from DNA at any stage of development of the organism. PCR-assisted molecular markers \u0026ndash; like random amplified polymorphic DNA (RAPD; Methods, such as, inter-simple sequence repeat (ISSR; Zietkiewicz \u003cem\u003eet al.\u003c/em\u003e, 1994), amplified fragment length polymorphism (AFLP; Vos et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), sequence-related amplified polymorphism (SRAP; Li \u0026amp; Quiros \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e)\u0026mdash;have emerged However, little work is known of the diversity and genetic structure of this species (Diquigiovanni \u003cem\u003eet al.\u003c/em\u003e,2017). Until recently the genetic diversity analysis on that of DNA markers viz. microsatellites, SRAP and RAPD (Sequence Related Amplified product and Randomly Amplified Polymorphic DNA) were used to evaluate genetic diversity in bixa. The current research attempts to evaluate the reliability of ISSR markers in the task of studying genetic variability of Bixa orellana which wasn\u0026rsquo;t realized earlier. ISSR comes to the dominant marker class; they are emerged as an alternative system, with reliability and benefits of SSR markers and therefore it is reliable, fast and very informative system for studying the diversity (Zhang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The present study made an attempt to study genetic diversity in the studied genotypes of Bixa orellana.\u003c/p\u003e"},{"header":"Materials and Methodology","content":"\u003cp\u003e \u003cb\u003ePlant material;\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess the genetic variation, seeds of Bixa were brought from field germplasm bank of Forest college \u0026amp; Research Institute(FCRI), (latitude 11 9\u0026rsquo;N and longitude 76 56\u0026rsquo;E) TNAU, Mettupalayam. The seeds were the individual collections from the progenies which are initially raised from the seeds of plus trees obtained from different parts of Southern India. For the purpose of this study, seeds from 24 such progenies (genotypes) were used to raise the seedlings \u0026amp; assembled at FCRI, Mulugu (17\u003csup\u003e\u0026ordm;\u003c/sup\u003e.728544 N to 78\u003csup\u003e\u0026ordm;\u003c/sup\u003e.63296 E), Telangana and were given accession codes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Genomic DNA was extracted from young leaves following the standard method of CTAB, based on the procedure given by Doyle and Doyle (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) with some minor changes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetails of accession number and genotypes of \u003cem\u003eBixa orellana\u003c/em\u003e.L\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccession No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotypes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSMLGB24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eG24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe leaf tissues of Bixa orellana (400mg) were grinded into to fine powder in liquid nitrogen and homogenized it with extraction buffer by adding 3ml of CTAB buffer,1%PVP, 250 \u0026micro;l of beta-mercapto-ethanol and incubated for 30min at By using the ratio of 24:1 v/v of chloroform and isoamyl-alcohol solution supernant was extracted twice. 25 \u0026micro;l of RNase A was introduced in the extract which was then kept for incubation at room temperature. With the chilled iso-propanol, the DNA was pelleted and washed 2 times with 70% ethanol. Then air dried pellet added with 200 \u0026micro;l of nuclease free grade water and stored at -20\u0026deg;C. The quality of DNA was tested in nanodrop (ND1000, Thermo Scientific, US) spectrophotometer at 260 and 280 nm and quantity was measured in ng/\u0026micro;l. Portions that had between 200 \u0026micro;l to 600 \u0026micro;l DNA were best suited for PCR process.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eISSR amplification\u003c/h2\u003e \u003cp\u003eTotal of 33 random UBC primers were screened initially to assess genetic diversity among 24 genotypes of \u003cem\u003eB.orellana.\u003c/em\u003e The 10 \u0026micro;l volume of ISSR PCR reaction mixture composed of 5 \u0026micro;l PCR emerald master mix, 2 \u0026micro;l water, 1.5 \u0026micro;l selected primer, 1.5 \u0026micro;l DNA sample. PCR reactions were performed as, i) denaturation was done at 94\u0026ordm; for 4 minutes followed by ii) Annealing of primer at which temperature was specific to each primer and run for 1 minute then iii) Extension was done at 72\u0026ordm;C for 2minutes.The reaction was repeated for 30 cycles. The amplified product was run for 1 hour at 100 V in 1.2% agarose gel (Himedia) immersed in 1X TBE buffer by placing the 1kb DNA ladder as reference. After running, the gel was scanned under UV Gel scanner and photographed in the system (Biorad).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eDNA fingerprint patterns received were transformed into binary data matrices consisting of arrays of 0 and 1 s. The bands were awarded points based on their presence (1) or lack (0) of bands of different molecular weight sizes. Polymorphic and reproducible bands were the only ones taken into account for the analysis. Data were analysed with SIMQUAL route for creating Jaccard\u0026rsquo;s similarity coefficient in NTSYS-pc version 2.02i (Numerical Taxonomy System) (Rohlf 1998). Dendrograms were done with using similarity matrices UPGMA (Unweighted Pair Group Method with Arithmetic Average) algorithm. The percentage polymorphism was done in Microsoft-excel. Some of the important measures which states the informativeness of marker was calculated using an online tool i.e.,marker efficiency calculator ( Amiryousefi et al ,2018;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://irscope.shinyapps.io/iMEC/\u003c/span\u003e\u003cspan address=\"https://irscope.shinyapps.io/iMEC/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Polymorphic Information Content (PIC) and Heterozygosity(H) were calculated using Botstein et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) and Liu (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePIC\u0026thinsp;=\u0026thinsp;1-(\u0026sum; pi2 \u0026ndash; Σ pi2 pj2)\u003c/p\u003e \u003cp\u003eWhen pi and pj are population frequency of the ith and jth allele.\u003c/p\u003e \u003cp\u003eH\u0026thinsp;=\u0026thinsp;1 \u0026ndash; Σ pi2, in this pi is the frequency of the allele for the ith allele.\u003c/p\u003e \u003cp\u003eEffective Multiplex Ratio (EMR) and Marker Index (MI) was calculated according to Powell \u003cem\u003eet al.\u003c/em\u003e (1996).\u003c/p\u003e \u003cp\u003eEMR\u0026thinsp;=\u0026thinsp;n β, β\u0026thinsp;=\u0026thinsp;np / (np\u0026thinsp;+\u0026thinsp;nnp), p and np represent polymorphic and nonpolymorphic fraction of the markers, hence np and nnp refer to the counting numbers of the same.\u003c/p\u003e \u003cp\u003eMI\u0026thinsp;=\u0026thinsp;EMR*Havp,\u003c/p\u003e \u003cp\u003eHavp refers to an average heterozygosity for the polymorphic makers. It is equal to Σ Hn/np, where, Hn represents the markers\u0026rsquo; polymorphic fraction\u0026rsquo;s heterozygosity, and the summation is done over all the polymorphic loci np.\u003c/p\u003e \u003cp\u003eThe discriminating power of a marker was determined by D\u0026thinsp;=\u0026thinsp;1-C (Tessier et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e ).\u003c/p\u003e \u003cp\u003eWhere, C\u0026thinsp;=\u0026thinsp;Σ pi (Npi-1)/(N-1),\u003c/p\u003e \u003cp\u003eN \u0026ndash; Individuals, C \u0026ndash; Totality of loci for all the patterns formed by the primer.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAmong the 33 ISSR primers used, 23 primers that yielded distinct,reproducible and polymorphic banding profiles were screened for subsequent molecular charachterization (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The total number of bands generated is 249 and per primer it is 10.8. Maximum number of bands were generated by UBC 826(9) while minimum was reported by ISSR-O4(6) with an average band size varied from 100bp to 3500bp. The polymorphism ranged from 50\u0026ndash;100% among all genotypes. Of the 23 primers used in the study, 15 primers have shown 100% polymorphism viz., UBC841, UBC857, UBC826, UBC815, UBC888, UBC891, UBC810, UBC845, UBC847, UBC864, UBC880, ISSR-O3, ISSR-O4, ISSR-O6, ISSR-F2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Minimum polymorphism was reported in UBC823 (50%) followed by UBC 864(62.5%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The extent of polymorphism by all primers was presented in Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Among all the bands generated, twenty-two bands were detected as unique among eight genotypes (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), out of which TSMLGB18 was recorded for maximum number of unique bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026amp;Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Molecular markers particular to 24 genotypes were identified; this can be transformed into SCAR primers, to increase accuracy and reliability of the marker assisted selection. Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e were presented to show the molecular marker characters. The highest PIC was showed by UBC 809(0.60) followed by ISSR O6(0.59) and the minimum was reported for the UBC 857(0.505). All the 23 markers had\u0026thinsp;\u0026gt;\u0026thinsp;0.50 PIC reporting the informative nature of the marker. The heterozygosity ranged from 0.2 to 0.6. In UBC 864 the highest values for PIC was 0.60 and that of heterozygosity was 0.66. The estimates of marker properties for ISSRs had wide range. The effective MP ratio is 1 for all the primers. Marker index (MI) was 0.66(UBC 864) to 0.24(ISSR O6). The maximum discriminating value was found in the case of UBC811 (0.86) and minimal in UBC 809(0.31).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers selected for amplification and their specific annealing temperatures\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS .NO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnnealing Temperature\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAGAGAGAGAGAYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 857\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACACACACACACACACG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACACACACACACACACC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTCTCTCTTCTCTCTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 888\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBDBCACACACACACACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 889\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDBDACACACACACACAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHVHTGTGTGTGTGTGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTCTCTCTCTCTCTCTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR M5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(GA)9-C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CT)8-GC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CA)6-AC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CT)6-AG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISLA(CT)8TG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CT)8-TG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR F2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(AG)8-CG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAGAGAGAGAGAGAGAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCTCTCTCTCTCTCTCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTCTCTCTCTCTCTCTRG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCACACACACACACACC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATGATGATGATGATGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGAGAGGAGAGGAGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49⁰C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage polymorphism of selected primers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS .NO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence 5\u0026rsquo;-3\u0026rsquo;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePer cent polymorphism\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAGAGAGAGAGAYC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 857\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACACACACACACACACG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACACACACACACACACC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTCTCTCTTCTCTCTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 888\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBDBCACACACACACACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 889\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDBDACACACACACACAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.6%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHVHTGTGTGTGTGTGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC 813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTCTCTCTCTCTCTCTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.7%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR M5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(GA)9-C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CT)8-GC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CA)6-AC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CT)6-AG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISLA(CT)8TG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(CT)8-TG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.3%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR F2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(AG)8-CG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAGAGAGAGAGAGAGAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.6%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCTCTCTCTCTCTCTCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAGAGAGAGAGAGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTCTCTCTCTCTCTCTRG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCACACACACACACACC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATGATGATGATGATGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUBC-880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGAGAGGAGAGGAGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetails of unique bands displayed by different primers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSL.NO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePRIMER\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGENOTYPE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUNIQUE BAND (base pairs)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e5\u003c/p\u003e \u003cp\u003e6\u003c/p\u003e \u003cp\u003e7\u003c/p\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e9\u003c/p\u003e \u003cp\u003e10\u003c/p\u003e \u003cp\u003e11\u003c/p\u003e \u003cp\u003e12\u003c/p\u003e \u003cp\u003e13\u003c/p\u003e \u003cp\u003e14\u003c/p\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e17\u003c/p\u003e \u003cp\u003e18\u003c/p\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISSR-03\u003c/p\u003e \u003cp\u003eISSR-04\u003c/p\u003e \u003cp\u003eUBC-810\u003c/p\u003e \u003cp\u003eUBC-823\u003c/p\u003e \u003cp\u003eISLA(CT)\u003csub\u003e8\u003c/sub\u003eTG\u003c/p\u003e \u003cp\u003eUBC-864\u003c/p\u003e \u003cp\u003eISSR-06\u003c/p\u003e \u003cp\u003eUBC-811\u003c/p\u003e \u003cp\u003eUBC-841\u003c/p\u003e \u003cp\u003eUBC-889\u003c/p\u003e \u003cp\u003eUBC-826\u003c/p\u003e \u003cp\u003eUBC-880\u003c/p\u003e \u003cp\u003eISSR M5\u003c/p\u003e \u003cp\u003eUBC-813\u003c/p\u003e \u003cp\u003eUBC-891\u003c/p\u003e \u003cp\u003eUBC-840\u003c/p\u003e \u003cp\u003eUBC-809\u003c/p\u003e \u003cp\u003eUBC-815\u003c/p\u003e \u003cp\u003eUBC-847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB19\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB19\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB24\u003c/p\u003e \u003cp\u003eTSMLGB17\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB1\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB17\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB24\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB1\u003c/p\u003e \u003cp\u003eTSMLGB2\u003c/p\u003e \u003cp\u003eTSMLGB17\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB6\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB16\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB2\u003c/p\u003e \u003cp\u003eTSMLGB13\u003c/p\u003e \u003cp\u003eTSMLGB2\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003cp\u003eTSMLGB8\u003c/p\u003e \u003cp\u003eTSMLGB18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003cp\u003e2500\u003c/p\u003e \u003cp\u003e800,750,600\u003c/p\u003e \u003cp\u003e1200,800\u003c/p\u003e \u003cp\u003e2100\u003c/p\u003e \u003cp\u003e1200,900,800,500\u003c/p\u003e \u003cp\u003e1300\u003c/p\u003e \u003cp\u003e1100,800\u003c/p\u003e \u003cp\u003e1400\u003c/p\u003e \u003cp\u003e1500\u003c/p\u003e \u003cp\u003e400\u003c/p\u003e \u003cp\u003e1300,1250,1000,900\u003c/p\u003e \u003cp\u003e650,300\u003c/p\u003e \u003cp\u003e1500,900\u003c/p\u003e \u003cp\u003e2000,1000,100\u003c/p\u003e \u003cp\u003e300\u003c/p\u003e \u003cp\u003e1250,800,550,400\u003c/p\u003e \u003cp\u003e250\u003c/p\u003e \u003cp\u003e1100\u003c/p\u003e \u003cp\u003e350\u003c/p\u003e \u003cp\u003e2300,1600,1250,180\u003c/p\u003e \u003cp\u003e1400\u003c/p\u003e \u003cp\u003e350\u003c/p\u003e \u003cp\u003e1400,900\u003c/p\u003e \u003cp\u003e1750,500\u003c/p\u003e \u003cp\u003e1500,1400,1000,750\u003c/p\u003e \u003cp\u003e800\u003c/p\u003e \u003cp\u003e1750\u003c/p\u003e \u003cp\u003e650\u003c/p\u003e \u003cp\u003e1200\u003c/p\u003e \u003cp\u003e3100,500\u003c/p\u003e \u003cp\u003e2000,500,300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of ISSR primers and their molecular characters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePIC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEMR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC 811\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC 841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC 857\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC 826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC 815\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC 888\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC 889\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC 891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC 813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISSR M5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISSR-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISSR-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISSR-06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISLA(CT)8TG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISSR F2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC-809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC-810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC-823\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC-840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC-845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC-847\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC-864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUBC-880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe Jaccard\u0026rsquo;s co-efficient of similarity varied from 0.57(between TSMLGB18 and TSMLGB1) to 0.88 (between TSMLGB10 and TSMLGB11) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A dendrogram based UPGMA analysis with ISSR data was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.The dendrogram divided the 24 genotypes into 7 clusters based on Jaccard\u0026rsquo;s coefficient similarity matrix at 0.75 similarity. Cluster I consisted of TSMLGB1, TSMLGB13 and cluster II includes maximum number (15) of genotypes viz., TSMLGB3, TSMLGB4, TSMLGB6, TSMLGB8, TSMLGB5, TSMLGB7, TSMLGB9, TSMLGB10, TSMLGB11, TSMLGB12, TSMLGB14, TSMLGB15, TSMLGB17, TSMLGB19 and TSMLGB20.Cluster III includes TSMLGB16, cluster IV has TSMLGB21, TSMLGB22, TSMLGB24.Cluster V, VI, VII consisted of single genotype each namely TSMLGB23, TSMLGB2, TSMLGB18 respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present investigation, ISSR markers were successfully employed in molecular diversity analysis uto assess the phylogenetic relationships at the intraspecific level amongst 24 genotypes of \u003cem\u003eB.orellana\u003c/em\u003e collected from the germplasm of TNAU, Mettupalayam, India. The 23 ISSR primers generated 249 bands among 24 genotypes, the molecular band size covered from 100 bp to 3500bp.The amplified fragment ranged from 6(ISSR-O4) to 13(UBC 826) with an average of 10.8 bands per primer. Similar results were reported in \u003cem\u003eElaeocarpus serratus\u003c/em\u003e (Raji R \u003cem\u003eet al\u003c/em\u003e., 2021), \u003cem\u003eRicinus communis\u003c/em\u003e (Gajera et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), \u003cem\u003ePistacia khinjuk\u003c/em\u003e (Karimi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and \u003cem\u003eCitrullus colocynthis\u003c/em\u003e (Verma et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) for the number of bands in ISSR markers. The primers have recorded high to moderate polymorphism indicating wider genetic diversity in the species. Similar results for ISSR markers have been reported in various woody species viz., J\u003cem\u003eatropha curcas\u003c/em\u003e (Singh \u003cem\u003eet al\u003c/em\u003e.2010), \u003cem\u003ePinus roxburghii\u003c/em\u003e (Rawat ,2014), \u003cem\u003ePongamia pinnata\u003c/em\u003e (Pradeep \u003cem\u003eet al.\u003c/em\u003e, 2010), \u003cem\u003eSimarouba glauca\u003c/em\u003e (Sekar \u003cem\u003eet al.\u003c/em\u003e, 2003), \u003cem\u003eAlbizia lebbeck\u003c/em\u003e (Radhakrishnan \u003cem\u003eet al,.\u003c/em\u003e 2001), \u003cem\u003eTectona grandis\u003c/em\u003e (Parthibhan 2001).\u003c/p\u003e \u003cp\u003eThe presence of unique bands facilitates the discrimination of the unknown genotypes, indicating that TSMLGB18 can be reliably identified based on its unique bands. The size of the amplified products varied from 100bp-3000bp and the PIC ranged between 0.6\u0026thinsp;\u0026minus;\u0026thinsp;0.5 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), signifying ISSR as an informative molecular marker. The high heterozygosity, multiplex ratio and discriminating power of markers revealed by the study exhibited the proficiency of ISSR markers for genetic diversity studies of \u003cem\u003eBixa orellana\u003c/em\u003e. The results were in line with the other species for marker values viz., \u003cem\u003eElaeocarpus serratus\u003c/em\u003e (Raji R \u003cem\u003eet al.\u003c/em\u003e,2021), \u003cem\u003eAcacia senegal\u003c/em\u003e (Josiah \u003cem\u003eet al.\u003c/em\u003e2008),and \u003cem\u003ePopulus cathayana\u003c/em\u003e (Z lu \u003cem\u003eet al.\u003c/em\u003e,2006).\u003c/p\u003e \u003cp\u003eThe Jaccard\u0026rsquo;s co-efficient of similarity varied from 0.57(between TSMLGB18 and TSMLGB1) to 0.88 (between TSMLGB10 and TSMLGB11) which indicated moderate level of genetic variation among the genotypes in the study. The highest JC value between TSMLGB10 and TSMLGB11 indicate their genetic similarity, whereas low JC between TSMLGB1 and TSMLGB18 denotes significant genetic differences. UPGMA cluster placed TSMLGB18 distantly from all other genotypes in the study possibly due to its distinctiveness whereas TSMLGB10 and TSMLGB11 placed closely at 0.88 similarity could rendered to its similarity in genetic structure. Generally, the individuals from different geographic origins have different genetic constitution due to its natural selection for different traits (Raji \u003cem\u003eet al.\u003c/em\u003e, 2021) and fall in same cluster based on their origin. The present study is a novel attempt to evaluate genetic diversity using ISSR markers in \u003cem\u003eBixa orellana\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe molecular analysis grouped the genotypes into 7 clusters. The wide divergence and a broad genetic base allow for identification and selection of divergent parents for any hybridization or improvement plans and contribute significantly to the future breeding and conservation initiatives in the species. The study also revealed considerable variation in banding patterns, high heterozygosity and wide range of polymorphism among the genotypes, which demonstrated the suitability of ISSR markers as powerful tools for genetic characterization, diversity assessment and population structure analysis of \u003cem\u003eBixa orellana\u003c/em\u003e genetic resources. Previous studies on genetic diversity analysis using ISSR markers were reported on many species with similar results including \u003cem\u003eSantalum album\u003c/em\u003e (Sandeep et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eEucalyptus cladocalyx\u003c/em\u003e (Ballesta \u003cem\u003eet al\u003c/em\u003e.,2015), \u003cem\u003eElaeocarpus serratus\u003c/em\u003e (Raji \u003cem\u003eet al\u003c/em\u003e.2021), Morus spp (Tikadar \u003cem\u003eet al.\u003c/em\u003e, 2003), \u003cem\u003eTectona grandis\u003c/em\u003e (Ansari et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), Bamboo spp (Amom et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) elucidating the effectiveness of ISSR markers in analysing the diversity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents a novel application of ISSR markers for assessing genetic diversity in \u003cem\u003eBixa orellana\u003c/em\u003e. The analysis revealed high levels of polymorphism, demonstrating the reliability and informativeness of ISSR markers for genetic characterization in this species. The substantial genetic variability observed highlights the heterogeneous nature of \u003cem\u003eB. orellana\u003c/em\u003e, indicating considerable potential for heterosis in future breeding efforts. A well-designed breeding strategy could facilitate the development of superior progenies with enhanced bixin content and associated agronomic traits. These findings provide a valuable genetic framework for the selection of diverse and promising genotypes for future genetic advancement programs targeting bixin yield improvement.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"left\" width=\"605\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eISSR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInter Simple Sequence Repeats\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSSR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSimple Sequence Repeats\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUPGMA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUnweighted pair group mean arthematics\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDe-oxy ribo Nucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCTAB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCetyl trimethyl ammonium bromide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUniversity of British Columbia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePolymerase Chain Reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eISSR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInter simple sequence repeats\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUltraviolet\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSIMQUAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSimilarity for Qualitative data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSCAR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSequence Characterized Amplified Region\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTNAU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTamilnadu Agriculture University\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTBE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTris/Borate/EDTA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ekb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKilobytes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eml\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMilli-litres\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eµl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMicro-litres\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003enm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNano-metres\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMinutes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePercentage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e°C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDegree-centigrade\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe infrastructure support provided by Forest College and Research Institute, Mulugu, is greatly appreciated by the authors. We are very grateful to FC\u0026amp;RI, Mettupalayam, Tamilnadu for supplying the seed material on schedule.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eAuthors do not have any conflict of interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical issues:\u003c/strong\u003e None.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eManasa conducted the entire research as part of her postgraduate studies. Reeja framed the research objectives, design and methodology and provided guidance. Lalitha supported the research execution, in field and lab, while Arjun contributed to the selection of primers and supported statistical interpretations. Shalini participated in design and methodology. Kumaran provided the seed material from germplasm bank, Tamil Nadu. Sreedhar assisted in statistical analysis and Chiranjeeva provided technical guidance during the seedling nursery stage. All authors have read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlc\u0026aacute;zar-Alay S. C., Osorio-Tob\u0026oacute;n J. F., Forster-Carneiro T., and Meireles M. A. A. (2017) Obtaining bixin from semi-defatted annatto seeds by a mechanical method and solvent extraction: process integration and economic evaluation. \u003cem\u003eFood research international\u003c/em\u003e,\u003cem\u003e99\u003c/em\u003e: 393\u0026ndash;402.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmiryousefi A, Hyv\u0026ouml;nen J, Poczai P. (2018) iMEC: Online Marker Efficiency Calculator. Appl Plant Sci. Jun 24;6(6):e01159. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/aps3.1159\u003c/span\u003e\u003cspan address=\"10.1002/aps3.1159\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 30131901; PMCID: PMC6025818.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmom T., Tikendra L., Apana N., Goutam M., Sonia P., Koijam A.S. and Nongdam P. (2020) Efficiency of RAPD, ISSR, iPBS, SCoT and phytochemical markers in the genetic relationship study of five native and economical important bamboos of North-East India. \u003cem\u003ePhytochemistry\u003c/em\u003e, \u003cem\u003e174\u003c/em\u003e: 112\u0026ndash;330.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnsari S. A., Narayanan C., Wali S. A., Kumar R., Shukla N. and Rahangdale S. K. (2012) ISSR markers for analysis of molecular diversity and genetic structure of Indian teak (\u003cem\u003eTectona grandis\u003c/em\u003e Lf) populations. \u003cem\u003eAnnals of Forest Research\u003c/em\u003e,\u003cem\u003e55\u003c/em\u003e(1): 11\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBallesta P., Mora F., Contreras-Soto R. I., Ruiz E. and Perret S. (2015)Analysis of the genetic diversity of \u003cem\u003eEucalyptus cladocalyx\u003c/em\u003e (sugar gum) using ISSR markers. \u003cem\u003eActa Scientiarum. Agronomy\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e:133\u0026ndash;140.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBotstein, D., White R. L., Skolnick M., and Davis R. W. (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics 32: 314\u0026ndash;331.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDequigiovani G., Ramos S. L. F., Alves-Pereira A., Fabri E. G., Carvalho P. R. N., da Silva M. G. and Veasey E. A. (2017) Genetic diversity and structure in a major Brazilian annatto (\u003cem\u003eBixa orellana\u003c/em\u003e) germplasm bank revealed by microsatellites and phytochemical compounds. \u003cem\u003eGenetic Resources and Crop Evolution\u003c/em\u003e, \u003cem\u003e64\u003c/em\u003e(7): 1775\u0026ndash;1788.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoyle, J. (1991) DNA protocols for plants. In Molecular techniques in taxonomy (pp. 283\u0026ndash;293). Berlin, Heidelberg: Springer Berlin Heidelberg.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranco C.F.O., Fabri E.G., Barreiro Neto M. (2008). Urucum: sistemas de produc\u0026cedil;a˜o para o Brasil. EMEPA, Joa˜o Pessoa\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGajera BB, Kumar N, Singh AS et al (2010) Assessment of genetic diversity in castor (\u003cem\u003eRicinus communis\u003c/em\u003e L.) using RAPD and ISSR markers. Ind Crops Prod 32(3):491\u0026ndash;498.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://irscope.shinyapps.io/iMEC/\u003c/span\u003e\u003cspan address=\"https://irscope.shinyapps.io/iMEC/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJosiah C. C., George D. O., Eleazar O. M. and Nyamu W. F. (2008) Genetic diversity in Kenyan populations of \u003cem\u003eAcacia senegal\u003c/em\u003e (L.) willd revealed by combined RAPD and ISSR markers. \u003cem\u003eAfrican Journal of Biotechnology\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(14).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarimi HR, Abadi MHH, Kohbanani AM (2012) Genetic diversity \u003cem\u003eof Pistacia khinjuk\u003c/em\u003e Stocks. using RAPD markers and leaf morphological characters. Plant Syst Evol 298(5):963\u0026ndash;968\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumaran, K. (2014) Production potential of annatto (\u003cem\u003eBixa orellana\u003c/em\u003e L.) as a source of natural edible dye. In \u003cem\u003ethe proceedings of the International workshop on natural dyes, Hyderabad, India\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeal F., de Clavijo CM.(2012).Annatto: botany and horticulture. In: Janick J (ed) Horticultural Reviews. Wiley, Hoboken. 389\u0026ndash;419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi G., and Quiros C. F. (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. \u003cem\u003eTheoretical and applied genetics\u003c/em\u003e, \u003cem\u003e103\u003c/em\u003e(2), 455\u0026ndash;461.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, B. H. (1998) Statistical genomics: Linkage, mapping and QTL analysis. CRC Press, Boca Raton, Florida, USA.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Z., Wang Y., Peng Y., Korpelainen H. and Li C. (2006) Genetic diversity of \u003cem\u003ePopulus cathayana\u003c/em\u003e Rehd populations in southwestern China revealed by ISSR markers. \u003cem\u003ePlant Science\u003c/em\u003e, \u003cem\u003e170\u003c/em\u003e(2): 407\u0026ndash;412.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParthiban K. T. (2001) Seed source variations, molecular characterization and clonal propagation in teak (\u003cem\u003eTectona grandis\u003c/em\u003e Linn. F.). \u003cem\u003ePhD, Tamil Nadu Agricultural University, Coimbatore, India\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePattanaik L., Naik S. N. and Hariprasad P. (2019) Valorization of waste \u003cem\u003eIndigofera tinctoria\u003c/em\u003e L. biomass generated from indigo dye extraction process\u0026mdash;potential towards biofuels and compost. \u003cem\u003eBiomass Conversion and Biorefinery\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(2): 445\u0026ndash;457.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePradeep kumar T., Hegde V., Sujatha R. and George T.(2010)Assessment of \u003cem\u003ePongamia pinnata\u003c/em\u003e (L.)\u0026ndash;a biodiesel producing tree species using ISSR markers. \u003cem\u003eCurrent Science\u003c/em\u003e, \u003cem\u003e99\u003c/em\u003e(10):13\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRadhakrishnan S. (2001) Genetic divergence and DNA based molecular characterization in \u003cem\u003eAlbizia lebbeck\u003c/em\u003e (L.) Benth (Doctoral dissertation, Ph. D. (For.) Thesis, Tamil Nadu Agricultural University, Coimbatore).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaji R. and Siril E. A.(2021) Genetic diversity analysis of promising Ceylon olive (\u003cem\u003eElaeocarpus serratus\u003c/em\u003e L.) genotypes using morphological traits and ISSR markers. \u003cem\u003eCurrent Plant Biology\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e: 100\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRawat A., Barthwal S., and Ginwal, H. S. (2014) Comparative assessment of SSR, ISSR and AFLP markers for characterization of selected genotypes of Himalayan Chir pine (\u003cem\u003ePinus roxburghii\u003c/em\u003e Sarg.) based on resin yield. \u003cem\u003eSilvae Genetica\u003c/em\u003e, \u003cem\u003e63\u003c/em\u003e;1\u0026ndash;6:94\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandeep C., Kumar A., Rodrigues V., Viswanath S., Shukla A. K. and Sundaresan V. (2020) Morpho-genetic divergence and population structure in Indian \u003cem\u003eSantalum album\u003c/em\u003e L. \u003cem\u003eTrees\u003c/em\u003e, \u003cem\u003e34\u003c/em\u003e(5): 1113\u0026ndash;1129.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSekar I. (2003) \u003cem\u003eSeed storage, seed source variations, molecular characterization and in vitro propagation in Simarouba glauca DC\u003c/em\u003e (Doctoral dissertation, Ph. D. Thesis, Tamil Nadu Agricultural University, Coimbatore, India).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh P., Singh S., Mishra S. P. and Bhatia S. K. (2010) Molecular characterization of genetic diversity in \u003cem\u003eJatropha curcas\u003c/em\u003e L. \u003cem\u003eGenes Genomes Genomics\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e:1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith J., and Wallin H. (2006) Annatto extracts. Chemical and technical assessment\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTessier, C., David J., This P., Boursiquot J. M., and Charrier A. (1999) Optimization of the choice of molecular markers for varietal identification in Vitis vinifera L. Theoretical and Applied Genetics 98: 171\u0026ndash;177.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTikader A., Rao A. A. and Thangavelu K. (2003) Genetic divergence in exotic mulberry (Morus spp.) germplasm. \u003cem\u003eSericologia\u003c/em\u003e, \u003cem\u003e43\u003c/em\u003e(4): 495\u0026ndash;501.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma, K. S., ul Haq, S., Kachhwaha, S., \u0026amp; Kothari, S. L. (2017) RAPD and ISSR marker assessment of genetic diversity in \u003cem\u003eCitrullus colocynthis\u003c/em\u003e (L.) Schrad: a unique source of germplasm highly adapted to drought and high-temperature stress. 3 Biotech, 7, 1\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVilares A.S., Sa˜o Jose\u0026acute; A.R., Rebouc T.N.H., Souza I.V.B. (1992) Estudo da biologia floral de urucuzeiro (\u003cem\u003eBixa orellana\u003c/em\u003e L.). Rev Bras Corantes Nat, 1:101\u0026ndash;105\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVos P., Hogers R., Bleeker M., Reijans M., Lee T. V. D., Hornes M., and Zabeau M. (1995) AFLP: a new technique for DNA fingerprinting. \u003cem\u003eNucleic acids research\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(21):4407\u0026ndash;4414.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams J.G., Kubelik A.R., Livak K.J., Rafalski J.A.,Tingey S.V. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. \u003cem\u003eNucleic acids research\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(22): 6531\u0026ndash;6535.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Y., Zhang, X., Wang, Y. H., \u0026amp; Shen, S. K. (2017) De novo assembly of transcriptome and development of novel EST-SSR markers in \u003cem\u003eRhododendron rex L\u0026eacute;vl\u003c/em\u003e. through Illumina sequencing. Frontiers in Plant Science, 8, 1664.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bixa orellana, PIC, unique bands, genetic diversity, UPGMA, ISSR markers","lastPublishedDoi":"10.21203/rs.3.rs-6660535/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6660535/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eBixa orellana\u003c/em\u003e.L is an important dye yielding tree. The bixin content in the seeds yields a red colour pigment in varying colours which is used as dye. The variation in bixin content could be attributed to both environmental and genetic changes. In this study a total of 24 genotypes of \u003cem\u003eBixa orellana\u003c/em\u003e.L were raised from FC\u0026amp;RI, Annatto gene bank of Mettupalayam, India and their genetic diversity were analysed using ISSR markers. Out of 33 primers used, 23 primers were effectively screened for amplification and their polymorphism ranged from 50\u0026ndash;100% indicating high genetic diversity among the genotypes. The values of polymorphic information content (PIC) ranged from 0.50\u0026ndash;0.60. The other marker traits viz., effective marker ratio, discriminating power and heterozygosity are in higher values revealing the effectiveness of the ISSR markers. The total number of bands varied from 6 to 13. Among all the bands generated, 22 unique bands were detected in eight genotypes, out of which TSMLGB18 produced maximum number of unique bands. The UPGMA clustering pattern revealed that 24 genotypes were divided into seven distinct clusters. The genetic relationship obtained through Jaccard\u0026rsquo;s similarity co-efficient ranged between 0.57 to 0.88. The result provided valid guidelines for collection, conservation and characterization of \u003cem\u003eBixa orellana\u003c/em\u003e genetic resources.\u003c/p\u003e","manuscriptTitle":"Genetic Diversity Assessment of Bixa orellana L., a Natural Seed Dye Species of the Tropics, using ISSR Markers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-05 17:59:37","doi":"10.21203/rs.3.rs-6660535/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8cb68933-d3e6-4bcc-8b48-4f142802387c","owner":[],"postedDate":"June 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-28T18:22:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-05 17:59:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6660535","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6660535","identity":"rs-6660535","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}