DNA methylation is involved in sex determination in spinach

DNA methylation is involved in sex determination in spinach | 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 DNA methylation is involved in sex determination in spinach Keli Jia, Jiaming Duan, Guangqian Cheng, Heng Li, Shufen Li, Miao Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2146618/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Nov, 2023 Read the published version in Biochemical Genetics → Version 1 posted 9 You are reading this latest preprint version Abstract To explore whether DNA methylation plays different roles in the spinach development of individual male and female dioecious plants, methylation sensitivity amplification polymorphism (MSAP) was used to assess differential cytosine CpG methylation profiles of CCGG motifs of the spinach. 16 pairs of amplification primers were selected, A total of 434 DNA fragments, of which 134 methylated fragments were detected in the male and female plants. The relative ratios of the methylated sites did have sex-specific differences, which was 28.8% and 33% in the male and female. In addition, 32 sex-specific cytosine methylation bands were obtained, 15 fragments were male specific and 17 fragments were female specific. The level of cytosine methylation on CCGG was higher in female genomes than male genomes. The methylation level of CG island was involved in the sex determination and the differentiation process of spinach, which provided information for revealing the sex determination of spinach. spinach dioecious DNA methylation MSAP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Simple Summary DNA methylation, an important epigenetic modification, regulates the expression of genes and is therefore involved in the sex determination and sex differentiation of plants. spinach ( (Spinacia oleracea L.), an important vegetable crop, was chosen as the research object. The results showed that: there was a difference in the degree of DNA methylation between male and female plants,which females had a higher degree of methylation. Introduction DNA methylation (DNAm) is an important epigenetic event involving the reversible addition of a methyl group primarily to cytosines, without changing genomic sequences. Recent research shows that DNA methylation, especially cytosine methylation in plants, is important in fundamental cellular activities, including control of gene expression, maintenance of genomic integrity, regulation of plant development, immune response, determination of chromatin structure, and control of genomic imprinting. About 2–12% of cytosine residues will be methylated in the higher animals genome, which almost all of them occur in symmetric “CpG” dinucleotide pairs . [ 3 ] ( Belanger and Hepburn 1990) The proportion of DNA methylation in the genomes of higher plants is higher than the higher animal genomes, which about about 20%~50% of cytosine residues are methylated. In plants, 5mC is mainly located in the symmetric "CpG" dinucleotide or "CpNpG" trinucleotide pair, which can also occur in asymmetric CpNpN, preventing their transcription and transposition, and therefore is essential for genome stability and gene silencing. ( Xiao et al. 2006) For example, in the CCG trinucleotide pair of the Arabidopsis genome, nearly 20%-30% of the outer cytosine and 20% of the cytosine are methylated. [ 5 ] ( Jeddeloh and Richards 1996) . [ 6 – 10 ] DNA methylation participates in sex determination and differentiation. Most of papers studied the sex chromosomes of human and other mammals. Only a few studies investigated the relationship between DNA methylation and plant sex determination or sex chromosome evolution. The method to study the level and pattern of genome methylation is as follows, such as HPLC (High-performance liquid chromatography, high performance liquid chromatography ), bisulfite sequencing method, etc. The methylation-sensitive amplified polymorphism(MSAP, also referred as MS-AFLP) technique (Reyna-Lpezet al.1997) is a modified version of the Amplified Fragment Length Polymorphism (AFLP) DNA fingerprinting technique (Voset al.1995), which can effectively monitor a large number of methylation sites in the sample DNA, with high polymorphism. Without the need to know the sequence of the analyzed DNA, which can detect changes in the cytosine methylation status of the 5'-CCGG-3' site in the whole genome. MSAP has been widely used to assess differentialcytosine CpG methylation profiles of CCGG motifs,mostly in plant cultures. [ 11 – 13 ] ( Xiong et al. 1999 ; Song et al. 2012 ; Abid et al. 2018 ). Research have shown that DNA methylation plays a vital role in the sex determination and differentiation process of plants and animals . [ 14 – 16 ] ( Tachibana 2016 ; Lai et al. 2017 ; Akagi et al. 2016 ) The spinach seeds were processed with the demethylation reagent 5-azaC (5-azacytidine), the results show that 5-azaC can significantly increase the proportion of hermaphrodite individuals in the population, revealing that DNA methylation may play a role in the sex determination and differentiation process of spinach [ 17 ] (Li et al. 2015). On the basis of this research, our research group aims to analyze the methylation level of spinach male and female individuals through MSAP technology, and provide a scientific basis for in-depth exploration of the role of DNA methylation in the process of spinach sex development. Materials And Methods 2.1 Plant material The Japanese large-leaf spinach was planted in the experimental field of the College of Life Sciences, Henan Normal University, and it grew naturally. After blooming, the sex of the plant was identified through the flower organ. 2.2 Methods 2.2.1 Extraction and Detection of spinach Flower DNA Extracted of genomic DNA via the modified CTAB method. Whole genome DNA was extracted from the floral organs of 4 male and female Japanese spinach. Detected its concentration and purity by spectrophotometer, stored at -20℃ for later use. 2.2.2 MSAP analysis An MSAP analysis was performed to detect methylation-sensitive restriction sites in spinach samples. Most of the processes were performed as described by Xiong et al. (1999) with appropriate modifications. The ‘rare-cutter’ enzyme Eco RI, combined with two‘frequent cutter’ isoschizomers, Hpa II and Msp I were employed to digest the DNA. Two consecutive PCRs were conducted to generate a specific DNA fragment fingerprint. The first-round PCR (pre-selective amplification) product were transferred to 1.5% agarose gel to check for the presence of fragments between 100 and 1000 bp in length by electrophoresis. Before initiating the MSAP analysis, 10 pairs of EcoR I- Hpa II and EcoR I- Msp I selective-amplification primer combinations which could generate quantity-rich and clear fingerprints were selected from 64 pairs of primer combinations through a trial test. The sequences of the 10 pairs of primer combinations are summarized in Supplemental Table 2. The PCR products were then separated by electrophoresis on 6% denaturing polyacrylamide sequencing gels in 1×TBE buffer. The MSAP used two groups of endonucleases EcoR I- Hpa II and EcoR I- Msp I with different sensitivities to methylation sites, including the following steps: digestion, ligation, pre-amplification, selective amplification, PAGE gel electrophoresis detection, Silver dye development, tape reading and data collation. The EcoR I linker and primers were synthesized by BGI . Table 1 MSAP adaptor and primer sequence Primer/Connector sequence Connector(5’-3’) EcoRⅠ- adapterF 5’-CTCGTAGACTGCGTACC-3’ EcoRⅠ- adapterR 5’-AATTGGTACGCAGTC-3’ HpaⅡ/MspⅠ- adapterF 5’-GATCATGAGTCCTGCT-3’ HpaⅡ/MspⅠ- adapterR 5’-CGAGCAGGACTCATGA-3’ Pre-amplification primer(5’-3’) EcoRⅠ+A 5’-GACTGCGTACCAATTC-3’ HpaⅡ/MspⅠ+0 5’-ATCATGAGTCCTGCTCGG-3’ Selective amplification primer(5’-3’) E1 5’-GACTGCGTACCAATTCAGG-3’ E2 5’-GACTGCGTACCAATTCACG-3’ E3 5’-GACTGCGTACCAATTCAAC-3’ E4 5’-GACTGCGTACCAATTCACA-3’ HM1 5’-ATCATGAGTCCTGCTCGGAAT-3’ HM2 5’-ATCATGAGTCCTGCTCGGACG-3’ HM3 5’-ATCATGAGTCCTGCTCGGATC-3’ HM4 5’-ATCATGAGTCCTGCTCGGATT-3’ Double enzyme digestion of genomic DNA of spinach male and female: The DNA of four male and female strains was mixed in equal amounts to construct a male and female genomic DNA pool, and the genomic DNA of spinach male and female was double digested, and using the restriction endonuclease EcoRI in combination with Hpa II/ Msp I with the same cleavage site. The 100 μL reaction system contained : 10×Buffer 10 μL, BSA 10 μL, EcoR I 2 μL, Hpa II/ Msp I 3 μL, genomic DNA 2 μg, deionized water to make up to 100 μL. After mixing, it was placed in a metal bath at 37 °C for 12 h, then placed at 65 °C for 10 min to inactivate the enzyme, finally stored at -20 °C for later use. Linker ligation: The product obtained by enzyme digestion and the linker diluted to 10 μmol/L were ligated, and the ligation reaction was carried out at 16°C for 12 h after the ligation system was mixed. The ligation system (20 μL) was as follows: 2 μL of 10× Ligation Buffer, 0.5 μL each of EcoR I linker and Hpa II/Msp I linker, 0.5 μL of T4 DNA ligase (10 U/μL), 10 μL of digestion product, deionized Water 6.5 μL. Pre-amplification reaction: The optimized pre-amplification reaction system is: 10×Buffer 2 μL, EcoR I and Hpa II/Msp I pre-amplification primers 3 μL each, 10 mmol/L dNTPs 0.6 μL, Taq DNA polymerase ( 10 U/μL) 0.5 μL, ligation product 1.5 μL, and deionized water to make up 20 μL. Mixed the pre-amplification system well and put it into the PCR machine. The pre-amplification reaction was carried out according to the following procedure: denaturation at 94°C for 2 min, amplification according to the following parameters for 30 cycles, 94°C for 30 s, 56°C for 30 s, 72°C for 80 s, and a final extension at 72°C for 10 min. Pre-amplification products were detected by 1% agarose gel electrophoresis. Selective amplification: Except for the use of selective amplification primers, the rest of the components were the same as the pre-amplification system. Mixed the selective amplification products into the PCR machine, and performed the Touchdown PCR reaction according to the following procedure: denaturation at 94°C for 2 min, the first cycle of amplification parameters was 94°C for 30 s, 65°C for 30 s, 72°C for 80 s, Then, the renaturation temperature in each cycle was successively decreased by 0.7°C for a total of 12 cycles of amplification; then the amplification was performed for 23 cycles according to the following steps, 94°C for 30 s, 56°C for 30 s, and 72°C for 80 s. Finally, which extended at 72 ℃ for 5 min. Polyacrylamide gel electrophoresis: The selective amplification products were mixed with 6×loading buffer and then spotted on a 6% non-denaturing polyacrylamide gel, and electrophoresed in TBE buffer. The gel was electrophoresed at 180 V for about 4 hours. After electrophoresis, the gel was fixed in fixative (500 mL double distilled water, 50 mL ethanol, 2.5 mL glacial acetic acid) for 6 min, and then the gel was transferred to silver stain to stain with silver in solution (500 mL double distilled water, 1 g silver nitrate) for 12 min, then transferred to 500 mL double distilled water for washing for 1 min, and then the gel was transferred to chromogenic solution (500 mL double distilled water, 7.5 g sodium hydroxide), 1.5 mL formaldehyde) for 8 min, and finally the gel was transfered to fixative for storage. At last, the gel was placed on a light box to observe and photograph. 2.3 Data analysis Both the isocutases Hpa II and Msp I can recognize and cleave the same site (5'-CCGG-3'), but these two enzymes show different sensitivities to DNA methylation. Methylation patterns can be classified into four types based on the gel banding patterns of selective amplification products (Table 2 ). Type I: Both the Hpa II and Msp I lanes can show bands, indicating either cytosine methylation or no methylation within the single strand of the DNA fragment. Type II: A band appeared in the Hpa II lane, but not in the Msp I lane, indicating that the outer cytosine of one strand in the DNA fragment was methylated. Type III: A band appeared in the Msp I lane, but not in the Hpa II lane, indicating that the cytosines in both strands of the DNA fragment were methylated. Type IV: No bands appeared in the Hpa II and Msp I lanes, indicating that the outer cytosines of both strands of the DNA fragment were methylated. For the whole genome methylation analysis, we usually consider that type I is that no methylation has occurred, type II as hemimethylation, and type III as full methylation. For methylation statistics in a sample, hemimethylation ratio = number of hemimethylated fragments/number of all fragments, full methylation ratio = number of fully methylated fragments/number of all fragments, all Methylation ratio = hemimethylation ratio + permethylation ratio. Table 2 Different methylation patterns and enzyme digestion results Type Methylation Enzyme sensitivity Bands status HpaⅡ MspⅠ EcoRⅠ/HpaⅡ EcoRⅠ/HpaⅡ Ⅰ CCGG CCGG GGCC CmCGG Yes Yes + + Ⅱ mCCGG GGCC Yes No + - Ⅲ CmCGG GGCmC No Yes - + Ⅳ mCCGG GGCCm No No - - Note: "+" means bands appear in the gel;"-" means that no methylation sites appear in the gel;“mC” stands for 5-methylcytosine. Results 3.1. MSAP amplification 3.1.1. Genomic DNA extraction, Enzyme digestion, Connect with, Pre-amplification A high-quality DNA template is the first condition for a smooth MSAP. In this study, the CTAB method was used to extract the genomic DNA of the male and female inflorescences of Japanese spinach, and then detected by 1.0% agarose gel electrophoresis. The results showed that the DNA bands were clear and basically did not degrade (Fig. 1 ). The OD260/280 values were all between 1.7 and 1.9, and the OD260/230 values were between 2.0 and 2.3, which indicated that the DNA was of high purity and could be used in subsequent experiments. Genomic DNA was double-digested with EcoR I- Hpa II and EcoR I- Msp I, and detected on a 1% agarose gel after digestion at 37℃ for 12 h. The results showed that the ligation reaction could be carried out completely.The results were shown in Fig. 2 . Pre-amplification was a key link in this experiment. The results showed that the obtained pre-amplification products were of good quality(Fig. 3 ). 3.1.2. Selective amplification 16 pairs of selective stains were used for amplification, and the results showed good quality(Figure 4 ). 3.2. PCR amplification and data statistics MSAP is a PCR technology based on AFLP technology to detect genomic methylation variation. Which can detect the difference in methylation degree between male and female spinach, and describe the cytosine methylation pattern of male and female spinach. Four types of fragments obtained after EcoR I- Hpa II/ EcoR I- Msp I double enzyme digestion, ligation, pre-amplification, selective amplification (Table 2 and Fig. 2 ) . The methylation patterns of male and female spinach were analyzed by 16 pairs of primers which consist of 4 EcoR I primers and 4 Hpa II/ Msp I primers.A total of 434 clear bands were amplified, most of the fragments were between 100–700 bp in size, and only a few of them were over 1000 bp. Among them, 222 fragments were obtained from the male genome, and an average of 6.94 fragments could be obtained from each prime, while the female genome obtained 212 fragments, with an average of 6.63 fragments per primer. (Table 3 and Table 4 ) . Table 3 Number of bands amplified by each pair of primers in male E1 E2 E3 E4 H M H M H M H M HM1 3 7 6 5 5 7 11 6 HM2 5 8 7 6 9 5 6 3 HM3 8 7 11 9 6 4 6 10 HM4 10 8 5 4 8 10 8 9 Note: H = EcoR I- Hpa II, M = EcoR I- Msp I Table 4 Number of bands amplified by each pair of primers in female E1 E2 E3 E4 H M H M H M H M HM1 10 7 9 8 5 3 6 10 HM2 2 1 9 6 9 4 6 10 HM3 8 7 9 8 4 4 7 9 HM4 9 9 4 4 8 9 7 9 Note: H = EcoR I- Hpa II, M = EcoR I- Msp I 3.3. Methylation fragment pattern analysis 222 fragments were obtained from the male spinach, among of them 26 were permethylated, 38 were hemimethylated and 158 were unmethylated, thus the ratio of permethylation, hemimethylation and unmethylated was 11.7%, 17.1% and 71.2%; On the contrary, in female spinach, we got a total of 212 fragments, including 32 fully methylated, 38 hemimethylated and 142 unmethylated fragments, so the ratio of permethylation, hemimethylation and unmethylated was 15.1%, 17.9% and 67%, respectively. Although the full methylation ratio of female spinach was much higher than the male spinach, while the percentage of hemimethylation was not much different(Table 5 ). Table 5 Statistics of MSAP Amplification Results of spinach Female and Male Genomes Type permethylated hemi-methylated unmethylated rate ratio Total methylation The male spinach 158 38(17.1%) 26(11.7%) 28.8% The female spinach 142 38(17.9%) 32(15.1%) 33% 3.4. DNA methylation specific fragments Thirty-two methylated bands which only exist in males or females were found from the bands amplified by 16 pairs of selective amplification primers. Among them, 15 methylated bands were only present in male spinach, including 8 hemimethylated bands and 7 fully methylated bands; 17 methylated bands were only exist in female spinach, including 7 hemimethylated bands and 10 fully methylated bands ( Table 6 ). These specific DNA methylation fragments would be very good candidate genes for future studies on the regulation of sex determination and differentiation by DNA methylation in spinach. Table 6 Specific DNA methylation fragments in spinach Specific DNA methylation fragments hemimethylated bands permethylated bands male spinach 8 7 female spinach 7 10 Discussion spinach (Spinacia oleracea L.) is a diploid dioecious vegetable in the Chenopodiaceae spinach genus (Spinacia) that has been cultivated an ideal material for studying the sex determination and differentiation mechanism of plants.spinach has 2n = 12 chromosomes, and the sex of this species is controlled by the X and Y chromosomes(S. F. Lia andG. J. Zhang 2015).The genome size of spinach is 989 Mbp (Arumuganathan and Earle 1991). The evolution of sex chromosomes was divided by Ming et al. (Sister Wei Li, reference 11) into 5 stages, of which the second stage is the formation of relatively obvious MSY on the Y chromosome.The spinach and papaya are turn out to be sterile at this stage of YY-type plants as the representative plants [ 18 ] .The sex of this species is controlled by the X and Y chromosomes, and the area of MSY is on the number 1 chromosome which is the largest sex chromosome(Iizuka and Janick 1962; Deng et al. 2012). Although the spinach genome sequence has been resolved (Xu et al. 2017), there are no other reports on study of sex-determining genes and sex-determining molecular mechanisms in spinach. The experimental results showed that 15 specific DNA methylation fragments were found in male spinach, and the ratios of hemimethylation and full methylation were 17.1% and 11.7%, respectively. In contrast, in female spinach, there were 17 specific DNA methylation segments with hemi- and full-methylation ratios of 17.9% and 15.1%, respectively (Tables 5 and 6 ). It turned out that the sex expression in spinach was influenced by the DNA methylation, and the female spinach plants have higherlevels of methylation, which also indirectly indicated that spinach genomic DNA methylation may be involved in the process of sex determination and differentiation. Similar phenomena have been found in other species. In plants, by studying the genomics of hermaphroditic Arabidopsis and maize, Momoko Ikeuchi et al found that histones are modified and DNA methylation profiles change dynamically as cells differentiate [ 21 ] ; In genetically modified papaya, the non-recombination region of sex chromosome Y was marked by DNA hypermethylation and heterozygosity. Methylation variants in the MSY region of the Y chromosome caused sex reversal [ 22 ] .In the model plant Silene latifolia L, and the gender was determined to be heterozygous XY with a higher degree of methylation of alleles in the original Y chromosome. Therefore, DNA methylation on the Y chromosome was closely related to sex differentiation in plants.In animals, studies had shown that methylation plays an important role in the sex differentiation of aphids. Compared with female individuals, the expression levels of DNMT1a and DNMT1b enzymes in males were relatively low, while the expression of DNMT3a was relatively high. The three enzymes mentioned above were involved in regulating methylation [ 23 ] .Huang Dejun et al found that the increase in the concentration of 2,4-DCP in the environment could cause the expression of sex-related genes (sox9a, amh and dmrt1) to be down-regulated in zebrafish, and the experimental results found that the methylation level of the promoter of sox9a was significantly increased, which resulted in down-regulation of sox9a expression and ultimately resulted in an increased proportion of female individuals. In summary, the methylation played a decisive role in the sex evolution of animals and plants. This paper aimed to study the differences in the methylation levels of male and female spinach genomes, provide a theoretical basis for studying the epigenetic mechanism of spinach sex differentiation, and provide an experimental basis for revealing the sex determination and sex differentiation of spinach. Our study found that the level of total methylation in female plants was significantly higher than that in male plants, which indicated that DNA methylation was closely related to the sex differentiation of spinach.These results may be helpful for investigating the molecular mechanisms that alter DNA methylation for further research the relationship between methylation and sex determination development of this dioecious plant spinach. Declarations Author Contributions Keli Jia was responsible for the extraction of experimental materials, experimental design, experimental operation, and preparation of manuscripts; Jiaming Duan was responsible for manuscript review and editing； Guangqian Cheng was responsible for the collection, sorting and analysis of experimental data ; Shufen Li was responsible for experimental project management. Data availability Date sharing is not applicable to this article as no new date were created or analyzed in this study. Conflict of interest The authors declare no competing interests. Funding: “This research was funded by The 2020 Excellent Young Teacher Training Plan of Sanquan College of Xinxiang Medical College , grant number SQ2021YQJHO3 . References Bender J. DNA methylation and epigenetics. Annual Review of Plant Biology, 2004, 55: 41-68. Pavlopoulou A, Kossida S. Plant cytosine-5 DNA methyltransferases: structure, function, and molecular evolution. Genomics, 2007, 90: 530-541. 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Molecular ecology, 2019, 28(18): 4228-4241. Supplementary Tables Supplementary Tables are not available with this version Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 11 Nov, 2023 Read the published version in Biochemical Genetics → Version 1 posted Editorial decision: Major revision 09 May, 2023 Reviewers agreed at journal 27 Apr, 2023 Reviewers agreed at journal 24 Apr, 2023 Reviews received at journal 24 Apr, 2023 Reviewers agreed at journal 18 Apr, 2023 Reviewers invited by journal 14 Apr, 2023 Editor assigned by journal 10 Oct, 2022 Submission checks completed at journal 10 Oct, 2022 First submitted to journal 09 Oct, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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University","correspondingAuthor":false,"prefix":"","firstName":"Jiaming","middleName":"","lastName":"Duan","suffix":""},{"id":143253651,"identity":"15a3ed82-9b45-4cf6-9efc-7f7d5d735bb1","order_by":2,"name":"Guangqian Cheng","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Guangqian","middleName":"","lastName":"Cheng","suffix":""},{"id":143253652,"identity":"2ce075dc-3352-4e5a-ac68-5e76d3b496bb","order_by":3,"name":"Heng Li","email":"","orcid":"","institution":"Sanquan College of Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Heng","middleName":"","lastName":"Li","suffix":""},{"id":143253653,"identity":"5c55ccd2-7327-4b0e-a347-95155d44f50d","order_by":4,"name":"Shufen Li","email":"","orcid":"","institution":"Henan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Shufen","middleName":"","lastName":"Li","suffix":""},{"id":143253654,"identity":"6104eadc-1064-4299-8ebd-92f085ed4008","order_by":5,"name":"Miao Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBACfmaG5Icf/9nw8BOtRbK94ZmxBFuanGQDsVoMzhx8IMHDdtjY4ADRtsxITjCQ4ElL3Hw8eQPDj4pthLXwS6QlPCiQsEncduZZAWPPmdvE2JIDtMUgLXHbjRwDZsY2IrQY3Mj/IMGTcDhx8wyitZw5kCDBcwDofQlitQADOc1YsiFNTgLol4NE+QUSlQ3AqGxP3vjgRwURWpBAAvFRg9BCqo5RMApGwSgYIQAAYeRAq9qjhw0AAAAASUVORK5CYII=","orcid":"","institution":"Sanquan College of Xinxiang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Miao","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2022-10-09 04:14:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2146618/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2146618/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10528-023-10524-4","type":"published","date":"2023-11-11T15:01:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":27792505,"identity":"eeb1e911-14c7-43da-8a45-6df5b2c88271","added_by":"auto","created_at":"2022-10-14 18:25:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20677,"visible":true,"origin":"","legend":"\u003cp\u003eThe electropherogram of Genomic DNA\u003c/p\u003e\n\u003cp\u003eNote: 1, 2 represents male spinach,3 and 4 represent female spinach.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2146618/v1/11a2c8e6bdce76f6cdaf1918.png"},{"id":27792506,"identity":"acb87b73-d0c7-41e1-b227-58ea6c0dc553","added_by":"auto","created_at":"2022-10-14 18:25:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29458,"visible":true,"origin":"","legend":"\u003cp\u003eTheelectropherogram of DNA digestion\u003c/p\u003e\n\u003cp\u003eNote：H=\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII，M=\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2146618/v1/80b8c0c1dacb14d323b0e1c5.png"},{"id":27792508,"identity":"bc22007f-aa31-4829-9efc-13f8046576ed","added_by":"auto","created_at":"2022-10-14 18:25:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40291,"visible":true,"origin":"","legend":"\u003cp\u003eThe electropherogram of pre-amplification\u003c/p\u003e\n\u003cp\u003eNote：H=\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII，M=\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2146618/v1/50b5fc122e5c67b2653af440.png"},{"id":27792504,"identity":"bb438c91-9846-4c34-afc7-6708ac145ccb","added_by":"auto","created_at":"2022-10-14 18:25:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":64853,"visible":true,"origin":"","legend":"\u003cp\u003eThe electropherogram of Selective amplification\u003c/p\u003e\n\u003cp\u003eNote：H=\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII，M=\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2146618/v1/6d1a073692057e651efe5023.png"},{"id":27792507,"identity":"f40f058b-0e88-4940-894a-efbe035fcbcf","added_by":"auto","created_at":"2022-10-14 18:25:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":182429,"visible":true,"origin":"","legend":"\u003cp\u003eThe electropherogram of Selective amplification polyacrylamide gel\u003c/p\u003e\n\u003cp\u003eNote: H=\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII, M=\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI; Which the number of 1 represents male spinach, 2 represents female spinach; The number of 3 represents no methylation, 4 represents hemimethylation, and 5 represents fully methylated\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2146618/v1/f7dc1d6ffd855255bf8fb095.png"},{"id":46348996,"identity":"a29f915e-a0d2-4466-90c9-3689ed14111e","added_by":"auto","created_at":"2023-11-13 15:07:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":842876,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2146618/v1/d87d3818-29d4-42d4-8a7e-ea181d1daeb4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"DNA methylation is involved in sex determination in spinach","fulltext":[{"header":"Simple Summary","content":"\u003cp\u003eDNA methylation, an important epigenetic modification,\u0026nbsp;regulates the expression of genes\u0026nbsp;and is therefore involved in the sex determination and sex differentiation of plants. spinach (\u003cem\u003e(Spinacia oleracea\u0026nbsp;\u003c/em\u003eL.), an important vegetable crop, was chosen as the research object. The results showed that: there was a difference in the degree of DNA methylation between male and female plants,which females had a higher degree of methylation.\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDNA methylation (DNAm) is an important epigenetic event involving the reversible addition of a methyl group primarily to cytosines, without changing genomic sequences. Recent research shows that DNA methylation, especially cytosine methylation in plants, is important in fundamental cellular activities, including control of gene expression, maintenance of genomic integrity, regulation of plant development, immune response, determination of chromatin structure, and control of genomic imprinting.\u003c/p\u003e \u003cp\u003eAbout 2\u0026ndash;12% of cytosine residues will be methylated in the higher animals genome, which almost all of them occur in symmetric \u0026ldquo;CpG\u0026rdquo; dinucleotide pairs .\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e ( Belanger and Hepburn 1990) The proportion of DNA methylation in the genomes of higher plants is higher than the higher animal genomes, which about about 20%~50% of cytosine residues are methylated. In plants, 5mC is mainly located in the symmetric \"CpG\" dinucleotide or \"CpNpG\" trinucleotide pair, which can also occur in asymmetric CpNpN, preventing their transcription and transposition, and therefore is essential for genome stability and gene silencing. ( Xiao et al. 2006) For example, in the CCG trinucleotide pair of the Arabidopsis genome, nearly 20%-30% of the outer cytosine and 20% of the cytosine are methylated.\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e ( Jeddeloh and Richards 1996) .\u003csup\u003e[\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e DNA methylation participates in sex determination and differentiation. Most of papers studied the sex chromosomes of human and other mammals. Only a few studies investigated the relationship between DNA methylation and plant sex determination or sex chromosome evolution.\u003c/p\u003e \u003cp\u003eThe method to study the level and pattern of genome methylation is as follows, such as HPLC (High-performance liquid chromatography, high performance liquid chromatography ), bisulfite sequencing method, etc. The methylation-sensitive amplified polymorphism(MSAP, also referred as MS-AFLP) technique (Reyna-Lpezet al.1997) is a modified version of the Amplified Fragment Length Polymorphism (AFLP) DNA fingerprinting technique (Voset al.1995), which can effectively monitor a large number of methylation sites in the sample DNA, with high polymorphism. Without the need to know the sequence of the analyzed DNA, which can detect changes in the cytosine methylation status of the 5'-CCGG-3' site in the whole genome. MSAP has been widely used to assess differentialcytosine CpG methylation profiles of CCGG motifs,mostly in plant cultures. \u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e( Xiong et al. 1999 ; Song et al. 2012 ; Abid et al. 2018 ).\u003c/p\u003e \u003cp\u003eResearch have shown that DNA methylation plays a vital role in the sex determination and differentiation process of plants and animals .\u003csup\u003e[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e( Tachibana 2016 ; Lai et al. 2017 ; Akagi et al. 2016 ) The spinach seeds were processed with the demethylation reagent 5-azaC (5-azacytidine), the results show that 5-azaC can significantly increase the proportion of hermaphrodite individuals in the population, revealing that DNA methylation may play a role in the sex determination and differentiation process of spinach \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e(Li et al. 2015). On the basis of this research, our research group aims to analyze the methylation level of spinach male and female individuals through MSAP technology, and provide a scientific basis for in-depth exploration of the role of DNA methylation in the process of spinach sex development.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Plant material\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe Japanese large-leaf spinach was planted in the experimental field of the College of Life Sciences, Henan Normal University, and it grew naturally. After blooming, the sex of the plant was identified through the flower organ.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Methods\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.2.1 Extraction and Detection of spinach Flower DNA\u003c/h2\u003e\n \u003cp\u003eExtracted of genomic DNA via the modified CTAB method. Whole genome DNA was extracted from the floral organs of 4 male and female Japanese spinach. Detected its concentration and purity by spectrophotometer, stored at -20℃ for later use.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.2.2 MSAP analysis\u003c/h2\u003e\n \u003cp\u003eAn MSAP analysis was performed to detect methylation-sensitive restriction sites in spinach samples. Most of the processes were performed as described by Xiong et al. (1999) with appropriate modifications. The \u0026lsquo;rare-cutter\u0026rsquo; enzyme \u003cem\u003eEco\u003c/em\u003eRI, combined with two\u0026lsquo;frequent cutter\u0026rsquo; isoschizomers, \u003cem\u003eHpa\u003c/em\u003eII and \u003cem\u003eMsp\u003c/em\u003eI were employed to digest the DNA. Two consecutive PCRs were conducted to generate a specific DNA fragment fingerprint. The first-round PCR (pre-selective amplification) product were transferred to 1.5% agarose gel to check for the presence of fragments between 100 and 1000 bp in length by electrophoresis. Before initiating the MSAP analysis, 10 pairs of \u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII and \u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI\u003c/p\u003e\n \u003cp\u003eselective-amplification primer combinations which could generate quantity-rich and clear fingerprints were selected from 64 pairs of primer combinations through a trial test. The sequences of the 10 pairs of primer combinations are summarized in Supplemental Table\u0026nbsp;2. The PCR products were then separated by electrophoresis on 6% denaturing polyacrylamide sequencing gels in 1\u0026times;TBE buffer.\u003c/p\u003e\n \u003cp\u003eThe MSAP used two groups of endonucleases \u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII and \u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI with different sensitivities to methylation sites, including the following steps: digestion, ligation, pre-amplification, selective amplification, PAGE gel electrophoresis detection, Silver dye development, tape reading and data collation. The \u003cem\u003eEcoR\u003c/em\u003eI linker and primers were synthesized by BGI .\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMSAP adaptor and primer sequence\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimer/Connector\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003esequence\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConnector(5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEcoRⅠ-\u0026nbsp;adapterF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-CTCGTAGACTGCGTACC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEcoRⅠ-\u0026nbsp;adapterR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-AATTGGTACGCAGTC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHpaⅡ/MspⅠ-\u0026nbsp;adapterF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-GATCATGAGTCCTGCT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHpaⅡ/MspⅠ-\u0026nbsp;adapterR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-CGAGCAGGACTCATGA-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePre-amplification primer(5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEcoRⅠ+A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-GACTGCGTACCAATTC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHpaⅡ/MspⅠ+0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-ATCATGAGTCCTGCTCGG-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSelective amplification primer(5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-GACTGCGTACCAATTCAGG-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-GACTGCGTACCAATTCACG-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-GACTGCGTACCAATTCAAC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-GACTGCGTACCAATTCACA-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-ATCATGAGTCCTGCTCGGAAT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-ATCATGAGTCCTGCTCGGACG-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-ATCATGAGTCCTGCTCGGATC-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026rsquo;-ATCATGAGTCCTGCTCGGATT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eDouble enzyme digestion of genomic DNA of spinach male and female: The DNA of four male and female strains was mixed in equal amounts to construct a male and female genomic DNA pool, and the genomic DNA of spinach male and female was double digested, and using the restriction endonuclease EcoRI in combination with \u003cem\u003eHpa\u003c/em\u003eII/\u003cem\u003eMsp\u003c/em\u003eI with the same cleavage site. The 100 \u0026mu;L reaction system contained : 10\u0026times;Buffer 10 \u0026mu;L, BSA 10 \u0026mu;L, EcoR I 2 \u0026mu;L, \u003cem\u003eHpa\u003c/em\u003eII/\u003cem\u003eMsp\u003c/em\u003eI 3 \u0026mu;L, genomic DNA 2 \u0026mu;g, deionized water to make up to 100 \u0026mu;L. After mixing, it was placed in a metal bath at 37 \u0026deg;C for 12 h, then placed at 65 \u0026deg;C for 10 min to inactivate the enzyme, finally stored at -20 \u0026deg;C for later use.\u003c/p\u003e\n \u003cp\u003eLinker ligation: The product obtained by enzyme digestion and the linker diluted to 10 \u0026mu;mol/L were ligated, and the ligation reaction was carried out at 16\u0026deg;C for 12 h after the ligation system was mixed. The ligation system (20 \u0026mu;L) was as follows: 2 \u0026mu;L of 10\u0026times; Ligation Buffer, 0.5 \u0026mu;L each of EcoR I linker and Hpa II/Msp I linker, 0.5 \u0026mu;L of T4 DNA ligase (10 U/\u0026mu;L), 10 \u0026mu;L of digestion product, deionized Water 6.5 \u0026mu;L.\u003c/p\u003ePre-amplification reaction: The optimized pre-amplification reaction system is: 10\u0026times;Buffer 2 \u0026mu;L, EcoR I and Hpa II/Msp I pre-amplification primers 3 \u0026mu;L each, 10 mmol/L dNTPs 0.6 \u0026mu;L, Taq DNA polymerase ( 10 U/\u0026mu;L) 0.5 \u0026mu;L, ligation product 1.5 \u0026mu;L, and deionized water to make up 20 \u0026mu;L. Mixed the pre-amplification system well and put it into the PCR machine. The pre-amplification reaction was carried out according to the following procedure: denaturation at 94\u0026deg;C for 2 min, amplification according to the following parameters for 30 cycles, 94\u0026deg;C for 30 s, 56\u0026deg;C for 30 s, 72\u0026deg;C for 80 s, and a final extension at 72\u0026deg;C for 10 min. Pre-amplification products were detected by 1% agarose gel electrophoresis.\u003cp\u003eSelective amplification: Except for the use of selective amplification primers, the rest of the components were the same as the pre-amplification system. Mixed the selective amplification products into the PCR machine, and performed the Touchdown PCR reaction according to the following procedure: denaturation at 94\u0026deg;C for 2 min, the first cycle of amplification parameters was 94\u0026deg;C for 30 s, 65\u0026deg;C for 30 s, 72\u0026deg;C for 80 s, Then, the renaturation temperature in each cycle was successively decreased by 0.7\u0026deg;C for a total of 12 cycles of amplification; then the amplification was performed for 23 cycles according to the following steps, 94\u0026deg;C for 30 s, 56\u0026deg;C for 30 s, and 72\u0026deg;C for 80 s. Finally, which extended at 72 ℃ for 5 min.\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003ePolyacrylamide gel electrophoresis: The selective amplification products were mixed with 6\u0026times;loading buffer and then spotted on a 6% non-denaturing polyacrylamide gel, and electrophoresed in TBE buffer. The gel was electrophoresed at 180 V for about 4 hours. After electrophoresis, the gel was fixed in fixative (500 mL double distilled water, 50 mL ethanol, 2.5 mL glacial acetic acid) for 6 min, and then the gel was transferred to silver stain to stain with silver in solution (500 mL double distilled water, 1 g silver nitrate) for 12 min, then transferred to 500 mL double distilled water for washing for 1 min, and then the gel was transferred to chromogenic solution (500 mL double distilled water, 7.5 g sodium hydroxide), 1.5 mL formaldehyde) for 8 min, and finally the gel was transfered to fixative for storage. At last, the gel was placed on a light box to observe and photograph.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.3 Data analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eBoth the isocutases \u003cem\u003eHpa\u003c/em\u003eII and \u003cem\u003eMsp\u003c/em\u003eI can recognize and cleave the same site (5\u0026apos;-CCGG-3\u0026apos;), but these two enzymes show different sensitivities to DNA methylation. Methylation patterns can be classified into four types based on the gel banding patterns of selective amplification products (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Type I: Both the \u003cem\u003eHpa\u003c/em\u003eII and \u003cem\u003eMsp\u003c/em\u003eI lanes can show bands, indicating either cytosine methylation or no methylation within the single strand of the DNA fragment. Type II: A band appeared in the \u003cem\u003eHpa\u003c/em\u003eII lane, but not in the \u003cem\u003eMsp\u003c/em\u003eI lane, indicating that the outer cytosine of one strand in the DNA fragment was methylated. Type III: A band appeared in the \u003cem\u003eMsp\u003c/em\u003eI lane, but not in the \u003cem\u003eHpa\u003c/em\u003eII lane, indicating that the cytosines in both strands of the DNA fragment were methylated. Type IV: No bands appeared in the \u003cem\u003eHpa\u003c/em\u003eII and \u003cem\u003eMsp\u003c/em\u003eI lanes, indicating that the outer cytosines of both strands of the DNA fragment were methylated. For the whole genome methylation analysis, we usually consider that type I is that no methylation has occurred, type II as hemimethylation, and type III as full methylation. For methylation statistics in a sample, hemimethylation ratio\u0026thinsp;=\u0026thinsp;number of hemimethylated fragments/number of all fragments, full methylation ratio\u0026thinsp;=\u0026thinsp;number of fully methylated fragments/number of all fragments, all Methylation ratio\u0026thinsp;=\u0026thinsp;hemimethylation ratio\u0026thinsp;+\u0026thinsp;permethylation ratio.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDifferent methylation patterns and enzyme digestion results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eType\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMethylation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eEnzyme sensitivity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eBands\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003estatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHpaⅡ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eMspⅠ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEcoRⅠ/HpaⅡ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEcoRⅠ/HpaⅡ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eⅠ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCCGG CCGG\u003c/p\u003e\n \u003cp\u003eGGCC CmCGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eⅡ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emCCGG\u003c/p\u003e\n \u003cp\u003eGGCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eⅢ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCmCGG\u003c/p\u003e\n \u003cp\u003eGGCmC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eⅣ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emCCGG\u003c/p\u003e\n \u003cp\u003eGGCCm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eNote: \u0026quot;+\u0026quot; means bands appear in the gel;\u0026quot;-\u0026quot; means that no methylation sites appear in the gel;\u0026ldquo;mC\u0026rdquo; stands for 5-methylcytosine.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e3.1. MSAP amplification\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec10\"\u003e\n \u003ch2\u003e3.1.1. Genomic DNA extraction, Enzyme digestion, Connect with, Pre-amplification\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eA high-quality DNA template is the first condition for a smooth MSAP. In this study, the CTAB method was used to extract the genomic DNA of the male and female inflorescences of Japanese spinach, and then detected by 1.0% agarose gel electrophoresis. The results showed that the DNA bands were clear and basically did not degrade (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eThe OD260/280 values were all between 1.7 and 1.9, and the OD260/230 values were between 2.0 and 2.3, which indicated that the DNA was of high purity and could be used in subsequent experiments. Genomic DNA was double-digested with \u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII and \u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI, and detected on a 1% agarose gel after digestion at 37℃ for 12 h. The results showed that the ligation reaction could be carried out completely.The results were shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003ePre-amplification was a key link in this experiment. The results showed that the obtained pre-amplification products were of good quality(Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec11\"\u003e\n \u003ch2\u003e3.1.2. Selective amplification\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e16 pairs of selective stains were used for amplification, and the results showed good quality(Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003e3.2. PCR amplification and data statistics\u003c/h2\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eMSAP is a PCR technology based on AFLP technology to detect genomic methylation variation. Which can detect the difference in methylation degree between male and female spinach, and describe the cytosine methylation pattern of male and female spinach. Four types of fragments obtained after \u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII/\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI double enzyme digestion, ligation, pre-amplification, selective amplification (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) .\u003c/p\u003e\n \u003cp\u003eThe methylation patterns of male and female spinach were analyzed by 16 pairs of primers which consist of 4 \u003cem\u003eEcoR\u003c/em\u003eI primers and 4 \u003cem\u003eHpa\u003c/em\u003eII/\u003cem\u003eMsp\u003c/em\u003eI primers.A total of 434 clear bands were amplified, most of the fragments were between 100\u0026ndash;700 bp in size, and only a few of them were over 1000 bp. Among them, 222 fragments were obtained from the male genome, and an average of 6.94 fragments could be obtained from each prime, while the female genome obtained 212 fragments, with an average of 6.63 fragments per primer. (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) .\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber of bands amplified by each pair of primers in male\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eE1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eE2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eE3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eE4\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003eNote: H\u0026thinsp;=\u0026thinsp;\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII, M\u0026thinsp;=\u0026thinsp;\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003ctable border=\"1\" id=\"Tab4\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber of bands amplified by each pair of primers in female\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eE1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eE2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eE3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eE4\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHM4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003eNote: H\u0026thinsp;=\u0026thinsp;\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eHpa\u003c/em\u003eII, M\u0026thinsp;=\u0026thinsp;\u003cem\u003eEcoR\u003c/em\u003eI-\u003cem\u003eMsp\u003c/em\u003eI\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003e3.3. Methylation fragment pattern analysis\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e222 fragments were obtained from the male spinach, among of them 26 were permethylated, 38 were hemimethylated and 158 were unmethylated, thus the ratio of permethylation, hemimethylation and unmethylated was 11.7%, 17.1% and 71.2%; On the contrary, in female spinach, we got a total of 212 fragments, including 32 fully methylated, 38 hemimethylated and 142 unmethylated fragments, so the ratio of permethylation, hemimethylation and unmethylated was 15.1%, 17.9% and 67%, respectively. Although the full methylation ratio of female spinach was much higher than the male spinach, while the percentage of hemimethylation was not much different(Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab5\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStatistics of MSAP Amplification Results of spinach Female and Male Genomes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eType\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epermethylated\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ehemi-methylated\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eunmethylated rate ratio\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal methylation\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe male\u003c/p\u003e\n \u003cp\u003espinach\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38(17.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26(11.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe female spinach\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38(17.9%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32(15.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.4. DNA methylation specific fragments\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThirty-two methylated bands which only exist in males or females were found from the bands amplified by 16 pairs of selective amplification primers. Among them, 15 methylated bands were only present in male spinach, including 8 hemimethylated bands and 7 fully methylated bands; 17 methylated bands were only exist in female spinach, including 7 hemimethylated bands and 10 fully methylated bands ( Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). These specific DNA methylation fragments would be very good candidate genes for future studies on the regulation of sex determination and differentiation by DNA methylation in spinach.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab6\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSpecific DNA methylation fragments in spinach\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecific DNA\u003c/p\u003e\n \u003cp\u003emethylation fragments\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ehemimethylated bands\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epermethylated bands\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emale spinach\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efemale spinach\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003espinach (Spinacia oleracea L.) is a diploid dioecious vegetable in the Chenopodiaceae spinach genus (Spinacia) that has been cultivated an ideal material for studying the sex determination and differentiation mechanism of plants.spinach has 2n\u0026thinsp;=\u0026thinsp;12 chromosomes, and the sex of this species is controlled by the X and Y chromosomes(S. F. Lia andG. J. Zhang 2015).The genome size of spinach is 989 Mbp (Arumuganathan and Earle 1991).\u003c/p\u003e \u003cp\u003eThe evolution of sex chromosomes was divided by Ming et al. (Sister Wei Li, reference 11) into 5 stages, of which the second stage is the formation of relatively obvious MSY on the Y chromosome.The spinach and papaya are turn out to be sterile at this stage of YY-type plants as the representative plants\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.The sex of this species is controlled by the X and Y chromosomes, and the area of MSY is on the number 1 chromosome which is the largest sex chromosome(Iizuka and Janick 1962; Deng et al. 2012). Although the spinach genome sequence has been resolved (Xu et al. 2017), there are no other reports on study of sex-determining genes and sex-determining molecular mechanisms in spinach.\u003c/p\u003e \u003cp\u003eThe experimental results showed that 15 specific DNA methylation fragments were found in male spinach, and the ratios of hemimethylation and full methylation were 17.1% and 11.7%, respectively. In contrast, in female spinach, there were 17 specific DNA methylation segments with hemi- and full-methylation ratios of 17.9% and 15.1%, respectively (Tables\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). It turned out that the sex expression in spinach was influenced by the DNA methylation, and the female spinach plants have higherlevels of methylation, which also indirectly indicated that spinach genomic DNA methylation may be involved in the process of sex determination and differentiation.\u003c/p\u003e \u003cp\u003eSimilar phenomena have been found in other species. In plants, by studying the genomics of hermaphroditic Arabidopsis and maize, Momoko Ikeuchi et al found that histones are modified and DNA methylation profiles change dynamically as cells differentiate\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e; In genetically modified papaya, the non-recombination region of sex chromosome Y was marked by DNA hypermethylation and heterozygosity. Methylation variants in the MSY region of the Y chromosome caused sex reversal\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.In the model plant Silene latifolia L, and the gender was determined to be heterozygous XY with a higher degree of methylation of alleles in the original Y chromosome. Therefore, DNA methylation on the Y chromosome was closely related to sex differentiation in plants.In animals, studies had shown that methylation plays an important role in the sex differentiation of aphids. Compared with female individuals, the expression levels of DNMT1a and DNMT1b enzymes in males were relatively low, while the expression of DNMT3a was relatively high. The three enzymes mentioned above were involved in regulating methylation \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.Huang Dejun et al found that the increase in the concentration of 2,4-DCP in the environment could cause the expression of sex-related genes (sox9a, amh and dmrt1) to be down-regulated in zebrafish, and the experimental results found that the methylation level of the promoter of sox9a was significantly increased, which resulted in down-regulation of sox9a expression and ultimately resulted in an increased proportion of female individuals.\u003c/p\u003e \u003cp\u003eIn summary, the methylation played a decisive role in the sex evolution of animals and plants. This paper aimed to study the differences in the methylation levels of male and female spinach genomes, provide a theoretical basis for studying the epigenetic mechanism of spinach sex differentiation, and provide an experimental basis for revealing the sex determination and sex differentiation of spinach. Our study found that the level of total methylation in female plants was significantly higher than that in male plants, which indicated that DNA methylation was closely related to the sex differentiation of spinach.These results may be helpful for investigating the molecular mechanisms that alter DNA methylation for further research the relationship between methylation and sex determination development of this dioecious plant spinach.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKeli Jia\u0026nbsp;was responsible for the extraction of experimental materials, experimental design, experimental operation, and preparation of manuscripts;\u0026nbsp;Jiaming Duan was responsible for manuscript review and editing；\u0026nbsp;Guangqian Cheng was responsible for the collection, sorting and analysis of experimental data ; Shufen Li was responsible for experimental project management.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDate sharing is not applicable to this article as no new date were created or analyzed in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e \u0026ldquo;This research was funded by The 2020 Excellent Young Teacher Training Plan of Sanquan College of Xinxiang Medical College , grant number SQ2021YQJHO3 .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBender J. 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Archives of Dermatological Research, 2018, 310(2):107-115.\u003c/li\u003e\n\u003cli\u003eXu W, Yang T, Dong X, Li DZ, Liu A. Genomic DNA methylation analyses reveal the distinct profiles in castor bean seeds with persistent endosperms. Plant Physiol, 2016, 171(2): 1242-1258.\u003c/li\u003e\n\u003cli\u003eDinh TT, Gao L, Liu X, Li S, Zhao Y, O\u0026rsquo;Leary M, Le B, Schmitz RJ, Manavella PA, Li S, Weigel D, Pontes O, Ecker JR, Chen X. DNA topoisomerase 1\u0026alpha; promotes transcriptional silencing of transposable elements through DNA methylation and histone lysine 9 dimethylation in Arabidopsis. PLoS Genet, 2014, 10(7): e1004446.\u003c/li\u003e\n\u003cli\u003eAlvarez-Errico D, Vento-Tormo R, Sieweke M, Ballestar E. Epigenetic control of myeloid cell differentiation, identity and function. Nature Reviews Immunology, 2015, 15(1): 7-17.\u003c/li\u003e\n\u003cli\u003eKim M, Costello J. DNA methylation: an epigenetic mark of cellular memory. 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Cytogenetic analysis of sex determination in \u003cem\u003eSpinacia oleracea\u003c/em\u003e. Genetics, 1962, 47: 1225-1241.\u003c/li\u003e\n\u003cli\u003eDeng CL, Qin RY, Gao J, Cao Y, Li SF, Gao WJ, Lu LD. Identification of sex chromosome of spinach by physical mapping of 45s rDNAs by FISH. Caryologia, 2012, 65(4): 322-327.\u003c/li\u003e\n\u003cli\u003eMomoko Ikeuchi, Michitaro Shibata, Bart Rymen, Akira Iwase, Anne-Maarit B\u0026aring;gman, Lewis Watt, Duncan Coleman, David S Favero, Tatsuya Takahashi, Sebastian E Ahnert, Siobhan M Brady, Keiko Sugimoto, A Gene Regulatory Network for Cellular Reprogramming in Plant Regeneration, \u003cem\u003ePlant and Cell Physiology\u003c/em\u003e, Volume 59, Issue 4, April 2018, Pages 770\u0026ndash;782\u003c/li\u003e\n\u003cli\u003eLin, H., Liao, Z., Zhang, L. \u0026amp; Yu, Q. Transcriptome analysis of the male-tohermaphrodite sex reversal induced by low temperature in papaya. Tree Genet.Genomes 12, 94 (2016).\u003c/li\u003e\n\u003cli\u003eMathers T C, Mugford S T, Percival‐Alwyn L, et al. Sex‐specific changes in the aphid DNA methylation landscape[J]. Molecular ecology, 2019, 28(18): 4228-4241.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Tables","content":"\u003cp\u003eSupplementary Tables are not available with this version\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biochemical-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bigi","sideBox":"Learn more about [Biochemical Genetics](http://link.springer.com/journal/10528)","snPcode":"10528","submissionUrl":"https://submission.nature.com/new-submission/10528/3","title":"Biochemical Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"spinach, dioecious, DNA methylation, MSAP ","lastPublishedDoi":"10.21203/rs.3.rs-2146618/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2146618/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo explore whether DNA methylation plays different roles in the spinach development of individual male and female dioecious plants, methylation sensitivity amplification polymorphism (MSAP) was used to assess differential cytosine CpG methylation profiles of CCGG motifs of the spinach. 16 pairs of amplification primers were selected, A total of 434 DNA fragments, of which 134 methylated fragments were detected in the male and female plants. The relative ratios of the methylated sites did have sex-specific differences, which was 28.8% and 33% in the male and female. In addition, 32 sex-specific cytosine methylation bands were obtained, 15 fragments were male specific and 17 fragments were female specific. The level of cytosine methylation on CCGG was higher in female genomes than male genomes. The methylation level of CG island was involved in the sex determination and the differentiation process of spinach, which provided information for revealing the sex determination of spinach.\u003c/p\u003e","manuscriptTitle":"DNA methylation is involved in sex determination in spinach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-14 18:25:23","doi":"10.21203/rs.3.rs-2146618/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-05-09T12:58:21+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"3326edd3-8562-4996-a152-9e3665161dcf_SNPRID","date":"2023-04-28T01:09:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a6689a7e-1216-418c-a0bf-b10022321e49","date":"2023-04-24T23:20:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-04-24T20:32:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253f37fa-7f7b-4330-8476-37993912abf7","date":"2023-04-18T19:15:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-04-14T21:23:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-11T01:26:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-10-11T01:26:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biochemical Genetics","date":"2022-10-09T04:08:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biochemical-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bigi","sideBox":"Learn more about [Biochemical Genetics](http://link.springer.com/journal/10528)","snPcode":"10528","submissionUrl":"https://submission.nature.com/new-submission/10528/3","title":"Biochemical Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4474d7f8-b569-49ed-9751-8cd00caa59ef","owner":[],"postedDate":"October 14th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-11-13T15:03:35+00:00","versionOfRecord":{"articleIdentity":"rs-2146618","link":"https://doi.org/10.1007/s10528-023-10524-4","journal":{"identity":"biochemical-genetics","isVorOnly":false,"title":"Biochemical Genetics"},"publishedOn":"2023-11-11 15:01:05","publishedOnDateReadable":"November 11th, 2023"},"versionCreatedAt":"2022-10-14 18:25:23","video":"","vorDoi":"10.1007/s10528-023-10524-4","vorDoiUrl":"https://doi.org/10.1007/s10528-023-10524-4","workflowStages":[]},"version":"v1","identity":"rs-2146618","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2146618","identity":"rs-2146618","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}