WO2023207233A1 - 豌豆中性SNaPshot标记及其在群体遗传多样性分析中的应用 - Google Patents

豌豆中性SNaPshot标记及其在群体遗传多样性分析中的应用 Download PDF

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WO2023207233A1
WO2023207233A1 PCT/CN2023/073449 CN2023073449W WO2023207233A1 WO 2023207233 A1 WO2023207233 A1 WO 2023207233A1 CN 2023073449 W CN2023073449 W CN 2023073449W WO 2023207233 A1 WO2023207233 A1 WO 2023207233A1
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pea
snapshot
neutral
markers
analysis
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French (fr)
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丁汉凤
王栋
李娜娜
张小燕
李润芳
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山东省农业科学院
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q1/6869—Methods for sequencing
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to a set of pea neutral SNaPshot markers and their application in population genetic diversity analysis, and belongs to the technical field of plant genetics.
  • SNPs single nucleotide polymorphism markers
  • association mapping genome-wide association analysis
  • GWAS genome-wide association analysis
  • QTL identification candidate gene mining
  • genetic linkage map construction widely used.
  • SnaPshot micro-sequencing technology
  • ABSI Applied Biosystems
  • the bound primers and remaining dNTPs are purified from the PCR product to avoid interference with the subsequent SBE reaction; the 3′ end of the primer then binds directly to the target SNP and is extended by TaqDNA polymerase, which binds the fluorescently labeled ddNTP performs a PCR reaction with primers at the 5' end close to the SNP site; finally, a sequencer and software such as GeneScan are used for genotyping and data analysis.
  • SNaPshot has the characteristics of high sensitivity, good repeatability, and no need for additional equipment. It has been widely used in research fields such as forensic identification and SNP detection of human genes.
  • the present invention provides a set of pea neutral SNaPshot markers and their application in analysis of genetic diversity and population genetic structure.
  • the present invention uses 432 pea germplasms as test materials and analyzes the genetic diversity and population genetic structure of pea through a set of pea neutral SNaPshot markers (46 neutral markers). Since neutral markers and heat-resistant and other functional genes Regardless, pea germplasm can be better grouped according to geographical origin, and has a high degree of consistency with sowing date types.
  • the first object of the present invention is to provide a set of pea neutral SNaPshot markers, which is characterized in that it consists of 46 neutral SNaPshot markers as shown in Table 2, the peripheral amplification primer sequences of the 46 neutral SNaPshot markers and the single The base extension primer sequences are shown in Table 3.
  • the second purpose of the present invention is to provide the above 46 neutral SNaPshot markers in the genetic diversity of pea populations. analysis and its application in the analysis of population genetic structure.
  • the third object of the present invention is to provide a method for analyzing pea genetic diversity using the above 46 neutral SNaPshot markers, which is characterized by:
  • Gene mapper 4.1 was used to analyze SNP site data. Each sample was genotyped according to the corresponding peak value of the SNP site. The analysis results were obtained as Excel format files and PDF format peak charts; PowerMarker 3.25 was used to calculate the genetic diversity of the two sets of SNP markers. Parameters include genotype number (NG), major allele frequency (MAF), allele number (NA), gene diversity (GD), expected heterozygosity (He), polymorphism information content (PIC), etc.
  • NG genotype number
  • MAF major allele frequency
  • NA allele number
  • GD gene diversity
  • He expected heterozygosity
  • PIC polymorphism information content
  • the total peripheral amplification amplification system is 35 ⁇ l: 1.1 ⁇ T3 Super PCR Mix, 30 ⁇ l; 10 ⁇ M Primer F, 2 ⁇ l; 10 ⁇ M Primer R, 2 ⁇ l; Template (gDNA), 1 ⁇ l.
  • Amplification program 98°C for 3 minutes; 98°C for 10 seconds, 57°C for 10 seconds, 72°C for 15 seconds, 35 cycles; 72°C for 2 minutes; store at 4°C.
  • the single base extension primer was used for SNaPshot PCR.
  • the total PCR system was 5 ⁇ l: ABI SnapShot multiplex Mix (Applied Biosystems, Foster City, CA, USA), 2 ⁇ l; Primers, 1 ⁇ l; purified PCR Template, 1 ⁇ l; ddH 2 O, 1 ⁇ l.
  • Amplification program 96°C for 2min; 96°C for 10s, 50°C for 5s, 60°C for 30s, 30cycles; 60°C for 30s; store at 4°C.
  • the present invention also provides a method for analyzing the genetic structure of pea populations, which is characterized in that, based on the above step 2), first, use Structure 2.3.4 to perform Bayesian clustering analysis, and determine the best value according to the Delta K ( ⁇ K) value Population structure and population number; secondly, principal coordinate analysis (PCoA) was performed to check whether the Structure analysis results of peas were reasonable; finally, UPGMA cluster analysis was used to construct a phylogenetic tree to visually display the analysis results.
  • ⁇ K Delta K
  • PCoA principal coordinate analysis
  • pea germplasm is divided into two genetic subpopulations A and B.
  • the present invention introduces the SNaPshot method into the identification and evaluation of pea germplasm for the first time, develops a set of pea neutral SNaPshot markers (46 neutral markers), and analyzes the genetic diversity and population genetic structure of pea. Due to the neutrality
  • the markers have nothing to do with functional genes such as heat resistance, and can better group pea germplasm according to geographical origin, and are highly consistent with sowing date types.
  • the present invention uses neutral SNaPshot markers to conduct genetic diversity evaluation and population genetic structure analysis on 432 pea germplasms. After analysis of neutral SNaPshot markers, it was found that the number of markers significantly affected the total amount of NG and NA, and had a certain impact on the mean values of MAF, GD and PIC, but had almost no impact on the mean value of He. As the number of markers increases, the total amounts of NG and NA increase, but the mean MAF decreases. The mean values of GD and PIC increase, and the proportion of high and moderate PIC markers increases; vice versa. From within the markers, the population size has little impact on the total amount of NG and NA, indicating that the neutral markers are scientifically selected and evenly distributed on the chromosomes.
  • Figure 1 shows the ⁇ K in Structure analysis of neutral SNaPshot markers in pea
  • Figure 2 shows the population genetic structure analysis of neutral SNaPshot markers in 432 pea germplasm; A: Structure analysis of 46 neutral SNaPshot markers; Figure B: PCoA of 46 neutral SNaPshot markers; Figure C: Nei-based genetics Distance and UPGMA phylogenetic tree of 46 neutral SNaPshot markers;
  • Genomic DNA came from 432 pea germplasms. The young leaves of 3 plants from each accession were collected 4 weeks after sowing, and mixed and extracted using the TSINGKE plant DNA extraction kit (Beijing Qingke Biotechnology Co., Ltd.).
  • peripheral primers follows the following principles: the primer length is 15-30 bp, and its effective length is generally not greater than 38 bp.
  • the GC content should be 40%-60%, and the optimal Tm value is 58-60°C.
  • the primer itself cannot contain its own complementary sequence. There should not be more than 4 complementary or homologous bases between primers, especially complementary overlap at the 3’ end should be avoided.
  • primer length is 15-30 bp
  • GC content is 40%-60%
  • optimal Tm value is 58-60°C.
  • the shortest length of the tailed primer is designed to be 36 bp, and the length of the primers for two adjacent SNP sites generally differs by 4-6 nucleotides.
  • Tayeh et al. were used to develop the GenoPea13.2K SNP chip, and 46 sites were selected, all of which were neutral mutations.
  • Premier5 was used to design a pair of peripheral amplification primers and a single-base extension primer. See Table 2 and Table 3 for details of SNP sites and SNaPshot primers.
  • the extracted DNA sample was diluted to 20ng/ ⁇ l and used as a PCR template.
  • 1.1 ⁇ T3 Super PCR Mix (Beijing Qingke Biotechnology Co., Ltd.) was used for peripheral amplification. A single amplification was performed for each site.
  • Each pair of primers was as follows: Amplification systems and procedures for amplification.
  • the total amplification system is 35 ⁇ l: 1.1 ⁇ T3 Super PCR Mix, 30 ⁇ l; 10 ⁇ M Primer F, 2 ⁇ l; 10 ⁇ M Primer R, 2 ⁇ l; Template (gDNA), 1 ⁇ l.
  • Amplification program 98°C for 3 minutes; 98°C for 10 seconds, 57°C for 10 seconds, 72°C for 15 seconds, 35 cycles; 72°C for 2 minutes; store at 4°C.
  • the amplified PCR product was subjected to agarose gel electrophoresis (2 ⁇ l sample + 6 ⁇ l bromophenol blue) at 300V for 12 minutes to obtain an identification gel map and determine the size of the target band through the gel map.
  • PCR products were purified using MagS magnetic bead gel recovery kit (Beijing Qingke Biotechnology Co., Ltd.).
  • the purified single PCR product is ready for use.
  • the single-base extension primer is diluted to 10 ⁇ M and SNaPshot PCR is performed.
  • the total PCR system is 5 ⁇ l: ABI SnapShot multiplex Mix (Applied Biosystems, Foster City, CA, USA), 2 ⁇ l; Primers, 1 ⁇ l; purification Post-PCR Template, 1 ⁇ l; ddH 2 O, 1 ⁇ l.
  • Amplification program 96°C for 2min; 96°C for 10s, 50°C for 5s, 60°C for 30s, 30cycles; 60°C for 30s; store at 4°C.
  • the SNaPshot PCR reaction product was detected by capillary electrophoresis using an ABI3730XL DNA analyzer (Applied Biosystems, Foster City, USA).
  • Gene mapper 4.1 was used to analyze SNP site data. Each sample was genotyped according to the peak value corresponding to the SNP site. The analysis results were obtained in Excel format files and PDF format peak charts. Use PowerMarker 3.25 to calculate the genetic diversity parameters of the two sets of SNP markers, including genotype number (NG), major allele frequency (MAF), allele number (NA), gene diversity (GD), expected heterozygosity ( He), polymorphic information content (PIC), etc.
  • NG genotype number
  • MAF major allele frequency
  • NA allele number
  • GD gene diversity
  • He expected heterozygosity
  • PIC polymorphic information content
  • PowerMarker3.25 was used to construct a phylogenetic tree based on the UPGMA (unweighted pair-group method) method for the pea population, and Figtree 1.4.3 (https://github.com/rambaut/figtree/releases/tag/v1. 4.3) Display it.
  • NG Number of genotypes
  • NA Number of alleles
  • MAF Major allele frequency
  • GD Gene diversity
  • He Expected heterozygosity
  • PIC Polymorphic information content, high (PIC ⁇ 0.5), medium (0.25 ⁇ PIC ⁇ 0.5), low (PIC ⁇ 0.25).
  • Green (light gray in the black and white picture, the same below) represents subgroup B, with a total of 263 copies, including 118 spring sowing types. (44.9%), 145 copies (55.1%) of winter sowing type, with slightly more winter sowing types; among subgroup B, 111 copies (42.2%) came from southern China, 87 copies (33.1%) from northern China, and 57 copies (21.7%) from abroad. ) and 8 copies (3.0%) from unknown sources (Table 6). Neutral SNaPshot markers divide two subgroups with large differences in quantity and composition.
  • PCoA Principal coordinate analysis
  • UPGMA cluster analysis is used to construct a phylogenetic tree, which can display the analysis results more intuitively.
  • the UPGMA dendrogram based on neutral markers divided all 432 pea accessions into two groups of tree branches. As shown in Figure 2C, the dark red tree branches are subgroup A, and the green tree branches are subgroup B. There are individual accessions within both subpopulations that are within the other's subpopulation, which is consistent with the PCoA analysis.
  • Neutral SNaPshot markers were used to evaluate the genetic diversity and analyze the population genetic structure of 432 pea germplasm. After analysis of neutral SNaPshot markers, it was found that the number of markers significantly affected the total amount of NG and NA, and had a certain impact on the mean values of MAF, GD and PIC, but had almost no impact on the mean value of He. As the number of markers increases, the total amounts of NG and NA increase, but the mean MAF decreases. The mean values of GD and PIC increase, and the proportion of high and moderate PIC markers increases; vice versa.
  • the population size has little impact on the total amount of NG and NA, indicating that the neutral markers are scientifically selected and evenly distributed on the chromosomes.
  • the mean MAF value increases, He changes little, the mean values of GD and PIC decrease, and the proportion of high and moderate PIC markers decreases; vice versa.
  • PCoA Principal coordinate analysis
  • UPGMA cluster analysis dendrogram can verify the Structure analysis results more intuitively.
  • the reason for this result is that neutral markers have nothing to do with functional genes such as heat resistance, which can better group pea germplasm according to geographical origin and have a high degree of consistency with sowing date types.

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Abstract

本发明公开了一套豌豆中性SNaPshot标记及其在群体遗传多样性分析中的应用。本发明以432份豌豆种质为试验材料，通过一套豌豆中性SNaPshot标记（46个中性标记），对豌豆进行遗传多样性和群体遗传结构分析。

Description

豌豆中性SNaPshot标记及其在群体遗传多样性分析中的应用 技术领域
本发明涉及一套豌豆中性SNaPshot标记及其在群体遗传多样性分析中的应用，属于植物遗传学技术领域。
背景技术
豌豆(Pisum sativum L.，2n＝14)是一种广泛种植于温带地区的冷季食用豆类作物，其营养价值丰富，是蛋白质、淀粉、糖类、粗纤维、维生素和低脂肪的重要来源。同时，豌豆根系中的根瘤菌可以固定大气中的氮，以便增加土壤肥力，减少环境污染。豌豆是粮、菜、饲、肥兼用的优良作物。中国的干豌豆产量居世界第三位，青豌豆产量居世界第一位，无疑是世界豌豆生产大国，但是每年仍需从加拿大等国进口大量的豌豆来满足日益增长的消费需求，因此，了解各份豌豆种质之间的遗传多样性和群体遗传关系对于豌豆遗传改良研究和在育种中选择合适的亲本至关重要。
近年来，随着单核苷酸多态性标记(SNPs)的兴起，在豌豆诸多研究领域如关联作图、全基因组关联分析(GWAS)、QTL鉴定、候选基因挖掘、遗传连锁图谱构建等得到广泛应用。SnaPshot(微测序技术)，是由美国应用生物系统公司(Applied Biosystems，ABI)开发的一种SNPs多重分析技术，可实现中通量的SNPs分型。其基本原理和流程：首先，对DNA模板进行多重PCR，以产生目标SNP扩增片段；然后通过添加核酸外切酶I(Exo I)和碱性磷酸酶(Shrimp Alkaline Phosphatase，SAP)来降解未结合的引物和剩余的dNTPs，从而对PCR产物进行纯化，以免干扰随后的SBE反应；随后引物的3′端直接与目标SNP结合，并由TaqDNA聚合酶延伸，该酶结合了带有荧光标记的ddNTP与5′端靠近SNP位点的引物进行PCR反应；最后，使用测序仪和GeneScan等软件进行基因分型和数据分析。SNaPshot具有灵敏度高，重复性好，无需额外设备等特点，已广泛应用于法医学鉴定和人类基因的SNP检测等研究领域。在植物遗传学研究领域如SNP分型及标记开发、分子标记辅助育种和遗传多样性分析等方面都有报道，展示了SNaPshot技术在植物遗传学研究方面的广阔应用前景。到目前为止，SNP标记在豌豆遗传多样性评价和群体遗传结构研究方面应用不多，仅有零星报道，而利用基于SNaPshot技术开发的SNP标记对豌豆进行遗传学方面的研究更是鲜有报道。
发明内容
本发明提供了一套豌豆中性SNaPshot标记及其在遗传多样性和群体遗传结构分析中的应用。本发明以432份豌豆种质为试验材料，通过一套豌豆中性SNaPshot标记(46个中性标记)，对豌豆进行遗传多样性和群体遗传结构分析，由于中性标记与耐热等功能基因无关，可以更好地将豌豆种质按照地理来源予以分组，且与播期类型一致度较高。
本发明的第一个目的是提供一套豌豆中性SNaPshot标记，其特征是，由如表2所示的46个中性SNaPshot标记组成，46个中性SNaPshot标记的外围扩增引物序列和单碱基延伸引物序列如表3所示。
本发明的第二个目的是提供上述46个中性SNaPshot标记在豌豆群体遗传多样性 分析以及群体遗传结构分析中的应用。
本发明第三个目的是提供采用上述46个中性SNaPshot标记进行豌豆遗传多样性分析的方法，其特征是，
1)SNaPshot PCR反应
以待测豌豆种质群体的DNA作为PCR模板，进行外围扩增，每个位点进行单一扩增，PCR产物纯化后再采用单碱基延伸引物进行SNaPshot PCR，SNaPshot PCR反应产物通过ABI3730XL DNA分析仪进行毛细管电泳检测；
2)数据分析
利用Gene mapper 4.1进行SNP位点数据分析，每个样品按照SNP位点对应峰值进行基因分型，所得分析结果为Excel格式文件和PDF格式峰图；利用PowerMarker 3.25计算两组SNP标记的遗传多样性参数，包括基因型数(NG)、主要等位基因频率(MAF)、等位基因数(NA)、基因多样性(GD)、期望杂合度(He)、多态信息含量(PIC)等。
上述步骤1)中外围扩增扩增体系共35μl：其中1.1×T3 Super PCR Mix，30μl；10μM Primer F，2μl；10μM Primer R，2μl；Template(gDNA)，1μl。扩增程序：98℃3min；98℃10s，57℃10s，72℃15s，35cycles；72℃2min；4℃保存。
上述步骤1)中单碱基延伸引物进行SNaPshot PCR，PCR体系共5μl：ABI SnapShot multiplex Mix(Applied Biosystems,Foster City,CA,USA)，2μl；Primers，1μl；纯化后PCR Template，1μl；ddH2O，1μl。扩增程序：96℃2min；96℃10s，50℃5s，60℃30s，30cycles；60℃30s；4℃保存。
本发明还提供了豌豆群体遗传结构分析的方法，其特征是，在上述步骤2)基础上，首先，利用Structure 2.3.4进行贝叶斯聚类分析，根据Delta K(ΔK)值确定最佳群体结构和群体数量；其次，进行主坐标分析(PCoA)，以检查豌豆的Structure分析结果是否合理；最后，利用UPGMA聚类分析构建系统发育树，直观地展示分析结果。
在本发明中，豌豆种质分成了两个遗传亚群A和B。
本发明的技术效果是：
1、本发明首次将SNaPshot方法引入到豌豆种质的鉴定评价中，开发了一套豌豆中性SNaPshot标记(46个中性标记)，对豌豆进行遗传多样性和群体遗传结构分析，由于中性标记与耐热等功能基因无关，可以更好地将豌豆种质按照地理来源予以分组，且与播期类型一致度较高。
2、本发明对432份豌豆种质利用中性SNaPshot标记进行遗传多样性评价及群体遗传结构分析。中性SNaPshot标记分析后发现，标记数量显著影响NG和NA总量，对MAF、GD和PIC的均值有一定影响，但对He均值几乎没有影响。标记数量增多，NG和NA总量升高，MAF均值却降低，GD和PIC均值升高，高度和中度PIC标记的比例增多；反之亦然。从标记内部看，群体量对NG和NA总量影响较小，表明中性标记选择科学，在染色体上的分布均匀。
附图说明
图1为豌豆中性SNaPshot标记Structure分析中的ΔK；
图2为432份豌豆种质的中性SNaPshot标记的群体遗传结构分析；A：46个中性SNaPshot标记的Structure分析；B图：46个中性SNaPshot标记的PCoA；C图：基于Nei的遗传 距离和46个中性SNaPshot标记的UPGMA系统发育树；
图3为432份豌豆种质的群体遗传组成；基于46个中性SNaPshot标记的(A图)春播豌豆种质(n＝246)和(B图)冬播豌豆种质(n＝186)的遗传组成。
具体实施方式
实施例1
1材料与方法
1.1植物材料
选取来自中国农业科学院作物科学研究所国家作物种质库(中国北京)的432份豌豆种质为试验材料，其中363(84.0％)份来自中国的22个省市自治区，61(14.1％)份来自中国以外的10个国家和组织，剩余8(1.9％)份来源地不明，归为“未知”类。所有豌豆种质按播期类型分为两类，246(56.9％)份为春播类型，186(43.1％)份为冬播类型(表1)。
表1 432份豌豆种质的来源和播期类型

1.2SNaPshot分析
基因组DNA来自432份豌豆种质，每份材料在播种后4周时采集3株的幼嫩叶片，使用TSINGKE植物DNA提取试剂盒(北京擎科生物科技有限公司)混合提取。
外围引物设计遵循以下原则：引物长度在15-30bp，其有效长度一般不大于38bp。GC含量应在40％-60％，最适Tm值在58-60℃。引物自身不能含有自身互补序列。引物之间不应有多于4个的互补或同源碱基，尤其应避免3’端的互补重叠。
单碱基延伸引物设计原则：引物长度在15-30bp，GC含量在40％-60％，最适Tm值在58-60℃。在引物的5'末端加上不同长度的PolyC或PolyT，使各条引物以长度区分。加尾后的引物最短设计为36bp，相邻两个SNP位点引物的长度一般相差4-6个核苷酸。
利用Tayeh等开发GenoPea13.2K SNP芯片，从中选出46个位点全部为中性突变。针对每个SNP位点序列，利用Premier5设计一对外围扩增引物和一条单碱基延伸引物。SNP位点及SNaPshot引物信息详见表2、表3。
表2SNP位点信息




表3SNaPshot引物信息



提取的DNA样品稀释至20ng/μl后作为PCR模板，以1.1×T3 Super PCR Mix(北京擎科生物科技有限公司)进行外围扩增，每个位点进行单一扩增，每对引物均按以下扩增体系和程序进行扩增。扩增体系共35μl：其中1.1×T3 Super PCR Mix，30μl；10μM Primer F，2μl；10μM Primer R，2μl；Template(gDNA)，1μl。扩增程序：98℃3min；98℃10s，57℃10s，72℃15s，35cycles；72℃2min；4℃保存。将扩增好的PCR产物进行琼脂糖凝胶电泳(2μl样品+6μl溴酚蓝)，300V电压下12分钟，获取鉴定胶图，通过胶图确定目的条带大小。PCR产物利用MagS磁珠凝胶回收试剂盒(北京擎科生物科技有限公司)进行纯化。
纯化好的单一PCR产物待用，单碱基延伸引物稀释到10μM，进行SNaPshot PCR，PCR体系共5μl：ABI SnapShot multiplex Mix(Applied Biosystems,Foster City,CA,USA)，2μl；Primers，1μl；纯化后PCR Template，1μl；ddH2O，1μl。扩增程序：96℃2min；96℃10s，50℃5s，60℃30s，30cycles；60℃30s；4℃保存。SNaPshot PCR反应产物通过ABI3730XL DNA分析仪(Applied Biosystems,Foster City,USA)进行毛细管电泳检测。
1.3数据分析
利用Gene mapper 4.1进行SNP位点数据分析，每个样品按照SNP位点对应峰值进行基因分型，所得分析结果为Excel格式文件和PDF格式峰图。利用PowerMarker 3.25计算两组SNP标记的遗传多样性参数，包括基因型数(NG)、主要等位基因频率(MAF)、等位基因数(NA)、基因多样性(GD)、期望杂合度(He)、多态信息含量(PIC)等。
利用不同的群体遗传结构分析方法，对豌豆群体进行了SNP标记的遗传结构分析。首先，利用Structure 2.3.4进行贝叶斯聚类分析。参数设定如下：Length of Burnin Period＝10000，Number of MCMC Reps after Burnin＝100000，群体数量K(Number of population)＝1-10，循环数(Number of Iterations)＝10。根据Evanno等提出的算法，根据Delta K(ΔK)值确定最佳群体结构和群体数量(在线分析网址为http://taylor0.biology.ucla.edu/struct_harvest/)。其次，利用GenAlEx6.5进行主坐标分析(PCoA)，以检查豌豆的群体遗传分析是否合理。最后，利用PowerMarker3.25对豌豆群体进行基于UPGMA(unweighted pair-group method)法的系统发育树构建，并用Figtree 1.4.3(https://github.com/rambaut/figtree/releases/tag/v1.4.3)加以展示。
2.结果
2.1豌豆群体遗传多样性分析
利用46个中性SNaPshot标记对豌豆种质群体进行遗传多样性评价。NG和NA总数分别为140和94(表4)。MAF、GD、He和PIC的均值分别是0.705，0.371，0.155和0.293(表4)，范围是分别是0.505–0.988，0.023–0.628，0.005–0.539和0.023–0.577(表5)。根据PIC值大小，可将SNaPshot标记分为高信息量(PIC≥0.5)、中信息量(0.25≤PIC<0.5)和低信息量(PIC<0.25)。按照此标准，共有1个高PIC，34个中PIC和11低PIC的SNaPshot标记(表4)。中性SNaPshot标记分析结果表明，432份豌豆种质群体具有较高的遗传多样性。
表4豌豆种质群体SNP标记遗传多样性参数总结
备注：NG：基因型数；NA：等位基因数；MAF：主要等位基因频率；GD：基因多样性；He：期望杂合度；PIC：多态信息含量，高(PIC≥0.5)，中(0.25≤PIC<0.5)，低(PIC<0.25)。
表5豌豆中性SNaPshot标记的遗传多样性指数

2.2豌豆种质群体遗传结构分析
为了研究432份豌豆种质的群体遗传结构，利用Structure 2.3.4计算432份豌豆种质的遗传组成，并确定其最佳的遗传亚群分组数(K)。Evanno’ΔK值在遗传亚群分组数K＝2时最高，且远高于其他K取值(图1)。图2A中，深红色(黑白图中为黑色，下同)代表亚群A，共计169份，其中春播类型128份(75.7％)，冬播类型41份(24.3％)，春播类型占大多数；亚群A中154份(91.1％)来自中国北方，少数来自中国南方和国外，分别为11份(6.5％)和4份(2.4％)。绿色(黑白图中为浅灰色，下同)代表亚群B，共计263份，其中春播类型118份 (44.9％)，冬播类型145份(55.1％)，冬播类型略多；亚群B中来自中国南方111份(42.2％)，中国北方87份(33.1％)，国外57份(21.7％)及未知来源8份(3.0％)(表6)。中性SNaPshot标记划分两个亚群在数量和组成上差别较大。
表6基于中性SNaPshot标记Structure分析的豌豆种质遗传亚群分组
利用主坐标分析(PCoA)对Structure分析结果结果加以验证。基于中性标记的PCoA将筛选后豌豆种质分成了两个遗传亚群A和B。如图2B所示，蓝色椭圆(右侧椭圆)中的亚群A与红色椭圆(左侧椭圆)中的亚群B清楚地分开，但有个别种质例外而在对方的亚群内，其中深红色方形代表亚群A春播类型，深红色圆形代表亚群A冬播类型；绿色方形代表亚群B春播类型，绿色圆形代表亚群B冬播类型，其群体组成与Structure分析一致。中性标记PCoA前三成分贡献率为34.56％。以上结果表明，PCoA较好地验证了Structure分析对于豌豆种质的遗传亚群分组。
利用UPGMA聚类分析构建系统发育树，可以更加直观地加以展示分析结果。基于中性标记的UPGMA树状图将432份豌豆种质都划分为两组树状分支。如图2C所示，深红色树状分支为亚群A，绿色树状分支为亚群B。两个亚群内都有个别种质在对方的亚群内部，这与PCoA分析一致。
432份豌豆种质可分为春播类型(n＝246)和冬播类型(n＝186)。通过中性SNaPshot标记的群体遗传结构分析后获得了2个亚群，可以对播期类型进行遗传组成解析。如图3所示，246份春播类型中属于亚群A的128份(52.0％)，略多于亚群B的118份(48.0％)；186份冬播类型中属于亚群A的仅有41份(22.0％)，远少于亚群B的145份(78.0％)，表明春播类型超过半数属于亚群A，而冬播类型大多数属于亚群B。
3.讨论
本研究首次将SNaPshot方法引入到豌豆种质的鉴定评价中，对432份豌豆种质利用中性SNaPshot标记进行遗传多样性评价及群体遗传结构分析。中性SNaPshot标记分析后发现，标记数量显著影响NG和NA总量，对MAF、GD和PIC的均值有一定影响，但对He均值几乎没有影响。标记数量增多，NG和NA总量升高，MAF均值却降低，GD和PIC均值升高，高度和中度PIC标记的比例增多；反之亦然。从标记内部看，群体量对NG和NA总量影响较小，表明中性标记选择科学，在染色体上的分布均匀。群体量减少，MAF均值升高，He变化不大，GD和PIC均值降低，高度和中度PIC标记的比例随之降低；反之亦然。
对于中性标记，Structure分析将432分豌豆种质划分为两个遗传亚群A和B。亚群A中共有种质169份，其中中国北方春播类型就有120份(71.0％)，占大多数；亚群B中共有种质263份，其中排在前三位的是中国南方冬播类型99份(37.6％)、中国北方春播类型60份(22.8％)和国外春播类型42份(16.0％)。这与豌豆生产实际高度吻合，因中国北方属豌豆 春播区，而中国南方则属豌豆冬播区，且国外种质来源地大半为欧洲和北美，其纬度较高而气温较低，属豌豆春播区。主坐标分析(PCoA)和UPGMA聚类分析树状图可以更加直观地验证Structure分析结果。之所以出现这种结果，是因为中性标记与耐热等功能基因无关，可以更好地将豌豆种质按照地理来源予以分组，且与播期类型一致度较高。

Claims (10)

  1. 一套豌豆中性SNaPshot标记，其特征是，其由下表所示的46个中性SNaPshot标记组成：


  2. 如权利要求1所述的一套豌豆中性SNaPshot标记，其特征是，所述46个中性SNaPshot标记的外围扩增引物序列和单碱基延伸引物序列如下表所示：




  3. 权利要求1或2所述的一套豌豆中性SNaPshot标记在豌豆群体遗传多样性分析中的应用。
  4. 权利要求1或2所述的一套豌豆中性SNaPshot标记在豌豆群体遗传结构分析中的应用。
  5. 采用权利要求2所述的一套豌豆中性SNaPshot标记进行豌豆遗传多样性分析的方法，其特征是，
    1)SNaPshot PCR反应
    以待测豌豆种质群体的DNA作为PCR模板，进行外围扩增，每个位点进行单一扩增，PCR产物纯化后再采用单碱基延伸引物进行SNaPshot PCR，SNaPshot PCR反应产物通过ABI3730XL DNA分析仪进行毛细管电泳检测；
    2)数据分析
    利用Gene mapper 4.1进行SNP位点数据分析，每个样品按照SNP位点对应峰值进行基因分型，所得分析结果为Excel格式文件和PDF格式峰图；利用PowerMarker 3.25计算两组SNP标记的遗传多样性参数。
  6. 如权利要求5所述的豌豆遗传多样性分析的方法，其特征是，所述两组SNP标记的遗传多样性参数包括基因型数NG、主要等位基因频率MAF、等位基因数NA、基因多样性GD、期望杂合度He、多态信息含量PIC。
  7. 如权利要求5或6所述的豌豆遗传多样性分析的方法，其特征是，所述外围扩增的扩增体系共35μl：其中1.1×T3 Super PCR Mix，30μl；10μM Primer F，2μl；10μM Primer R，2μl；Template，1μl；扩增程序：98℃3min；98℃10s，57℃10s，72℃15s，35cycles；72℃2min；4℃保存。
  8. 如权利要求5或6所述的豌豆遗传多样性分析的方法，其特征是，所述单碱基延伸引物进行SNaPshot PCR，PCR体系共5μl：ABI SnapShot multiplex Mix，2μl；Primers，1μl；纯化后PCR Template，1μl；ddH2O，1μl；扩增程序：96℃2min；96℃10s，50℃5s，60℃30s，30cycles；60℃30s；4℃保存。
  9. 一种采用豌豆中性SNaPshot标记进行豌豆群体遗传结构分析的方法，其特征是，在权利要求5的步骤2)基础上，首先，利用Structure 2.3.4进行贝叶斯聚类分析，根据ΔK值 确定最佳群体结构和群体数量；其次，进行主坐标分析PCoA，以检查豌豆的Structure分析结果是否合理；最后，利用UPGMA聚类分析构建系统发育树，直观地展示分析结果。
  10. 如权利要求9所述的豌豆群体遗传结构分析的方法，其特征是，根据ΔK值，将豌豆种质分成了两个遗传亚群A和B。
PCT/CN2023/073449 2022-04-25 2023-01-21 豌豆中性SNaPshot标记及其在群体遗传多样性分析中的应用 WO2023207233A1 (zh)

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