WO2021226806A1 - 甘蓝型油菜高密度全基因组snp芯片及其应用 - Google Patents

甘蓝型油菜高密度全基因组snp芯片及其应用 Download PDF

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WO2021226806A1
WO2021226806A1 PCT/CN2020/089681 CN2020089681W WO2021226806A1 WO 2021226806 A1 WO2021226806 A1 WO 2021226806A1 CN 2020089681 W CN2020089681 W CN 2020089681W WO 2021226806 A1 WO2021226806 A1 WO 2021226806A1
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brassica napus
snp
chip
genome
analysis
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周发松
易斌
邱树青
肖清
丁怡雯
沈金雄
冯芳
涂金星
谢为博
傅廷栋
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武汉双绿源创芯科技研究院有限公司
华中农业大学
双绿源创芯科技(佛山)有限公司
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Priority to PCT/CN2020/089681 priority Critical patent/WO2021226806A1/zh
Priority to CN202080001504.9A priority patent/CN115867667A/zh
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  • the invention relates to the fields of molecular biology, genomics, bioinformatics and plant molecular breeding, and specifically relates to a high-density whole genome SNP chip of Brassica napus and its application.
  • SNP markers are based on DNA sequencing (Davey et al., Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nat Rev Genet. 12:499-510.), since the advent of the 454 sequencer in 2005
  • the second-generation sequencing technology has been continuously improved, the efficiency of genome sequencing has been greatly improved, the cost of sequencing has been drastically reduced, and the complete genome sequence of a large number of species has been completed, which has greatly promoted the progress of functional genome research.
  • Re-sequencing different varieties using the SNP markers found in re-sequencing, to construct a high-density cabbage-type oil menu ploidy map (HapMap), and using Genome-wide Association Mapping (GWAS) to analyze important agronomic traits Association analysis, identify candidate gene loci related to important agronomic traits, and establish a set of efficient, rapid, mature and stable, low-cost, high-throughput genotyping methods (WEI DaYong, etc., Genome-Wide Association Study of the Fertility Restorer Loci for polCMS in Rapeseed (Brassica napus L.). Scientia Agricultura Sinica.2017, Issue(5):802-810), is the priority of molecular biology researchers.
  • Illumina Infinium Rice SNP50 (Chen et al., A High-Density SNP Genotyping Array for RiceBiology and Molecular Breeding.Mo lecular Plant,2014,7(3):541-553) has been widely used in germplasm resource screening, variety authenticity and purity identification, genetic background analysis of breeding materials, etc.; Ganal et al. (Alargemaize(ZeamaysL.
  • SNP genotyping array development and germplasm genotyping, and genetic mapping to compare with the B73 reference genome.PLos One.2011, 6:e28334)
  • the Illumina Infinium maize SNP50 gene chip was used to analyze two maize recombinant inbred line populations and obtained High-density genetic linkage maps containing 20913 and 14524 markers respectively; in the domestication history of soybeans, Affymetrix Axiom genome-wide SNP chip NJAU 355K SoySNP analyzed 105 wild and 262 cultivars, and concluded that soybean cultivars originated in China
  • the conclusions of the central and northern regions Wang et al., Development and application of a novel gene-wide SNP array reveals domestication history in soybean. Sci Rep.
  • the present invention designs a 50K DNA chip based on 32,216,304 SNPs, which are extracted from resequencing data of 510 rape inbred lines.
  • 1,618 functional probes targeting published functional genes have been designed, such as mitochondrial-specific genes from the cytoplasmic male sterility mitochondrial genome and corresponding restorer genes, as well as the specificity of genetically modified materials. Sex probe.
  • the experimental results show that, compared with the Brassica60K chip of cabbage, the probes of the Bnapus50K chip have more polymorphisms in the tested varieties. Therefore, the Bnapus50K chip has better applications in the genome breeding and genome research of Brassica napus. prospect.
  • the purpose of the present invention is to provide a high-density whole-genome SNP chip for Brassica napus, the SNP chip includes 42,090
  • SNP molecular marker its nucleotide sequence is shown in SEQID No. 1 ⁇ 42,090.
  • Another object of the present invention is to provide the application of a high-density whole genome SNP chip of Brassica napus.
  • the present invention adopts the following technical measures:
  • the suballelic frequency is greater than 0.05 and the mass score is greater than or equal to 30, a total of 11,249,037; SNPs that are less than 50bp away from the left and right SNPs are removed , Remaining 1,040,415; Extract the uniquely aligned SNP:
  • the 50bp sequence on the left and right sides of the SNP has a unique match on one side (the matching degree of the sequence at other positions is less than 85%), a total of 286,921; these 286,921 sites are submitted to the scoring system for scoring , And integrated the QTL loci (including flowering time, oil content, self-incompatibility, etc.) reported in 25 rape GWAS literatures reviewed, and finally selected 45,707 polynucleotides.
  • the application of the Brassica napus high-density whole genome SNP chip includes the use of the SNP chip provided by the present invention to identify or locate the functional gene of Brassica napus; the use of the SNP chip provided by the present invention to analyze the genetic diversity of Brassica napus; and the use of the present invention provides The SNP chip is used for the analysis of Brassica napus varieties.
  • the present invention has the following advantages:
  • the invention Compared with the same type of Brassica 60K chips that integrates 52,157 SNP markers, the invention has a better variety representation than the same type of Brassica 60K chips that are publicly released by 16 international academic and commercial institutions.
  • the SNP sites of the Brassica 60K chip were identified and screened from more than 70 Brassica napus sequencing data.
  • the Bnapus50K Brassica napus genome chip SNP sites of the present invention were derived from the resequencing results of 510 Brassica napus varieties.
  • the Bnapus 50K probe of the present invention is more evenly distributed on the genome than Brassica60K ( Figure 1, left).
  • Figure 1 is a schematic diagram of the distribution of all SNP sites on the Brassica 60K chip and Bnapus50K whole genome breeding chip on the whole genome.
  • the left picture is the Brassica60K chip, and the right picture is the Bnapus 50K chip.
  • Figure 2 is a typing diagram of Brassica napus P4, 1L238 and their hybrid progeny P4/1L238.
  • Figure 3 shows the Manhattan chart of the GWAS association results.
  • Figure 4 shows the BSA positioning results.
  • the present invention uses 510 cabbage rape varieties (89.8% of the varieties are from China, 10.2% of the varieties are from East Asia, Europe, America, Australia and other countries, which are very representative of Chinese rape varieties) re-sequencing results to identify A total of 32,216,304 original mutation sites (SNP+INDEL) were retained, and only two genotypes were retained.
  • the suballelic frequency was greater than 0.05 and the mass score was greater than or equal to 30, a total of 11,249,037 sites; the distance between the left and right SNPs was less than 50bp SNP, 1,040,415 remaining; extract the uniquely aligned SNP: the 50bp sequence on the left and right sides of the SNP has a unique match on one side (the matching degree of the sequence at other positions is less than 85%), a total of 286,921; these 286,921 sites are submitted to the scoring system Score and integrate the QTL loci reported in 25 rape GWAS literatures reviewed, and finally screened 45707 polynucleotides. These sequences were submitted to Illumina, and the Infinium chip manufacturing technology was used to make SNP chips. Among them, 42,090 high-quality probes were successfully synthesized and named Bnapus50K; the nucleotide sequences of the 42,090 probes are as SEQ ID NO.1 ⁇ 42,090 shown.
  • the method of using Bnapus50K Brassica napus whole genome breeding chip to detect Brassica napus samples includes the following steps:
  • Bnapus50K chip is applied to the genetic background analysis of Brassica napus:
  • the Bnapus50K chip was used to detect P4/1L238 and its parents P4 and 1L238 to determine whether it can be used for the background selection of the progeny population of the hybrid combination.
  • Bnapus50K chip is used in genome-wide association study (Genome-wide association study; GWAS):
  • Genome-wide association analysis is to use millions of single-nucleotide polymorphisms (SNPs) in the genome as molecular genetic markers to conduct a control analysis or correlation analysis at the whole-genome level, and to find out the impact through comparison A new strategy for gene mutation of complex traits.
  • SNPs single-nucleotide polymorphisms
  • the Bnapus50K chip was used to detect 356 Brassica napus materials, and the same quantitative trait RT2 (root weight at seedling stage) of the corresponding materials was determined, and the p value associated with each point and the phenotype was calculated. Draw the corresponding Manhattan map according to the p value.
  • Bnapus50K chip is used in mixed grouping analysis (Bulkedsegregation analysis; BSA):
  • BSA Bitsegregation analysis
  • segregated group grouping analysis is a method of analysis by selecting individuals with extreme or representative traits in a group to form a mixed pool. By studying the difference of allele/molecular marker frequency between mixed pools, the locus related to the trait is located on the genome.
  • the Bnapus50K chip was used to detect two cabbage rape mixed pools with extreme phenotypes (flowering and closed flowers) (20 samples of each were mixed), and the genes of each locus were tested. The type is compared, and the position of the difference site is marked in the Brassica napus genome.

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Abstract

本发明公开了甘蓝型油菜高密度全基因组SNP芯片及其应用，该芯片命名为Bnapus50 K，包括42,090个SNP标记的探针，这些SNP均匀分布在甘蓝型油菜基因组的19条染色体上，平均密度为每100Kb 5个SNP，所述探针在所有测试品种中均具有丰富的多态性。与现有的甘蓝型油菜SNP芯片相比，Bnapus50K芯片的SNP标记来源于510份甘蓝型油菜品种的重测序结果，具有更好的品种代表性，并增加了新的功能基因探针，能够更有效地用于功能基因鉴定或定位、遗传多样性分析以及品种分析。

Description

甘蓝型油菜高密度全基因组SNP芯片及其应用 技术领域
本发明涉及分子生物学、基因组学、生物信息学及植物分子育种领域，具体地，涉及甘蓝型油菜高密度全基因组SNP芯片及其应用。
背景技术
SNP标记的开发是基于DNA测序基础上的(Davey等，Genome-wide genetic marker discovery and genotyping using next-generation sequencing.Nat Rev Genet.12:499-510.)，自从2005年454测序仪问世以来的十多年时间，二代测序技术不断完善，基因组测序效率大大提高，测序成本大幅度下降，大量物种的全基因组序列完成，极大地推进了功能基因组研究的进展。
甘蓝型油菜为十字花科草本植物，同时也是三种油用油菜(白菜型油菜、芥菜型油菜、甘蓝型油菜)中籽粒产量最高的种类，由白菜(AA,n＝10)与甘蓝(CC,n＝9)通过自然种间杂交后双二倍化进化而来的一种复合种。对不同的品种进行重测序，利用重测序发现的SNP标记，构建高密度的甘蓝型油菜单倍型图谱(HapMap)，利用全基因组关联分析(Genome-wide Association Mapping,GWAS)对重要农艺性状进行关联分析，确定重要农艺性状相关的候选基因位点，建立起一套高效快速、成熟稳定、成本低、通量高的基因型鉴定方法(WEI DaYong等，Genome-Wide Association Study of the Fertility Restorer Loci for polCMS in Rapeseed(Brassica napus L.).Scientia Agricultura Sinica.2017,Issue(5):802-810)，是分子生物学研究人员优先考虑的方向。
目前多个作物物种中都开发出了SNP基因芯片，甘蓝型油菜的Illumina InfiniumBrassica 60K(Wayne E.Clarke等，A high-density SNP genotyping array for Brassica napus and its ancestral diploid species based on optimised selection of single-locus markers in the allotetraploid genome.Theoretical and Applied Genetics,2016,1887–1899)、水稻的Illumina Infinium Rice6K(Yu等，A whole genome SNP array(RICE6K)for genomic breeding in rice.Plant Biotech J,2014,12:28-37)、Cornell_6K_Array_Infinium_Rice(Thomson等，Large-scale deployment of a rice 6K SNP array for genetics and breeding applications.Rice(NY),2017,10:40)、Illumina GoldenGate(Parida等，SNPs in stress-responsive rice genes:validation,genotyping,functional relevance and population structure.BMC Genomics.2012,13:426-443；)、Affymetrix GeneChip Rice 44K(Zhao等，Genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa.Nat Commun.2011,13:467)、Illumina Infinium RiceSNP50(Chen等，A High-Density SNP Genotyping Array for RiceBiology and Molecular Breeding.Molecular Plant,2014,7(3):541-553)已经广泛的应用于种质资源筛选、品种真实性和纯度鉴定、育种材料遗传背景分析等工作；Ganal等(A large maize(Zea mays L.)SNP genotyping array:development and germplasm genotyping,and genetic mapping to compare with the B73reference genome.PLos One.2011, 6:e28334)利用Illumina Infinium maize SNP50基因芯片对两个玉米重组自交系群体进行分析，获得了分别包含20913和14524个标记的高密度遗传连锁图；在大豆的驯化历史研究中，Affymetrix Axiom全基因组SNP芯片NJAU 355K SoySNP分析了105个野生和262个栽培品种，得出大豆栽培品种起源于中国的中部和北部的结论(Wang等，Development and ap plication of a novel genome-wide SNP array reveals domestication history in soybean.Sci Rep.2016,6:20728-20737)；为构建小麦的高密度遗传连锁图，以小麦90KInfinium iSelect SNP芯片对4个小麦群体进行了扫描，将29692个SNP标记定位到了6倍体小麦的21条染色体上(Wen等，A high-density consensus map of common wheat integrating four mapping populations scanned by the 90K SNP array.Front Plant Sci.2017,doi:10.3389/fpls.2017.01389)。
本发明基于32,216,304个SNP设计了50K DNA芯片，这些SNP是从510个油菜自交系的重测序数据中提取的。考虑到在育种中的应用，设计了1,618种针对已发表的功能基因的功能探针，例如来自细胞质雄性不育线粒体基因组的线粒体特异性基因和相应的恢复基因，以及用于检测转基因材料的特异性探针。实验结果表明，与甘蓝型Brassica60K芯片相比，Bnapus50K芯片的这些探针在测试品种中具有更丰富的多态性，因此，Bnapus50K芯片在甘蓝型油菜的基因组育种和基因组研究中具有更好的应用前景。
发明内容
本发明的目的在于提供了甘蓝型油菜高密度全基因组SNP芯片，所述的SNP芯片包括42,090个
SNP分子标记，其核苷酸序列如SEQID No.1～42,090所示。
本发明的另一个目的在于提供了甘蓝型油菜高密度全基因组SNP芯片的应用。
为了达到上述目的，本发明采取以下技术措施：
申请人利用510份自然群体二代重测序数据，其中89.8％的品种来自中国，10.2％的品种来自东亚、欧洲、美洲、澳洲等国，对中国的油菜品种具有非常好的代表性，鉴定出原始变异位点(SNP+INDEL)共32,216,304，保留只存在两种基因型，次等位基因频率大于0.05且质量分数大于等于30的位点共11,249,037；去除与左右两侧SNP距离小于50bp的SNP，剩余1,040,415；提取唯一比对的SNP：SNP左右两侧50bp序列有一侧唯一匹配(序列在其他位置的匹配度小于85％)的位点共286,921；将这286,921个位点提交给打分***打分，并整合从查阅的25篇油菜GWAS文献中报道的QTL位点(包括开花时间，含油量，自交不亲和性等)，最终筛选得到45,707个多核苷酸。将这些序列提交给Illumina公司，利用Infinium芯片制造技术制作SNP芯片，按照Illumina公司对探针的条件要求，成功合成42,090个高质量探针，命名为Bnapus50K；所述的42,090个多核苷酸为SEQ ID NO.1～42,090所示。
甘蓝型油菜高密度全基因组SNP芯片的应用，包括利用本发明提供的SNP芯片进行甘 蓝型油菜功能基因鉴定或定位；利用本发明提供的SNP芯片进行甘蓝型油菜遗传多样性分析；利用本发明提供的SNP芯片进行甘蓝型油菜品种分析。
与现有技术相比，本发明具有以下优点：
本发明与同类型的由16家国际学术和商业机构公开发布的一套整合了52157个SNP标记的甘蓝型油菜Brassica 60K芯片相比，具有更好的品种代表性。Brassica 60K芯片的SNP位点是从70多份甘蓝型油菜测序数据中鉴定筛选出来的，本发明的Bnapus50K甘蓝型油菜全基因组芯片SNP位点来源于510份甘蓝型油菜品种的重测序结果。本发明的Bnapus 50K探针比Brassica60K在基因组上分布更均匀(图1，左)。
除此之外，利用Brassica60K时，研究人员在GWAS分析中只能有效使用大约40％的探针；本发明对标记进行了优选，优异标记的比例更多，标记密度分布更合理，增加了已发表的功能基因探针，能够更有效地鉴定功能基因和育种材料遗传背景分析。
附图说明
图1为Brassica 60K芯片和Bnapus50K全基因组育种芯片所有SNP位点在全基因组上的分布示意图，左图为Brassica60K芯片，右图为Bnapus 50K芯片。
图2为甘蓝型油菜P4、1L238和其杂交后代P4/1L238的分型图。
图3为GWAS关联结果曼哈顿图展示图。
图4为BSA定位结果。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
甘蓝型油菜全基因组育种芯片Bnapus50K的获得：
本发明利用510份甘蓝型油菜品种(其中89.8％的品种来自中国，10.2％的品种来自东亚、欧洲、美洲、澳洲等国，对中国的油菜品种具有非常好的代表性)重测序结果，鉴定出原始变异位点(SNP+INDEL)共32,216,304，保留只存在两种基因型，次等位基因频率大于0.05且质量分数大于等于30的位点共11,249,037；去除与左右两侧SNP距离小于50bp的SNP，剩余1,040,415；提取唯一比对的SNP：SNP左右两侧50bp序列有一侧唯一匹配(序列在其他位置的匹配度小于85％)的位点共286,921；将这286,921个位点提交给打分***打分，并整合从查阅的25篇油菜GWAS文献中报道的QTL位点，最终筛选得到45707条多核苷酸。将这些序列提交给Illumina公司，利用Infinium芯片制造技术制作SNP芯片，其中成功合成42,090个高质量探针，命名为Bnapus50K；所述的42,090个探针的核苷酸序列如SEQ ID NO.1～42,090所示。
对SNP位点的选择步骤如下：
1)利用510份甘蓝型油菜品种重测序结果，鉴定出原始变异位点；
2)保留只存在两种基因型，次等位基因频率大于0.05且质量分数大于等于30的位点；
3)去除与左右两侧SNP距离小于50bp的位点；
4)提取唯一比对的SNP：SNP左右两侧50bp序列有一侧唯一匹配(序列在其他位置的匹配度小于85％)的位点；
5)将Darmor基因组划分为每100kb的区段，计算每区段内SNP两两间的相关系数，以相关系数(R 2≥0.65)为阈值将SNP分组；
6)构建一种综合评分策略，对剩余SNP效能进行评价，具体如下：
(1)从GWAS文献(共查阅文献25篇)中报道的QTL位点，提取了极显著的位点共304个，包含的性状有：开花期，早熟，株型(株高，分枝角度等)，产量(千粒重等)，种子品质(含油量，芥酸含量，硫苷含量等)，抗病性(根肿病，黑茎病，菌核病)，抗非生物胁迫(水，热，盐，镉)；在每个位点上下游150K内计划各增加3个探针；
(2)确定标记密度：chr*上每100K低于6个，random上每100K低于2个探针；
(3)统计每100K内需要增加的探针个数，根据以下优先级对286,921个SNP进行筛选：在每100K内，打分≥0.6；MAF较高；尽可能来自不同的bin；
(4)最终选择出45707个SNP位点。
将这些序列提交给Illumina公司，利用Infinium芯片制造技术制作SNP芯片Bnapus50K。Bnapus50K所有SNP位点在全基因组上的分布如图1(右)所示，总体上在全基因组均匀分布，同时在重要功能基因区域分布密集，能满足甘蓝型油菜种质资源遗传多样性分析和亲缘关系分析、满足甘蓝型油菜的功能基因鉴定和基因定位、满足甘蓝型油菜品种真实性鉴定和甘蓝型油菜育种材料遗传背景分析的需求。
实施例2：
利用Bnapus50K甘蓝型油菜全基因组育种芯片检测甘蓝型油菜样品的方法，包括以下步骤：
1)甘蓝型油菜样品DNA的制备：根据检测需要利用天根生化科技(北京)有限公司的植物基因组DNA抽提试剂盒，按照试剂盒标准流程抽提不同组织、器官或个体的DNA；
2)甘蓝型油菜样品DNA的质量控制：用1％的琼脂糖凝胶电泳，检测基因组DNA的完整性；按照仪器使用说明，用Nanodrop2000分光光度计测定DNA的浓度，判断蛋白质和RNA的污染程度；
3)基因芯片检测：按照Illumina Infinium基因芯片检测标准流程操作，芯片扫描使用Illumina HiScan芯片扫描仪；
4)基因芯片数据分析：Illumina HiScan扫描结果用Genome Studio软件分析基因型，并用R语言编程获得基因型比较结果。
实施例3：
Bnapus50K芯片应用于甘蓝型油菜遗传背景分析：
利用实施例2所述的方法，应用Bnapus50K芯片对P4/1L238及其亲本P4、1L238进行检测，确定是否可用于对上述杂交组合后代群体的背景选择。
分析结果显示P4、1L238亲本为差异的纯合位点区，杂种后代P4/1L238在该区域表现为杂合状态，符合杂交现象；且差异标记在全基因组上分布均匀，可有效满足分子育种中对背景进行精确选择的标记需求(图2)。
实施例4：
Bnapus50K芯片应用于全基因组关联分析(Genome-wide association study；GWAS)：
全基因组关联分析是应用基因组中数以百万计的单核苷酸多态性(single nucleotide ploymorphism，SNP)为分子遗传标记，进行全基因组水平上的对照分析或相关性分析，通过比较发现影响复杂性状的基因变异的一种新策略。
利用实施例2所述的方法，应用Bnapus50K芯片对356份甘蓝型油菜材料进行检测，并测定相应材料的同一数量性状RT2(苗期根的重量)，计算各位点与表型关联的p值，根据p值画出对应的曼哈顿图。
分析结果显示该表型与染色体C03的53Mb区域存在强烈的关联性，推测控制该表型性状的基因很有可能位于C03染色体的这一区段，从而有效的识别、鉴定新基因(图3)。
实施例5：
Bnapus50K芯片应用于混合分组分析(Bulkedsegregationanalysis；BSA)：
BSA(Bulkedsegregationanalysis)即混合分组分析，也称分离群体分组分析，是一种通过在群体中挑选极端或代表性性状的个体组成混池进行分析的方法。通过研究混池之间等位基因/分子标记频率的差异，将与性状相关的位点在基因组上进行定位。
利用实施例2所述的方法，应用Bnapus50K芯片对具有极端表型(开花和闭花)的两个甘蓝型油菜混池(各20株样品混合)进行检测，对检测的每一个位点的基因型进行比较，在甘蓝型油菜基因组中标记出差异位点的位置。
分析结果显示这两个混池在染色体A01的8Mb区域存在差异，推测控制该表型性状的基因很有可能位于A01染色体的这一区段，从而有效的识别、鉴定新基因(图4)。

Claims (4)

  1. 甘蓝型油菜高密度全基因组SNP芯片，其特征在于，所述的SNP芯片包括42090个SNP分子标记，其核苷酸序列如SEQID No.1～42090所示。
  2. 权利要求1所述的SNP芯片在甘蓝型油菜功能基因鉴定或定位中的应用。
  3. 权利要求1所述的SNP芯片在甘蓝型油菜遗传多样性分析中的应用。
  4. 权利要求1所述的SNP芯片在甘蓝型油菜品种分析中的应用。
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