WO2018085971A1 - 棉花全基因组snp芯片及其应用 - Google Patents

棉花全基因组snp芯片及其应用 Download PDF

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WO2018085971A1
WO2018085971A1 PCT/CN2016/105001 CN2016105001W WO2018085971A1 WO 2018085971 A1 WO2018085971 A1 WO 2018085971A1 CN 2016105001 W CN2016105001 W CN 2016105001W WO 2018085971 A1 WO2018085971 A1 WO 2018085971A1
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cotton
snp
chip
genome
seq
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PCT/CN2016/105001
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郭旺珍
张天真
蔡彩平
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南京农业大学
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    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof

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  • the present invention relates to the field of genomics, bioinformatics and molecular plant breeding, and in particular to a cotton genome-wide SNP chip (Cotton SNP80K) consisting of 82,259 SNP sites and uses thereof.
  • a cotton genome-wide SNP chip (Cotton SNP80K) consisting of 82,259 SNP sites and uses thereof.
  • SSR single nucleotide polymorphism
  • SSR markers still have problems such as uneven distribution in the genome, low genotyping polymorphism, and insufficient genome coverage. Flux molecular identification and selection needs. SNP markers are evenly distributed and most polymorphic DNA molecular markers in the genome. Using SNP chips covering the whole genome, tens of thousands, hundreds of thousands, or even millions of SNP loci can be classified by one hybridization. It has the advantages of low cost, high throughput and large amount of information obtained.
  • SNP chips have become the most ideal technology platform for genome-wide association analysis, fingerprint mapping construction, genotyping, molecular design breeding, etc., and have played an important role in the genetics and breeding of corn, rice and other crops.
  • Hulse-Kemp et al (2015) developed a SNP chip (CottonSNP63K) containing 63K sites using public database information from different cotton sources, and used the chip to pair 1156 materials from different sources (including two F 2 separate populations were tested and 38,822 polymorphic loci were obtained. Further, a F 2 segregating population of 93 individual plants (G. hirsutum lines Phytogen 72 ⁇ Stoneville 474) was constructed to construct a terrestrial cotton species.
  • Nanjing Agricultural University and the Cotton Research Institute of the Chinese Academy of Agricultural Sciences published the genomic information of the allotetraploid cotton upland cotton genetic standard TM-1 in Nat Biotechnol in 2015 (Zhang et al .2015; Li et al. 2015); Esquel Group and Huazhong Agricultural University both announced the island cotton Xinhai 21 and Sci Rep respectively. Genomic information of 3-79 (Liu et al. 2015; Yuan et al. 2015).
  • Nanjing Agricultural University constructed a genetic map of 4,999,048 SNP loci, 4,049 recombination bins covering 4,042cM ultra-high density SNP and SSR integration, and successfully used in heterogeneous tetraploid upland cotton genome assembly. Error correction and scaffolds direction and order determination (Genome Biol 2015). For the 100 varieties of upland cotton from different ecological sources in different cultivation years in China, Nanjing Agricultural University completed the resequencing analysis of the average genome coverage of 5 times. The genomic information of the terrestrial cotton genetic standard TM-1 was used as the reference sequence to carry out the genome-wide coverage of SNP loci in the whole genome, and the polymorphic sites of 1,372,195 SNPs with MAF>10% were obtained.
  • Illumina SNP genotyping platform is widely used in high-throughput SNP typing technology ( Technology) and Affymetrix Genotyping Platform (Affymetrix technology).
  • Technology high-throughput SNP typing technology
  • Affymetrix Genotyping Platform Affymetrix technology
  • Illumina's SNP typing technology uses BeadArray technology, which includes two series of products, Infinium and GoldenGate. Infinium technology is suitable for multi-site medium-high throughput (30,000 to 5 million sites) full-gene components.
  • the GoldenGate technology is suitable for medium to low-throughput (96 to 3072 sites) genotypes with fewer SNP sites, both available in off-the-shelf or fully customizable iSelect or semi-customized chips to meet different research and analytical needs.
  • each SNP site has been widely recognized in the industry for its high detection rate, good repeatability, and reliable detection quality, and has been widely used in human, animal and plant genomic analysis.
  • the invention is based on the SNP variation in the upland cotton species, and utilizes Assay's proven technology integrates a cotton genome-wide SNP chip for genotyping in upland cotton.
  • the present invention provides a cotton genome-wide set of SNP sites comprising 82,259 SNP sites. Each SNP site contains two different base variation sites for detecting allelic changes at that site.
  • the SNP site is located at position 61 of the nucleotide sequence set forth in SEQ ID NO: 1 - SEQ ID NO: 82, 259.
  • the invention also provides a set of probes for detecting a cotton genome-wide SNP site set, the nucleotide sequence of which is shown in SEQ ID NO: 1 - SEQ ID NO: 82, 259, wherein the SNP site is located The 61st position of the nucleotide sequence shown in SEQ ID NO: 1 - SEQ ID NO: 82, 259.
  • the invention also provides a cotton whole genome SNP typing chip, named CottonSNP80K.
  • the chip contains a set of nucleotide probes for detecting corresponding SNP sites; the nucleotide probe sequence is selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 82, 259 and can be used for the corresponding SNP position. Point genotyping.
  • the SNP site is located at position 61 of the nucleotide sequence set forth in SEQ ID NO: 1 - SEQ ID NO: 82, 259.
  • the experimental steps include: 1) making a genotyping nucleotide probe and a reagent; 2) hybridizing and scanning the probe and the sample on the genotyping system to obtain the analyzed sample in the Genotype at the SNP locus.
  • the above cotton genome-wide SNP locus collection is used in the preparation of a cotton genome-wide SNP chip.
  • the above probes are applied in the fingerprint construction of cotton germplasm resources, genotypic identification of cotton hybrid progeny, cotton genome-wide association analysis, cotton variety identification or cotton breeding material selection.
  • the present invention provides a cotton whole genome SNP typing chip which is produced by Illumina Corporation according to the nucleotide sequence shown in SEQ ID NO: 1 - SEQ ID NO: 82, 259 using the Infinium patented design and manufacturing technology, but is not limited thereto.
  • the genotyping system for providing a cotton whole genome SNP chip of the present invention utilizes Illumina's GenomeStudio genotyping module, but is not limited thereto.
  • the invention provides the application of the CottonSNP80K chip in cotton whole genome SNP typing, is applicable to the fingerprinting of cotton germplasm resources, the genotype identification of hybrid progeny, the genome-wide association analysis of important agronomic traits, the identification of varieties, the selection of breeding materials, and the molecular Design breeding, etc.
  • the invention utilizes the whole genome sequence information of the allogeneic tetraploid upland cotton genetic standard system TM-1 assembled by Nanjing Agricultural University, and the resequencing data of an average of 5 times genomic coverage of 100 different sources of upland cotton materials, and the research covers the whole genome, A SNP chip with high polymorphism and suitable for identification of different genotypes in upland cotton.
  • the 100 parts of the upland cotton material used for resequencing are shown in Table 1.
  • the SNP selection process for customizing the genome-wide covered SNP chip is shown in Figure 1.
  • the specific selection process for designing the whole-genome SNP chip of the upland cotton is as follows:
  • the probe length of the chip is about 50 bp, there is no other polymorphic site within 50 bp on both sides of the SNP site, and the target SNP is not in the SNP site in the Indel region. After filtration, 619,979 sites were obtained.
  • the distribution of the 82,259 locus set in the TM-1 genome of the present invention is shown in Figure 1.
  • the SNP locus involved in the chip covers the cotton genome, with an average of 23.5 Kb and a SNP locus designed on the chip.
  • the detection method by PCR detection, probe hybridization, chip detection and kit analysis is used. Plus All applications are within the scope of the present invention.
  • the SNP sites related to important agronomic traits excavated by the SNP chip disclosed by the present invention are applied by detection methods such as PCR detection, probe hybridization, chip detection and kit analysis, and are all within the protection scope of the present invention. .
  • the beneficial effects of the present invention are to construct a high density SNP site collection of cotton, which has high polymorphism, large flux, and a clear position on the chromosome, covering the whole genome of cotton.
  • 17,671 SNPs were distributed among 13,759 genes, involving 19.57% of the genes in the tetraploid cotton genome.
  • These cSNP loci located in the gene region can meet the needs of functional gene-related breeding and research.
  • Another beneficial effect of the present invention is to develop a SNP chip (Cotton SNP80K) for cotton genome-wide SNP typing, which can be applied to correlation analysis and localization research of important agronomic traits, fingerprint identification analysis of cotton germplasm resources, and breeding materials. Research on prospects and background selection, molecular design breeding and development of cotton breeding chips all have important economic value and application prospects.
  • the polymorphic marker of the present invention can be used for screening of sites/genes associated with important agronomic traits of cotton in a natural population of cotton (different cotton germplasm resources). According to different research and breeding objectives, different cotton germplasm resources were selected, and the SNP chip of the present invention was used to complete the whole genome SNP typing of different cotton materials. Combining the results of accurate phenotypic identification, GWAS analysis of important agronomic traits such as yield, quality, resistance (antibiotic stress or abiotic stress); excavation of important gene/main QTL and cotton germplasm materials carrying a large number of excellent genes.
  • the polymorphic marker in the present invention can be used for the construction of high-density genetic maps, as well as the mapping of target trait genes/QTLs based on genetic maps and map cloning.
  • different parents are selected, large isolated populations (including F 2 , BC 1 and RIL populations, etc.) are configured, and the SNP chips of the present invention are used to complete the typing of SNP sites in the population and construct High density, high quality genetic map. The localization of target trait-related genes and map cloning studies were completed.
  • the SNP chip of the invention can be used for genome-wide SNP typing of different cotton germplasm resources, and the detected polymorphic markers can be used for the construction of fingerprints of cotton germplasm resources.
  • the development of molecular fingerprints of cotton varieties is not only important for protecting the intellectual property rights of cotton cultivars and the rights of breeders, but also improving the seed quality of seed market. It also provides judicial identification of the authenticity and purity of cotton varieties, protection of new plant variety rights, and regulation. Variety management, controlling varieties, chaos, miscellaneous, combating counterfeiting, and further clarifying the genetic relationship of cotton germplasm in China are all necessary.
  • the invention can also be effectively used for intra- and inter-species genotyping analysis of other cotton species such as sea-island cotton.
  • polymorphic markers of the present invention can be used for assisted selection for correlation phenotypic analysis, and other sites are used for background analysis studies.
  • marker-assisted selection firstly, a high-density genetic map is constructed by separating populations, and molecular markers closely linked to the target traits are screened; or molecular markers stably associated with important agronomic traits are screened by natural populations. If the marker is more closely linked to the target trait, the higher the utilization value.
  • the association between specific marker alleles and target trait QTLs can also be used for prediction of population progeny genotypes, population size estimation.
  • the SNP chip of the present invention can be used for genotyping of desired sites in breeding foreground selection and background selection.
  • a site closely linked to a target trait or stably associated with an important agronomic trait is selected, that is, using the target SNP site contained in the present invention and other SNP background molecular markers in the vicinity region, and is effectively used for Molecular design breeding.
  • SNP chip of the present invention screening for a site closely linked to a target trait or stably associated with an important agronomic trait, extracting a target SNP site in the present invention, including adding a part of a new SNP site, and combining them into ⁇ 1 million A SNP set of sites to develop a SNP chip suitable for breeding utilization.
  • DNA extraction and quality detection The whole genome DNA of cotton young leaves was extracted by CTAB method, and the concentration and integrity of DNA were detected by 1% agarose gel electrophoresis and UV spectrophotometer. Ensure that each sample meets the following conditions: 260/280 between 1.7 and 2.1, sample concentration >50 ng/ ⁇ L, total DNA >1 ⁇ g, no macromolecular contamination, no degradation.
  • Chip Detection Operate in accordance with the standard protocol using the Illumina iScan chip scanner.
  • FIG. 1 Chromosome distribution of 82,259 SNP loci in the upland cotton genetic standard TM-1
  • FIG. 1 Cotton CottonSNP80K chip development flow chart
  • Figure 3 Cluster diagram of 344 varieties of upland cotton varieties from different sources
  • Figure 4 Manhattan map of SNP sites associated with cotton fiber strength traits
  • the invention utilizes the whole genome sequence information of the allogeneic tetraploid upland cotton genetic standard system TM-1 assembled by Nanjing Agricultural University, and the resequencing data of 100 times of genome coverage covered by 100 different sources of upland cotton materials, and the research covers the whole genome and polymorphism.
  • the specific selection process for designing the whole-genome SNP chip of the upland cotton is as follows:
  • the probe length of the chip is about 50 bp, there is no other polymorphic site within 50 bp on both sides of the SNP site, and the target SNP is not in the SNP site in the Indel region. After filtration, 619,979 sites were obtained.
  • CottonSNP80K The chip can detect 24 samples at the same time, which is more suitable for SNP typing in upland cotton. It can also be used for intra- and inter-species genotyping analysis of other cotton species such as sea-island cotton.
  • DNA extraction and quality detection The whole genome DNA of cotton young leaves was extracted by CTAB method, and the concentration and integrity of DNA were detected by 1% agarose gel electrophoresis and UV spectrophotometer. Ensure that each sample meets the following conditions: 260/280 between 1.7 and 2.1, sample concentration >50 ng/ ⁇ L, total DNA >1 ⁇ g, no macromolecular contamination, no degradation.
  • Chip Detection Operate in accordance with the standard protocol using the Illumina iScan chip scanner.
  • CottonSNP80K chip of the present invention 344 cotton materials of different origins in three cotton regions of China are numbered 1-344, of which 342 are land cotton materials and 2 are sea island cotton materials. Chip detection and SNP typing analysis were carried out in accordance with the method of Example 2. The names of 344 cotton materials are shown in Schedule 2, and the cluster diagram between materials is shown in Figure 3. The specific performance is as follows:
  • Each chip can detect 24 samples at the same time. Among the 82,259 sites tested, the actual number of sites in the chip is 77,774 (94.55%).
  • the average detection rate of the loci was 99.37%; a total of 59,324 loci showed polymorphism, accounting for 76.44% of the total locus.
  • the polymorphism rate between sea-island cotton and upland cotton was detected to be >30%.
  • the polymorphism rate between TM-1 and Hai7124 is 31.10%
  • the polymorphism rate between TM-1 and Junhai 1 is 31.21%.
  • the genetic background is similar to the Xinxiang Xiaoji velvet-free floc and the Xinxiang Xiaoji velvet-free genomic polymorphism rate is 15.35%
  • the polymorphism rate between Xuzhou 142 velvet-free and Xuzhou 142 wild-type genome is 17.51%, 7235 strain
  • the polymorphism rate was 20.56% with the 7235 strain mutant genome; the genome of the upland cotton genetic standard TM-1 and the series of upland cotton fiber development mutant materials (SL1-7-1, MD-17, N1, n2 and T586, etc.)
  • the inter-state polymorphism rate was between 22.17 and 31.86%; the polymorphism rate between the fiber mutant material imim and the TM-1 genome was 21.89%.
  • the chip has good repeatability.
  • different single plants of three transgenic receptor materials W0 were detected, two different individuals of the upland cotton genetic standard TM-1, and two different orders of the middle 12 and Xuzhou 142 of different sources. Plant material.
  • the results showed that the sites detected by the three W0 plants were identical, and the sites detected by the two TM-1 plants were identical.
  • the agreement among the 12 plants of the two different sources was 95.24%.
  • the agreement between Xuzhou 142 plants from two different sources was 90.43%. It shows that the CottonSNP80K chip has good repeatability and small difference site detection ability.
  • the SNP site on the CottonSNP80K chip has wide applicability, good repeatability, and high-efficiency differential detection capability. Therefore, the CottonSNP80K chip is very suitable for establishing cotton fingerprint database, identifying the genetic relationship between varieties and assisting the selection of breeding materials.
  • the SNP typing results of 242 upland cotton varieties in Example 3 were selected. Correlation analysis of cotton genome-wide fiber quality traits was carried out based on the data of fiber quality traits in three different environments at three and three years. The quality of the typing results was controlled, and the MAF ⁇ 0.05 and the genotype type 1 loci were removed. Finally, 54,408 polymorphic SNP loci were selected for the target trait correlation analysis, which was significantly correlated with fiber quality, and the reproducibility was good. The locus is further used for fine mapping and cloning of target trait genes. For example, by correlation analysis, a SNP interval significantly associated with fiber strength is detected on chromosome A11 (Fig. 4).
  • the SNP chip of the present invention comprises 82,259 SNP sites with well-defined chromosome positions for rapid, high-throughput, low-cost SNP typing of cotton varieties/materials. It can be applied to the correlation analysis and localization research of important agronomic traits, fingerprint classification analysis of cotton germplasm resources, prospects and background selection research of breeding materials, molecular design breeding and development of cotton breeding chips, etc., all of which have important economic value and application. prospect.

Abstract

一种棉花全基因组SNP芯片及其应用，所述芯片命名为CottonSNP80K，包含82,259个SNP位点（SEQ ID NO：1-SEQ ID NO：82,259），主要基于陆地棉种内SNP变异定制，非常适于陆地棉种内基因分型检测，可大大克服陆地棉种内遗传基础狭窄，遗传多样性低的瓶颈。该芯片可以对陆地棉品种资源进行分子标记指纹分析，品种纯度和真实性鉴定，育种材料遗传背景的分析和筛选、农艺性状重要基因位点关联分析等。同时，该芯片也将有效用于海岛棉等其他棉种的种内及种间基因分型分析。

Description

棉花全基因组SNP芯片及其应用 技术领域
本发明涉及基因组学、生物信息学和分子植物育种领域，具体地，涉及一种由82,259个SNP位点组成的棉花全基因组SNP芯片(CottonSNP80K)及其应用。
背景技术
迄今为止，棉花遗传育种研究中可选标记主要为SSR(simple sequence repeat)标记，已被广泛应用到遗传图谱构建、目标性状/QTL定位、关联分析等相关研究。但相比于全基因组覆盖的SNP(single nucleotide polymorphism)标记，SSR标记仍存在基因组中分布不均匀、基因分型多态性不高，以及基因组覆盖度不够等问题，尚不能满足不同基因型高通量分子鉴定及选择的需求。SNP标记是基因组中均匀分布且多态性最为丰富的DNA分子标记，利用覆盖全基因组的SNP芯片，可以通过一次杂交实现数以万计，十万计，乃至百万计的SNP位点分型，具有成本低，通量高，获得的信息量大等优势。目前，SNP芯片已成为全基因组关联分析、指纹图谱构建、基因分型、分子设计育种等研究中最理想的技术平台，已在玉米、水稻等作物遗传育种研究中发挥重要作用。在棉花上，Hulse-Kemp et al(2015)利用不同棉种来源的公共数据库信息，开发了包含63K位点的SNP芯片(CottonSNP63K)，并用该芯片对1156个不同来源的材料(包含两个F2分离群体单株)进行检测，共得到38,822个多态位点；进一步利用(G.hirsutum lines Phytogen 72×Stoneville 474)组配的93个单株的F2分离群体构建了一个陆地棉种内遗传图谱，含7171个SNP标记，覆盖3499cM遗传距离。利用(G.barbadense line 3-79×G.hirsutum standard line TM-1)组配的118个单株的F2分离群体，构建了一个海陆种间遗传图谱，含19,191个SNP标记，覆盖3854.3cM遗传距离。但由于该芯片所涉及的SNP位点主要来源于已释放的不同棉种基因组及转录组数据，位点的基因组覆盖度及具体染色体信息未知。
近年来，棉花基因组学相关研究取得令人瞩目的成果。2012年，美国佐治亚大学的Paterson实验室牵头在Nature发表了关于棉花基因组多倍化及纤维发育研究，同时释放了二倍体D基因组雷蒙德氏棉全基因组序列信息(Paterson et al.2012)。最近几年，来自中国不同研究单位的科学家在二倍体棉种亚洲棉和雷蒙德氏棉，四倍体棉种陆地棉和海岛棉的基因组信息解析上取得突出进展。中国农业科学研究院棉花研究所先后于2012年、2014年在Nat Genet上公布了二倍体雷蒙德氏棉(D基因组)、亚洲棉(A基因组)全基因组序列信息(Wang et al.2012；Li et al.2014)；南京农业大学和中国农科院棉花研究所于2015年均在Nat Biotechnol分别公布了异源四倍体棉种陆地棉遗传标准系TM-1基因组信息(Zhang et al.2015；Li et al.2015)；溢达集团和华中农业大学均在Sci Rep上分别公布了海岛棉新海21及 3-79的基因组信息(Liu et al.2015；Yuan et al.2015)。南京农业大学构建完成了包含4,999,048个SNP位点，4,049个重组bins，覆盖4,042cM的超高密度SNP和SSR整合的海陆种间遗传图谱，并成功用于异源四倍体陆地棉基因组组装中纠错及scaffolds方向和顺序确定(Genome Biol 2015)。针对中国不同培育年份，不同生态来源的100份陆地棉品种，南京农业大学完成平均基因组覆盖5倍的重测序分析。以陆地棉遗传标准系TM-1基因组信息为参考序列，进行全基因组覆盖的陆地棉种内SNP位点发掘研究，获得MAF>10％的1,372,195个SNPs多态位点。上述高密度的SNP遗传图谱、高质量的四倍体棉花基因组序列信息、以及不同陆地棉材料重测序序列信息等为棉花全基因组SNP芯片开发及利用奠定基础。
迄今为止，有近百种不同的基因分型技术用于满足多样化的研究需求。研究人员根据需检测的样本大小、位点多少等选择不同的检测平台及检测技术。在高通量的SNP分型技术中应用较多的有Illumina SNP基因分型平台(
Figure PCTCN2016105001-appb-000001
技术)和Affymetrix基因分型平台(Affymetrix
Figure PCTCN2016105001-appb-000002
技术)。其中Illumina公司的SNP分型技术采用微珠芯片技术(BeadArray)，包含Infinium和GoldenGate两大系列产品，Infinium技术适合于多位点的中高通量(3万到500万位点)的全基因组分型；而GoldenGate技术适合于SNP位点较少的中低通量(96到3072位点)基因组分型，均提供现成芯片或完全定制的iSelect或半定制芯片，以满足不同的研究和分析需求。例如，Illumina Infinium芯片中，每个SNP位点的被检测率高、重复性好、检测质量可靠等优点，得到业界的广泛认可，已被广泛应用到人类、动植物基因组分析相关研究中。
在植物中，玉米、水稻的芯片开发及应用较其他作物更为广泛。例如，以水稻为研究材料，已利用不同的平台开发了多个不同密度，多功能的芯片，包括：与Illumina公司合作生产的基于Infinium平台的RiceSNP6K、RiceSNP50K和HDRA700K，基于GoldenGate平台开发的1,536SNPs，基于BeadXpress开发的384-plex；与Affymetrix公司合作生产的Rice44KGeneChip和50K chipOsSNPnks等(Plant Biotech，Yu et al.2014；Molecular plant，Chen et al.2014；Nat Commu，McCouch et al.2015；Int JAgri Sci，Utami 2014；Theor Appl Genet，Chen et al.2011；Nat Commu，Zhao et al.2011；Sci Rep，Singh et al.2016)。上述不同用途芯片均取得较好研究进展。本发明基于陆地棉种内SNP变异，利用
Figure PCTCN2016105001-appb-000003
Assay这一成熟的技术集成一款适于陆地棉种内基因分型检测的棉花全基因组SNP芯片。
发明内容
本发明的目的在于提供一种棉花全基因组SNP芯片。
本发明的目的在于提供上述棉花全基因组SNP芯片的应用。
本发明的目的通过以下技术方案实现：
本发明提供一种棉花全基因组SNP位点集合，包含82,259个SNP位点。每个SNP位点包含两个不同碱基变异位点，用于检测该位点的等位基因变化。所述的SNP位点位于如SEQ ID NO：1-SEQ ID NO：82,259所示核苷酸序列的第61位。
本发明还提供一套用于检测棉花全基因组SNP位点集合的探针，所述探针的核苷酸序列如SEQ ID NO：1-SEQ ID NO：82,259所示，所述的SNP位点位于如SEQ ID NO：1-SEQ ID NO：82,259所示核苷酸序列的第61位。
本发明还提供棉花全基因组SNP分型芯片，命名为CottonSNP80K。所述的芯片含有一套用于检测相应SNP位点的核苷酸探针；所述核苷酸探针序列选自如SEQ ID NO：1-SEQ ID NO：82,259所示，能够用于相应SNP位点基因分型。所述的SNP位点位于如SEQ ID NO：1-SEQ ID NO：82,259所示核苷酸序列的第61位。
所述的基因分型，其实验步骤包括：1)制作基因分型核苷酸探针和试剂；2)在基因分型系统上把探针和样本杂交并扫描，获得所分析样本在所述SNP位点处的基因型。
上述的棉花全基因组SNP位点集合在制备棉花全基因组SNP芯片中的应用。
上述的探针在制备棉花全基因组SNP芯片中的应用。
上述的探针在棉花种质资源指纹图谱构建、棉花杂交后代基因型鉴定、棉花全基因组关联分析、棉花品种身份鉴定或棉花育种材料选择中的应用。
本发明提供棉花全基因组SNP分型芯片是根据SEQ ID NO：1-SEQ ID NO：82,259所示的核苷酸序列，利用Infinium专利设计制造技术由Illumina公司制作的，但不限于此。
本发明提供棉花全基因组SNP芯片的基因分型系统是利用Illumina公司的GenomeStudio基因分型模块，但不限于此。
本发明提供CottonSNP80K芯片在棉花全基因组SNP分型中的应用，适用于棉花种质资源指纹图谱构建、杂交后代基因型鉴定、重要农艺性状全基因组关联分析、品种身份鉴定、育种材料辅助选择、分子设计育种等。
本发明利用南京农业大学组装的异源四倍体陆地棉遗传标准系TM-1全基因组序列信息，及100份不同来源陆地棉材料平均5倍基因组覆盖度的重测序数据，研发覆盖全基因组、多态性高、适于陆地棉种内不同基因型鉴定的SNP芯片。用于重测序的100份陆地棉材料见附表1，用于定制全基因组覆盖的SNP芯片的SNP选点流程见附图1。设计陆地棉全基因组SNP芯片的具体挑选流程如下：
1.基于重测序信息，选取100份陆地棉材料间MAF>10％的1,372,195个SNPs多态位点，用于SNP芯片开发的初始SNP集合。
2.利用选择的SNP位点，回帖参考基因组，完成原始基因型的准确率鉴定，选择准确 率>99.12％的位点。通过repeat-masker寻找重复区域，过滤掉位于重复区域的SNP位点。综合上述分析，过滤后得到1,089,201个SNP位点。
3.通过模拟产生了24倍基因组覆盖，pair-end长度为120bp的reads数据。进一步和基因组比对检测每个基因位点的覆盖度。分别去掉位于染色体间重复区域内的12,385个SNP位点，以及覆盖度未达到24倍的基因位点。过滤后得到899,913个SNP位点。
4.通过实际的重测序覆盖度信息，进一步检测每个位点的覆盖度。发现大部分位点的覆盖率长度在331bp。我们选取覆盖率在231bp到431bp之间的SNP。得到了620,065个SNP位点。
5.由于芯片的探针长度为50bp左右，因此选择SNP位点两侧50bp内没有其他多态位点，且目标SNP也不在Indel区域内的SNP位点。过滤后得到619,979个位点。
6.由于棉花是常异花授粉作物，一些位点杂合率比较高，杂合位点在芯片中难以区分。因此基于重测序数据，去掉杂合率大于15％的位点。得到339,537个位点。
7.通过聚类分析，对于得到的339,537个位点，计算每个多态性位点的多态性值(π)。利用正态分布去掉显著性高于平均值5％的多态性位点和低于5％的多态性位点。利用随机抽样的方法，从339,537点中按照染色体比例抽取160,000个点形成一个数集。重复10000次。对抽样形成的数据集，计算个体相互之间的平均差异程度、最小差异程度和方差。选取10000次中平均差异最高，最小差异度在所有排名最高5％，同时方差也在排名最小20％以内的样本集作为最后的结果。最后筛选出175,192万个SNP位点作为定制SNP芯片的候选位点，送Illumina公司对探针位点进行打分评判。
8.根据探针设计分值，选取基因区>0.7，基因间区>0.9，且在基因组中间距>2500bp的SNP位点，最终确定82,259个位点用于后期芯片点制。
按照上述步骤筛选，最后获得82,259个SNP位点，将这些位点对应的DNA序列提交Illumina公司点制芯片，根据探针序列合成82,259种微珠，将该芯片命名为CottonSNP80K。该芯片可同时检测24个样品，更适宜于陆地棉种内SNP分型检测。
本发明所述的82,259个位点集合在TM-1基因组中的分布见附图1。芯片中涉及到的SNP位点覆盖棉花全基因组，平均23.5Kb有一个SNP位点设计在芯片上。在上述SNP位点的选择方法及其在染色体上的物理位置公开以后，任何人通过增加或减少部分不同的棉花材料测序结果分析，从而获得包含本发明所有公开的SNP位点集合或部分位点的集合，都在本发明保护范围之内。
在获得本发明所公开的SNP位点之后，通过确定具体的SNP位点所在的基因或与该基因连锁的SNP位点以后，通过PCR检测、探针杂交、芯片检测和试剂盒分析等检测方法加 以应用，都在本发明保护范围之内。
通过本发明所公开的SNP芯片挖掘出的与重要农艺性状相关的SNP位点，并通过PCR检测、探针杂交、芯片检测和试剂盒分析等检测方法加以应用，都在本发明保护范围之内。
本发明的有益效果：
本发明的有益效果是构建了棉花高密度的SNP位点集合，这些SNP位点具有多态性高，通量大，在染色体上具有明确的位置，覆盖棉花全基因组特点。其中17,671个SNP位点分布于13,759个基因中，涉及四倍体棉花基因组中19.57％的基因。这些位于基因区的cSNP位点(功能标记)可以满足功能基因相关的育种及研究需求。
本发明的另一个有益效果是开发了用于棉花全基因组SNP分型检测的SNP芯片(CottonSNP80K)，可以应用于重要农艺性状的关联分析及定位研究、棉花种质资源指纹分型分析、育种材料进行前景及背景选择研究，开展分子设计育种以及开发棉花育种芯片等，均具有重要的经济价值及应用前景。
1.重要农艺性状的关联分析：
本发明中的多态标记，可用于棉花自然群体(不同棉花种质资源)中与棉花重要农艺性状关联的位点/基因的筛选。根据研究及育种目标的不同，选择不同的棉花种质资源，利用本发明中的SNP芯片完成不同棉花材料全基因组SNP分型工作。结合精准表型鉴定结果，开展产量、品质、抗性(抗生物胁迫或非生物胁迫)等重要农艺性状GWAS分析；发掘重要性状基因/主效QTL及携带大量优异基因的棉花种质材料。
2.目标性状基因/QTLs的定位研究：
本发明中的多态标记，可用于高密度遗传图谱构建，以及基于遗传图谱的目标性状基因/QTLs的定位及图位克隆等研究。根据研究及育种目标的不同，选择不同的亲本，配置大的分离群体(包括：F2、BC1及RIL群体等)，利用本发明的SNP芯片，完成群体中SNP位点的分型，构建高密度、高质量的遗传图谱。完成目标性状相关基因的定位及图位克隆研究。
3.种质资源指纹图谱的构建：
本发明中的SNP芯片可用于不同棉花种质资源全基因组SNP分型工作，检测到的多态标记可用于棉花种质资源指纹图谱的构建。棉花品种分子指纹的研制不仅对保护棉花育成品种的知识产权和育种家的权益、提高种子市场的种子质量具有重要意义，而且对棉花品种真实性和纯度的司法鉴定，植物新品种权保护，规范品种管理，控制品种多、乱、杂，打击假冒伪劣，以及进一步理清我国棉花种质亲缘关系等都非常必要。本发明也可有效用于海岛棉等其他棉种的种内及种间基因分型分析。
4.育种材料的辅助选择：
利用本发明中的多态标记获得与目标性状紧密连锁或与重要农艺性状稳定关联的位点，可以利用这些位点针对相关性状表型分析进行辅助选择，其他位点用于背景分析研究。在标记辅助选择中，首先要通过分离群体构建高密度遗传图谱，筛选与目标性状紧密连锁的分子标记；或通过自然群体筛选与重要农艺性状稳定关联的分子标记。如果标记与目标性状连锁越紧密，则利用价值越高。特定标记等位基因与目标性状QTLs之间的关联还可用于杂交种后代基因型的预测、群体规模的估算。本发明中的SNP芯片可用于育种材料前景选择、背景选择过程中所需位点的基因分型。
5.分子设计育种：
利用本发明中的SNP芯片，筛选到与目标性状紧密连锁或与重要农艺性状稳定关联的位点，即利用本发明中包含的目标SNP位点及在附近区域其他SNP背景分子标记，有效用于分子设计育种。
6.育种芯片的开发：
利用本发明中的SNP芯片，筛选到与目标性状紧密连锁或与重要农艺性状稳定关联的位点，提取本发明中的目标SNP位点，包括增加部分新的SNP位点，组合成<1万位点的SNP集合，开发适于育种利用的SNP芯片。
具体的实验流程
1.DNA的提取及质量检测：利用CTAB法提取棉花幼嫩叶片全基因组DNA，用1％的琼脂糖凝胶电泳及紫外分光光度仪，检测DNA的浓度及完整性。保证每个样本满足以下条件：260/280在1.7～2.1之间，样品浓度>50ng/μL，总DNA>1μg，无大分子污染，无降解。
2.芯片检测：用Illumina iScan芯片扫描仪，按照标准的Protocol进行操作。
3.数据统计分析：利用Illumina官方软件(GenomeStudio)的基因分型模块完成原始数据的分型。
附图说明
图1：82,259个SNP位点在陆地棉遗传标准系TM-1中的染色体分布
图2：棉花CottonSNP80K芯片开发流程图
图3：344个不同来源陆地棉品种聚类图
图4：与棉花纤维强度性状关联的SNP位点曼哈顿图
具体实施方式
以下实施例用于说明本发明，但不限制本发明的范围。实例中所用到的技术手段均为本领域技术人员所熟悉的常技术。
实施例1CottonSNP80K陆地棉全基因组SNP芯片制备方法
本发明利用南京农业大学组装的异源四倍体陆地棉遗传标准系TM-1全基因组序列信息，及100份不同来源陆地棉材料5倍基因组覆盖的重测序数据，研发覆盖全基因组、多态性高、适于陆地棉种内基因型鉴定的SNP芯片。100份陆地棉材料见附表1，用于定制芯片的SNP选点流程见附图2。
设计陆地棉全基因组SNP芯片的具体挑选流程如下：
1.基于重测序信息，选取100份陆地棉材料间MAF>10％的1,372,195个SNPs多态位点，用于SNP芯片开发的初始SNP集合。
2.利用选择的SNP位点，回帖参考基因组，完成原始基因型的准确率鉴定，选择准确率>99.12％的位点。通过repeat-masker寻找重复区域，过滤掉位于重复区域的SNP位点。综合上述分析，过滤后得到1,089,201个SNP位点。
3.通过模拟产生了24倍基因组覆盖，pair-end长度为120bp的reads数据。进一步和基因组比对检测每个基因位点的覆盖度。分别去掉位于染色体间重复区域内的12,385个SNP位点，以及覆盖度未达到24倍的基因位点。过滤后得到899,913个SNP位点。
4.通过实际的重测序覆盖度信息，进一步检测每个位点的覆盖度。发现大部分位点的覆盖率长度在331bp。我们选取覆盖率在231bp到431bp之间的SNP。得到了620,065个SNP位点。
5.由于芯片的探针长度为50bp左右，因此选择SNP位点两侧50bp内没有其他多态位点，且目标SNP也不在Indel区域内的SNP位点。过滤后得到619,979个位点。
6.由于棉花是常异花授粉作物，一些位点杂合率比较高，杂合位点在芯片中难以区分。因此基于重测序数据，去掉杂合率大于15％的位点。得到339,537个位点。
7.通过聚类分析，对于得到的339,537个位点，计算每个多态性位点的多态性值(π)。利用正态分布去掉显著性高于平均值5％的多态性位点和低于5％的多态性位点。利用随机抽样的方法，从339,537点中按照染色体比例抽取160,000个点形成一个数集。重复10000次。对抽样形成的数据集，计算个体相互之间的平均差异程度、最小差异程度和方差。选取10000次中平均差异最高，最小差异度在所有排名最高5％，同时方差也在排名最小20％以内的样本集作为最后的结果。最后筛选出175,192万个SNP位点作为定制SNP芯片的候选位点，送Illumina公司对探针位点进行打分评判。
8.根据探针设计分值，选取基因区>0.7，基因间区>0.9，且在基因组中间距>2500bp的SNP位点，最终确定82,259个位点用于后期芯片点制。
按照上述步骤筛选，最后获得82,259个SNP位点，将这些位点对应的DNA序列提交Illumina公司点制芯片，根据探针序列合成82,259种微珠，将该芯片命名为CottonSNP80K。该芯片可同时检测24个样品，更适宜于陆地棉种内SNP分型检测。也可用于海岛棉等其他棉种的种内及种间基因分型分析。
实施例2CottonSNP80K陆地棉全基因组SNP芯片在棉花材料SNP分型中的应用
1.DNA的提取及质量检测：利用CTAB法提取棉花幼嫩叶片全基因组DNA，用1％的琼脂糖凝胶电泳及紫外分光光度仪，检测DNA的浓度及完整性。保证每个样本满足以下条件：260/280在1.7～2.1之间，样品浓度>50ng/μL，总DNA>1μg，无大分子污染，无降解。
2.芯片检测：用Illumina iScan芯片扫描仪，按照标准的Protocol进行操作。
3.数据统计分析：利用Illumina官方软件(GenomeStudio)的基因分型模块，结合手动调点检测，完成原始数据的分型。
实施例3CottonSNP80K陆地棉全基因组SNP芯片在不同棉花种质资源指纹分型中的应用
利用本发明CottonSNP80K芯片对我国三大棉区不同来源的344个棉花材料，编号为1～344，其中342份为陆地棉材料，2份为海岛棉材料。按照实施例2的方法进行芯片检测及SNP分型分析。344个棉花材料名称见附表2，材料间的聚类图见附图3。具体表现如下：
1.每张芯片可同时检测24个样本，在供试的82,259个位点中，实际点到芯片中的位点数为77,774个(94.55％)。
2.在344个棉花材料中，位点的平均检出率为99.37％；共有59,324个位点呈现多态性，占总位点的76.44％。利用该芯片，检测到海岛棉和陆地棉种间的多态率>30％。如：TM-1与Hai7124间多态率为31.10％，TM-1与军海1号间多态率为31.21％。
3.在遗传背景相似的陆地棉材料间也存在大量的多态位点。如：遗传背景相似的新乡小吉无绒无絮与新乡小吉无绒有絮基因组间多态率为15.35％，徐州142无绒无絮与徐州142野生型基因组间多态率为17.51％，7235品系与7235品系突变体基因组间多态率为20.56％；陆地棉遗传标准系TM-1与系列陆地棉纤维发育突变体材料(SL1-7-1、MD-17、N1、n2及T586等)基因组间的多态率在22.17-31.86％之间；纤维突变体材料imim与TM-1基因组间的多态率为21.89％。上述鉴定结果远远高于前期利用SSR位点检测到的多态率，如imim 与TM-1基因组间的SSR标记多态率为1.28％(Wang et al,2012)，表明利用CottonSNP80K芯片可以很好的区分遗传背景相似的陆地棉材料。
4.芯片具有很好的重复性。为了检测芯片的重复性，检测了3个转基因受体材料W0的不同单株，2个陆地棉遗传标准系TM-1的不同单株，及不同来源的中12及徐州142各两份不同单株材料。结果表明，3个W0单株间检测到的位点完全相同，2个TM-1单株间检测到的位点也完全相同，两个不同来源的中12单株间一致性为95.24％，两个不同来源的徐州142单株间的一致性为90.43％。表明CottonSNP80K芯片具有很好的重复性及微小差异位点检测能力。
综上所述，CottonSNP80K芯片上的SNP位点具有广泛的适用性，良好的重复性，高效差异检测能力。因此，CottonSNP80K芯片非常适用于建立棉花品种指纹数据库，进行品种间亲缘关系的鉴定及辅助育种材料选择等研究。
实施例4CottonSNP80K陆地棉全基因组SNP芯片在棉花重要农艺性状关联分析中的应用
选取实施例3中242个陆地棉品种SNP分型结果。结合前期测定的三年三点9个不同环境下纤维品质性状数据进行棉花全基因组纤维品质性状关联分析。对分型结果进行质控，去掉MAF<0.05以及基因型类型为1的位点，最终选取54,408个多态SNP位点用于目标性状关联分析，获得与纤维品质显著关联，且重复性好的位点，进一步用于目标性状基因精细定位及克隆研究。例如，通过关联分析，在染色体A11上检测到与纤维强度显著关联的一个SNP区间(附图4)。
工业实用性
本发明的SNP芯片，包含染色体位置明确的82,259个SNP位点，用于棉花品种/材料快速、高通量、低成本的SNP分型。可以应用于重要农艺性状的关联分析及定位研究、棉花种质资源指纹分型分析、育种材料进行前景及背景选择研究，开展分子设计育种以及开发棉花育种芯片等，均具有重要的经济价值及应用前景。
附表1:100份重测序陆地棉材料信息
Figure PCTCN2016105001-appb-000004
Figure PCTCN2016105001-appb-000005
附表2：用于芯片检测的344份棉花材料信息
Figure PCTCN2016105001-appb-000006
Figure PCTCN2016105001-appb-000007
Figure PCTCN2016105001-appb-000008
Figure PCTCN2016105001-appb-000009

Claims (13)

  1. 一种棉花全基因组SNP位点集合，包含82,259个SNP位点，每个SNP位点包含两个不同碱基变异位点，用于检测该位点的等位基因变化，其特征在于：所述的SNP位点位于如SEQ ID NO：1-SEQ ID NO：82,259所示核苷酸序列的第61位。
  2. 一组用于检测棉花全基因组SNP位点集合的探针，其特征在于所述探针的核苷酸序列如SEQ ID NO：1-SEQ ID NO：82,259所示，所述的SNP位点位于如SEQ ID NO：1-SEQ ID NO：82,259所示核苷酸序列的第61位。
  3. 一种用于检测棉花全基因组SNP位点集合的芯片，其特征在于：所述的芯片含有一套用于检测相应SNP位点的核苷酸探针；所述核苷酸探针的序列如SEQ ID NO：1-SEQ ID NO：82,259所示，能够用于相应SNP位点基因分型。
  4. 根据权利要求3所述的芯片，其特征在于：所述的SNP位点位于如SEQ ID NO：1-SEQ ID NO：82,259所示核苷酸序列的第61位。
  5. 根据权利要求3所述的芯片，其特征在于：所述的基因分型，其实验步骤包括：1)制作基因分型核苷酸探针和试剂；2)在基因分型系统上把探针和样本杂交并扫描，获得所分析样本在所述SNP位点处的基因型。
  6. 权利要求1所述的棉花全基因组SNP位点集合在制备棉花全基因组SNP芯片中的应用。
  7. 权利要求2所述的探针在制备棉花全基因组SNP芯片中的应用。
  8. 权利要求2所述的探针在棉花种质资源指纹图谱构建、棉花杂交后代基因型鉴定、棉花全基因组关联分析、棉花品种身份鉴定或棉花育种材料选择中的应用。
  9. 权利要求3～5所述的芯片在棉花种质资源指纹图谱构建中的应用。
  10. 权利要求3～5所述的芯片在棉花杂交后代基因型鉴定中的应用。
  11. 权利要求3～5所述的芯片在棉花全基因组关联分析中的应用。
  12. 权利要求3～5所述的芯片在棉花品种身份鉴定中的应用。
  13. 权利要求3～5所述的芯片在棉花育种材料选择中的应用。
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