WO2025001891A1 - 一种基于靶向捕获测序的陆地棉低密度液相芯片及其应用 - Google Patents

一种基于靶向捕获测序的陆地棉低密度液相芯片及其应用 Download PDF

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WO2025001891A1
WO2025001891A1 PCT/CN2024/099474 CN2024099474W WO2025001891A1 WO 2025001891 A1 WO2025001891 A1 WO 2025001891A1 CN 2024099474 W CN2024099474 W CN 2024099474W WO 2025001891 A1 WO2025001891 A1 WO 2025001891A1
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upland cotton
liquid phase
sites
snp
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司占峰
胡艳
张天真
方磊
韩泽刚
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Zhejiang University
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to the fields of genetics, molecular biology, bioinformatics and cotton molecular breeding, and in particular to a low-density liquid phase chip of upland cotton based on targeted capture sequencing and an application thereof.
  • Single Nucleotide Polymorphism refers to the variation of a single nucleotide in the genome, which is a variation form formed by the substitution, transversion, insertion or deletion of a single nucleotide pair.
  • SNPs are numerous, densely distributed, and easy to detect, making them ideal targets for genotyping.
  • SNPs single nucleotide polymorphisms based on DNA sequence variation have approached the ultimate standard for variation detection at the molecular level. It is expected that the development of molecular detection technology and platforms in the next few years will be roughly upgraded and improved on this basis. Therefore, efficient and low-cost SNP genotype detection technology has become the best choice for the development of shared technology and platforms.
  • the SNP-based genotyping by target sequencing (GBTS) technology is to select specific target sites from genomic DNA for sequencing and genotyping.
  • This targeted or fixed simplified genome sequencing greatly reduces the amount of DNA sequencing, simplifies bioinformatics analysis and data processing, and improves the adaptability to various genotyping detection platforms.
  • this technology is suitable for the detection of all marker sites, including sites with known functions (such as cloned genes), sites with unknown functions (candidate genes), and neutral sites.
  • the plasticity of the number of markers in its system provides huge flexibility for various applications, and is basically suitable for all scenarios that require different numbers of markers, such as marker-assisted major gene selection, backcross breeding and background selection, multi-gene aggregation breeding, seed purity detection, and identification of transgenic components. Therefore, GBTS will become the first choice for genotyping detection for a long time in the future.
  • the developed cotton SNP chips such as Illumina 80K and ZJU40K have been widely adopted by breeding companies and scientific research institutions for genotyping, genome selection, and analysis of genetic diversity of germplasm resources.
  • the SNP density of these chips is generally high, and the Illumina 80K solid phase chip has the disadvantage of being unable to be customized; the chip site design is generally based on sites evenly distributed throughout the genome, without considering the association with traits, so the typing cost is high and the selection purpose is not strong.
  • the traditional breeding cycle is long, phenotypic variation is unpredictable, and it mainly relies on the experience of breeders for screening. When improving the target traits of breeding, it is easy to cause the loss of other excellent traits.
  • Molecular marker-assisted breeding uses the characteristics of molecular markers being closely linked to the genes that determine the target traits. By detecting molecular markers, detecting target genes and sites, the purpose of selecting target traits is achieved. It has the advantages of being fast, accurate, and not affected by environmental conditions. Its key limiting factor is the accurate identification of sites associated with traits.
  • the purpose of the present invention is to discover and screen 894 SNP sites in view of the shortcomings of the existing technology, and to develop a liquid gene chip using the 894 SNP sites of upland cotton and 14 major transgenics for the detection and genotyping of transgenic components of upland cotton.
  • a low-density liquid phase chip of upland cotton based on targeted capture sequencing wherein the upland cotton whole genome low-density SNP chip is composed of probes for detecting 894 SNP sites and 14 major transgenics; the 14 major transgenics are specifically: A1Bt, A2CPTI, A3CP4-epsps, A4CP4-epsps, A5bar, A6pat, A7CdP450, A8cp4-epsps, B.1CaMV35S, B.2FMV35S, B.3NOS, B.4NOS, B.5CaMV35S and NPTII; the 894 SNP sites are shown in Table 1.
  • probes for detecting 14 major transgenic genes are shown as SEQ ID NO.1 to SEQ ID NO.14.
  • the gene chip of the present invention adopts a liquid phase chip, based on liquid phase probe capture technology, to design probes and synthesize a set of 908 site information, which can be used to detect whether upland cotton contains 14 common transgenic components, as well as upland cotton resource evaluation and kinship identification, seed purity identification and genetic improvement of the main agronomic traits of upland cotton varieties.
  • main agronomic traits include fiber quality traits, yield traits, and disease resistance.
  • the liquid chip technology system used in the present invention is the GenoBaits technology system.
  • the working principle is to carry out targeted sequence complementary binding and sequencing of the probe designed based on the target SNP marker and the DNA of the test sample, so as to achieve the purpose of detecting the genotype of the target SNP marker of the test sample.
  • all probes that can accurately detect 894 SNP sites and 14 major transgenics can achieve the same technical effect.
  • the application of the chip of the present invention can greatly improve the accuracy of genotype detection, improve the detection level, reduce the detection cost, and provide important technical support for cotton molecular breeding.
  • Figure 1 is a statistical diagram of the chromosome distribution of SNP markers; the statistical results of the number of SNP markers on different chromosomes: a.
  • the horizontal axis is the chromosome number; b.
  • the left vertical axis represents the number of sites and segments; c.
  • the right vertical axis represents the chromosome length (unit Mb);
  • Figure 2 shows the distribution of SNP sites on chromosomes.
  • Example 1 Design and preparation of a low-density liquid phase chip of upland cotton based on targeted capture sequencing.
  • VCF files obtained based on resequencing of three populations (Fang et al. Genomic analyses in cotton identify signatures of selection and loci associated with fiber quality and yield traits. Nature genetics, 2017, 49(7): 1089; Liu et al. Association mapping of seed oil and protein contents in upland cotton. Euphytica, 2015, 205(2): 637-645;), with cotton genome TM-1_V2.1 (Hu et al. Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton.
  • probes were designed.
  • the specific design principles are: select all probes with a length of 100-120bp that can cover the region; calculate the GC content of the probes that can cover the target region; calculate the number of homology regions of the probes. Among them, a. completely identical over 40bp; b. similarity of 85%, length of 80bp; c. similarity of 95%, length of 70bp are all considered as one homology region.
  • Probe selection principles select probes with a GC content between 30% and 70%; select probes with a number of homology regions ⁇ 5; select probe regions that do not contain SSR or N regions.
  • the 14 detection probes provided in this embodiment are shown in SEQ ID NO.1 to SEQ ID NO.14, and detect A1Bt, A2CPTI, A3CP4-epsps, A4CP4-epsps, A5bar, A6pat, A7CdP450, A8cp4-epsps, B.1CaMV35S, B.2FMV35S, B.3NOS, B.4NOS, B.5CaMV35S and NPTII genes in order; the amplification product of the primer pair combination can be subjected to one high-throughput sequencing and analysis to complete the detection of transgenic components in the sample.
  • the probes for detecting the A3CP4-epsps gene and the B.4NOS gene are shown in the table; wherein, I is hypoxant
  • a low-density liquid phase chip of upland cotton based on targeted capture sequencing will be composed of probes for detecting 894 SNP sites and 14 major transgenics; among them, 329 sites are significantly associated with important agronomic traits of upland cotton such as fiber quality, yield, and disease resistance, 14 common plant transgenic detection sites, and 565 other sites.
  • a capture probe is synthesized by Boridi Biotechnology Co., Ltd. to construct a chip, which is based on the targeted genotype detection (GBTS) technology of liquid phase probe hybridization: GenoBaits.
  • the working principle of GenoBaits is to perform fixed-point capture based on the complementary binding of the target probe and the target sequence.
  • a gDNA library is constructed for the material to be tested.
  • a probe covering the target SNP is designed at each site to be tested, and the target probe is labeled with biotin.
  • the biotin-labeled target probe is hybridized with the target region of the genome in a liquid state to form a double chain.
  • the target probe carrying the biotin label is molecularly adsorbed using streptavidin-coated magnetic beads to capture the target hybridized with the probe.
  • the captured target sequence is eluted, the target amplified, the library is built and sequenced, and the genotype of the target SNP is finally obtained.
  • Ref represents the base type of the SNP site in the reference genome
  • Alt represents the base type of the SNP site in the population.
  • Example 2 Application of upland cotton low-density liquid phase chip based on targeted capture sequencing in upland cotton genotyping and transgenic component detection.
  • the chip was tested using 200 varieties of upland cotton. The test results showed that the average site detection rate of the cotton functional marker chip was 94.17%, which met the requirements for upland cotton genotyping detection.
  • the entire experimental process is based on the GenoBaits technical system.
  • the specific experimental process is as follows:
  • the CTAB method was used to extract DNA from the samples.
  • the DNA concentration of the test sample DNA was measured by Qubit Fluorometric Quantitation (Thermo Fisher), and the integrity of the DNA was detected by 1wt% agarose gel electrophoresis.
  • the qualified samples were placed in a 4°C refrigerator for storage and standby use.
  • GenoBaits End Repair Buffer (GenoBaits, Shijiazhuang Boridi Biotechnology Co., Ltd.) and 2.7 ⁇ L GenoBaits End Repair Enzyme into the tube, make up to 20 ⁇ L with water, and place in ABI 9700 PCR instrument and incubate at 37°C for 20 minutes to complete the end repair and A addition process of the broken fragments.
  • Beackman AMPure XP Beads (Beackman) to the ligation product to purify it. After purification, use magnetic beads to screen the fragments and retain the ligation products with insert fragments between 200 and 300 bp.
  • the amplification program is as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; repeat denaturation, annealing, and extension for a total of 15 cycles; extension at 72°C for 5 min.
  • the DNA concentration of the library was measured using Qubit Fluorometric Quantitation (Thermo Fisher), and then agarose gel electrophoresis was used to detect whether the fragment size of the library DNA was between 300-400 bp.
  • the constructed DNA library was sequenced using the BGI MGISEQ2000 sequencer.

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Abstract

公开了一种基于靶向捕获测序的陆地棉低密度液相芯片及其应用。低密度芯片包括908个SNP位点，其中与陆地棉纤维品质、产量、抗病性等重要农艺性状显著关联的位点329个，常见植物转基因检测位点14个，其他位点565个。此芯片适合于陆地棉品种中转基因成分检测、资源评价和亲缘鉴定、种子纯度鉴定以及陆地棉品种主要农艺性状遗传改良中的应用。

Description

一种基于靶向捕获测序的陆地棉低密度液相芯片及其应用 技术领域
本发明涉及遗传学、分子生物学、生物信息学和棉花分子育种领域，尤其涉及一种基于靶向捕获测序的陆地棉低密度液相芯片及其应用。
背景技术
单核苷酸多态性(Single Nucleotide Polymorphism，SNP)指基因组单个核苷酸的变异，是由单个核苷酸对置换、颠换、插入或缺失所形成的变异形式。单核苷酸多态性作为第三代遗传标记，SNP数量众多、分布密集、易于检测，是较理想的基因分型目标。纵观分子标记技术的革新，以DNA序列变异为基础的单核苷酸多态性(SNP)，已经接近在分子水平进行变异检测的终极标准。预期今后若干年分子检测技术和平台的发展大致是在此基础上进行提升和改进。因此，高效、低成本的SNP基因型检测技术成为发展共享技术和平台的最佳选择。
基于SNP的靶向测序基因型检测(genotyping by target sequencing，GBTS)技术就是要从基因组DNA中挑选特定的靶向位点，进行测序和基因型检测。这种靶向或固定的简化基因组测序大大减少了DNA测序量，简化了生物信息分析和数据处理，提高了对各种基因型检测平台的适应性。且该技术适合所有标记位点的检测，包括功能已知位点(如已克隆的基因)、功能未知位点(候选基因)以及中性位点。其系统标记数量的可塑性为各种应用提供了巨大弹性空间，基本上适合需要不同标记数量的全部场景，如标记辅助的主基因选择、回交育种及其背景选择、多基因聚合育种、种子纯度检测、转基因成份鉴定等。因此GBTS将成为未来很长一段时间基因型检测的首选。
已开发的棉花SNP芯片如Illumina 80K，ZJU40K等已经被育种企业和科研单位广泛采用，应用于基因分型、基因组选择、种质资源遗传多样性分析等。这些芯片SNP密度普遍较高，其中Illumina 80K固相芯片还存在无法定制的缺点；芯片位点设计普遍以全基因组均匀分布的位点为基础，没有考虑与性状的关联，因而分型成本较高且选择的目的性不强。传统育种周期较长，表型变异不可预见，主要靠育种家的经验筛选。在对育种目标性状进行改良时容易造成其他优异性状的丢失。分子标记辅助育种是利用分子标记与决定目标性状基因紧密连锁的特点，通过检测分子标记，检测目的基因和位点，达到选择目标性状的目的，具有快速、准确、不受环境条件干扰的优点，其关键的限制因素是与性状关联的位点的准确鉴定。
综上，基于陆地棉基因组测序和全基因组关联分析挖掘的陆地棉纤维品质、产量、抗病性等重要农艺性状显著关联的位点，以及常见植物转基因检测位点，部分背景位点，开发一 款用于陆地棉基因分型、转基因检测、种质资源评价和分子标记辅助选择等的低密度液相SNP芯片迫在眉睫。
发明内容
本发明的目的在于针对现有技术的不足，发现及筛选出894个SNP位点，利用陆地棉894个SNP位点与14种主要转基因开发液相基因芯片，用于陆地棉转基因成分检测及基因分型。
本发明采用的技术方案具体如下：
一种基于靶向捕获测序的陆地棉低密度液相芯片，所述陆地棉全基因组低密度SNP芯片由检测894个SNP位点以及14种主要转基因的探针组成；所述14种主要转基因具体为：A1Bt、A2CPTI、A3CP4-epsps、A4CP4-epsps、A5bar、A6pat、A7CdP450、A8cp4-epsps、B.1CaMV35S、B.2FMV35S、B.3NOS、B.4NOS、B.5CaMV35S和NPTII；所述894个SNP位点如表1所示。
进一步地，所述检测14种主要转基因的探针如SEQ ID NO.1～SEQ ID NO.14所示。
本发明所述基因芯片采用液相芯片，基于液相探针捕获技术，设计探针并合成一套包含908位点信息，可用于检测陆地棉中是否含有常见的14种转基因成分以及陆地棉资源评价和亲缘鉴定、种子纯度鉴定以及陆地棉品种主要农艺性状遗传改良。
进一步地，主要农艺性状包括纤维品质性状、产量性状、抗病性。
本发明所用的液相芯片技术体系为GenoBaits技术体系。工作原理是基于目标SNP标记设计的探针与检测样本的DNA进行靶向序列互补结合以及测序，从而达到对检测样本的目标SNP标记基因型检测的目的。本发明中，凡是能准确检测894个SNP位点以及14种主要转基因的探针均可达到相同的技术效果。本发明芯片的应用可以极大提高基因型检测准确度、提高检测水平，降低检测成本，并为棉花分子育种提供重要的技术支撑。
附图说明
图1为SNP标记染色体分布统计图；SNP标记在不同染色体上的个数统计结果：a.横坐标为染色体编号；b.左纵坐标表示位点数和区段数；c.右纵坐标表示染色体长度(单位Mb)；
图2为SNP位点在染色体上的分布图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述。
实施例1、基于靶向捕获测序的陆地棉低密度液相芯片的设计和制备。
具体方法为：
1.基于三个群体重测序获得的VCF文件(Fang等.Genomic analyses in cotton identify signatures of selection and loci associated with fiber quality and yield traits.Nature genetics,2017,49(7):1089；Liu等.Association mapping of seed oil and protein contents in upland cotton. Euphytica,2015,205(2):637-645；)，以棉花基因组TM-1_V2.1(Hu等.Gossypium barbadense and Gossypium hirsutum genomes provide insights into the origin and evolution of allotetraploid cotton.Nature genetics,2019,51(4):739-748.)为参考，根据SNP位点在群体中的MAF>0.35，杂合率<15％，NA比例<10％，染色体均匀分布为原则，筛选出601个SNP位点(背景位点)。
2.根据主要农艺性状GWAS关联结果(Fang等.Genomic analyses in cotton identify signatures of selection and loci associated with fiber quality and yield traits.Nature genetics,2017,49(7):1089；Liu等.Association mapping of seed oil and protein contents in upland cotton.Euphytica,2015,205(2):637-645；Li等，Genomic Insights into the Genetic Basis of Cotton Breeding in China，Molecular Plant 16,662-677)，选择与农艺性状及抗病性显著关联的634个SNP位点(前景位点)。
3.以上共获得陆地棉基因组目标区段共1235个，通过检测备选SNP位置和位点基因型是否与陆地棉参考基因组TM-1V2.1(cotton.zju.edu.cn)一致，筛选后获得1148个候选目标区段。
4.根据1148个候选目标区段，进行探针设计，设计原则具体为：挑选出可覆盖区域的所有长度为100-120bp的探针；计算可覆盖目标区域探针的GC含量；计算探针的同源性区域个数。其中，a.40bp以上完全相同；b.相似度85％，长度80bp；c.相似度95％，长度70bp三种情况均视为一个同源性区域。探针挑选原则：选取探针GC含量在30％～70％之间的；选取同源性区域个数≤5的；选取探针区域不包含SSR、N区域的。根据以上这些设计及挑选原则，共设计挑选出1795个探针，能检测894个目标区段，894个目标区段对应的核心SNP位点信息如表1所示，SNP标记染色体分布统计如图1所示，分布如图2所示。
5.根据农业部公布的棉花转基因检测14个常见检测序列，设计出14条检测探针，本实施例中提供的14条检测探针如SEQ ID NO.1～SEQ ID NO.14所示，按顺序分别检测A1Bt、A2CPTI、A3CP4-epsps、A4CP4-epsps、A5bar、A6pat、A7CdP450、A8cp4-epsps、B.1CaMV35S、B.2FMV35S、B.3NOS、B.4NOS、B.5CaMV35S和NPTII基因；该引物对组合的扩增产物可以进行一次高通量测序和分析即可完成样本中转基因成分的检测。其中，检测A3CP4-epsps基因和B.4NOS基因的探针(SEQ ID NO.3、SEQ ID NO.12)如表所示；其中，I是次黄嘌呤。

6.将检测894个SNP位点以及14种主要转基因的探针组成基于靶向捕获测序的陆地棉低密度液相芯片；其中与陆地棉纤维品质、产量、抗病性等重要农艺性状显著关联的位点329个，常见植物转基因检测位点14个，其他位点565个。
本实施例中通过博瑞迪生物技术有限公司合成捕获探针构建芯片，该芯片基于液相探针杂交的靶向基因型检测(GBTS)技术：GenoBaits。GenoBaits工作原理是基于目标探针与靶向序列互补结合进行定点捕获。首先对要测试的材料进行gDNA文库构建。同时根据DNA互补原理，在每个待测位点设计覆盖目标SNP的探针，采用生物素(Biotin)标记对目标探针进行标记。然后，在液态中利用生物素标记的目标探针与基因组目标区域杂交形成双链。随后利用链霉亲和素包衣的磁珠对携有生物素标记的目标探针进行分子吸附，从而捕获与探针杂交的靶点。最后，对捕获的靶点序列进行洗脱、靶点扩增、建库和测序，最终获得目标SNP的基因型。
表1 894个核心SNP位点信息










其中，Ref表示SNP位点在参考基因组中的碱基类型，Alt表示SNP位点在群体中的碱基类型。
实施例2、基于靶向捕获测序的陆地棉低密度液相芯片在陆地棉基因分型以及转基因成分检测中的应用。
使用200个陆地棉品种对芯片进行测试。测试结果显示，棉花功能标记芯片的平均位点检出率为94.17％，满足陆地棉基因分型检测。
整个实验流程是基于GenoBaits技术体系完成的。具体实验流程如下：
1.提取待测样本的基因组DNA，并构建样品文库；
1.1样品DNA提取
采用CTAB法对样本DNA进行提取。
1.2样品DNA质检
将测试样品DNA，用Qubit Fluorometric Quantitation(Thermo Fisher)对DNA浓度进行测定，用1wt％琼脂糖凝胶电泳检测DNA的完整性。检测合格的样品放入4℃冰箱，保存、备用。
1.3样品DNA片段化
取12μL质检合格的DNA放置于0.2μL PCR管中，将管置于超声波破碎仪中对DNA进行随机物理破碎，片段破碎至200-400bp。
1.4样品末端修复
向管中加入4μL GenoBaits End Repair Buffer(GenoBaits，即石家庄博瑞迪生物科技有限公司)和2.7μL GenoBaits End Repair Enzyme，补水至20μL，放入ABI 9700 PCR仪中37℃温育20分钟，完成破碎片段的末端修复和加A过程。
1.5样品测序接头连接
从PCR仪中取出小管加入2μL GenoBaits Ultra DNA ligase、8μL GenoBaits Ultra DNA Ligase Buffer和2μL GenoBaits Adapter，补水至40μL，然后放置于ABI9700 PCR仪上22℃反应30分钟，完成测序接头的连接。
1.6样品DNA纯化
向连接产物中加入48μL的Beackman AMPure XP Beads(Beackman公司)对连接产物进行纯化，纯化后采用磁珠进行片段筛选，保留插入片段在200～300bp的连接产物。
1.7样品文库扩增
向上一步的PCR管中加入5μL带有Barcode序列的测序接头、1μLP5接头、10μL GenoBaits PCR Master Mix，并用纯水补至20μL；用ABI 9700 PCR仪进行扩增，扩增程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸30s；重复变性、退火、延伸，共8个循环；72℃延伸5min。不同的Barcode用于区分不同的样品。
1.8样品文库纯化
向第二轮PCR产物中加入24μL Beckmen AMPure XP Beads(Beackman公司)，用移液器上下吸打均匀后，将0.2μL的PCR管置于磁力架上至溶液澄清，弃去上清并用75vol％乙醇洗涤磁珠一次，用pH值为8.0的Tris-HCl将文库DNA洗脱下来。
2.利用基于靶向捕获测序的陆地棉低密度液相芯片测定目标样品在894个SNP位点和14个基因的基因型
2.1 DNA杂交
取500ng已完成构建的基因组DNA测序文库，加入5μL GenoBaits Block I和2μL GenoBaits Block II，置于Eppendorf Concentrator plus(Eppendorf公司)真空浓缩仪上，在≤70℃的温度下蒸干至干粉。向干粉管中加入8.5μL GenoBaits 2×Hyb Buffer、2.7μL GenoBaits Hyb Buffer Enhancer、2.8μL Nuclease-Free Water，用移液器吸打混匀后放置于ABI 9700 PCR仪上95℃温育10分钟，然后取出PCR管加入3μL已经合成的探针(探针的浓度为60ng/μL)，旋涡震荡混匀后放置于ABI 9700 PCR仪上65℃温育2小时，完成探针杂交反应。
2.2 DNA捕获
向上一步杂交完成的反应体系中加入100μL GenoBaits DNA Probe Beads，上下吸打10次，放入ABI 9700 PCR仪上65℃温育45分钟，使磁珠与探针结合。用100μL GenoBaits Wash Buffer I、150μL GenoBaits Wash BufferII分别对结合探针后的磁珠进行65℃热洗，然后再用100μL GenoBaits Wash Buffer I、150μL GenoBaits Wash Buffer II(和150μL GenoBaits Wash Buffer III分别对磁珠进行常温洗涤。洗涤完成的磁珠用20μL Nuclease-Free Water进行重悬。
取13μL重悬后的DNA(带磁珠)加入到新的0.2mL PCR管中，然后加入15μL GenoBaits PCR Master Mix、2μL GenoBaits Primer Mix配置post-PCR体系，用ABI 9700 PCR仪进行文库扩增，扩增程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸30s；重复变性、退火、延伸，共15个循环；72℃延伸5min。
向post-PCR产物中加入45μL Beckmen AMPure XP Beads(Beackman公司)并用移液器上下吸打均匀，然后将0.2mL PCR管置于磁力架上至溶液澄清，弃去上清并用75vol％乙醇洗涤磁珠两次，用pH为8.0的Tris-HCl将文库DNA洗脱下来。完成探针的杂交捕获工作。
2.3 DNA杂交捕获文库质检
用Qubit Fluorometric Quantitation(Thermo Fisher)对文库的DNA浓度进行测定，然后用琼脂糖凝胶电泳检测文库DNA的片段大小是否在300-400bp之间。
2.4 DNA杂交捕获文库测序
将构建好的DNA文库用华大MGISEQ2000测序仪进行测序。
2.5基因型数据分析
测序数据经过FastQC(www.bioinformatics.babraham.ac.uk/project)质控后，用BWA(bio-bwa.sourceforge.net)的默认参数将测序数据比对到参考基因组上，用GATK(software.broadinstitute.org/gatk)软件对测序数据进行SNP鉴定，用自编的Perl脚本对探针捕获测序的基因型分型信息进行提取，形成最终的基因型分型结果，如表2和表3所示。
表2低密度液相基因芯片在200个棉花品种材料中检出率




表3低密度液相基因芯片在200个棉花品种材料中转基因成分检出情况




以上结果证实低密度SNP液相芯片在200份陆地棉材料中的检出率为90.7％-96.5％，平均位点检出率为94％。在陆地棉品种中具有较高多态性，十分适合陆地棉种质资源评价、遗传改良以及转基因成分检测，检测准确性高。相比于已有的固相芯片和已开发的高密度液相芯片，可以以更低的成本开展棉花育种研究。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其他不同形式的变化或变动。这里无需也无法把所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围。

Claims (4)

  1. 一种基于靶向捕获测序的陆地棉低密度液相芯片，其特征在于，所述陆地棉全基因组低密度SNP芯片由检测894个SNP位点以及14种主要转基因的探针组成；所述14种主要转基因具体为：A1Bt、A2CPTI、A3CP4-epsps、A4CP4-epsps、A5bar、A6pat、A7CdP450、A8cp4-epsps、B.1CaMV35S、B.2FMV35S、B.3NOS、B.4NOS、B.5CaMV35S和NPTII；所述894个SNP位点如表1所示。
  2. 根据权利要求1所述的基于靶向捕获测序的陆地棉低密度液相芯片，其特征在于，所述检测14种主要转基因的探针如SEQ ID NO.1～SEQ ID NO.14所示。
  3. 权利要求1或所述的基于靶向捕获测序的陆地棉低密度液相芯片的应用，其特征在于，包括：陆地棉转基因成分检测及基因分型、陆地棉资源评价和亲缘鉴定、种子纯度鉴定以及陆地棉品种主要农艺性状遗传改良。
  4. 根据权利要求3所述的应用，其特征在于，主要农艺性状包括纤维品质性状、产量性状、抗病性。
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