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作物学报 ›› 2018, Vol. 44 ›› Issue (11): 1631-1639.doi: 10.3724/SP.J.1006.2018.01631

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

适于陆地棉品种身份鉴定的SNP核心位点筛选与评价

朱国忠,张芳,付洁,李乐晨,牛二利,郭旺珍()   

  1. 南京农业大学 / 作物遗传与种质创新国家重点实验室 / 杂交棉创制教育部工程研究中心, 江苏南京 210095
  • 收稿日期:2018-02-25 接受日期:2018-08-20 出版日期:2018-11-12 网络出版日期:2018-09-04
  • 通讯作者: 郭旺珍
  • 基金资助:
    本研究由国家重点研发计划项目(2017YFD0102000);江苏现代作物生产协同创新中心(No.10)

Genome-wide Screening and Evaluation of SNP Core Loci for Identification of Upland Cotton Varieties

Guo-Zhong ZHU,Fang ZHANG,Jie FU,Le-Chen LI,Er-Li NIU,Wang-Zhen GUO()   

  1. State Key Laboratory of Crop Genetics & Germplasm Enhancement / Hybrid Cotton R&D Engineering Research Center (the Ministry of Education) / Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
  • Received:2018-02-25 Accepted:2018-08-20 Published:2018-11-12 Published online:2018-09-04
  • Contact: Wang-Zhen GUO
  • Supported by:
    This study was supported by the National Key R&D Program for Crop Breeding(2017YFD0102000);Jiangsu Collaborative Innovation Center for Modern Crop Production Project(No.10)

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摘要：

利用全基因组SNP信息, 筛选陆地棉品种特异的核心SNP位点组合, 可为陆地棉品种身份鉴定提供准确高效的检测手段。本研究利用棉花CottonSNP80K芯片对326份不同来源的陆地棉种质进行SNP分型。以南京农业大学陆地棉TM-1基因组Gossypium hirsutum (AD1) genome NBI v1.1版本为参考序列, 对SNP位点进行注释。结果表明, 93.85% (72 990/77 774)的位点检出率超过99%, 61 595 (79.20%)个SNP位点具有多态性, 其中76.32% (47 009)的位点最小等位基因频率(MAF)大于0.1。基于位点检出率大于0.99、位点具多态性、MAF大于0.2、杂合率小于0.05、每条染色体的SNP密度为400 kb/SNP左右等要求, 最终获得4857个覆盖全基因组的高质量核心SNP位点组合。这些核心SNP位点组合平均检出率接近100%; 平均MAF值为0.34; 平均杂合率为0.02; 99%以上的陆地棉材料均能够被准确鉴定。统计分析表明利用核心SNP位点组合与CottonSNP80K的鉴定结果呈极显著相关。本研究提供了包含4857个SNP位点, 适于陆地棉品种指纹图谱绘制的核心SNP位点组合, 可实现陆地棉品种身份鉴定和品种确权。

关键词: DNA芯片, 指纹图谱, SNP, 核心位点, 陆地棉

Abstract:

Utilizing the genome-wide SNP information to screen the core SNP loci may provide an accurate and efficient method for the identification of upland cotton varieties. Using the CottonSNP80K array, SNP genotyping was performed within 326 upland cotton accessions. Then, the SNP loci were annotated with TM-1 genomic sequence of Gossypium hirsutum (AD1) genome NBI v1.1 Upland cotton of Nanjing Agricultural University as reference sequence. Statistical analysis of all loci in CottonSNP80K showed that the call rate of 93.85% loci (72 990 in 77 774) was more than 99%, and 61 595 (79.20%) SNPs were polymorphic loci among the tested upland cotton accessions. Among them, minor allele frequency (MAF) of 76.32% (47 009) loci was greater than 0.1. Based on call frequency for each locus > 0.99; loci with polymorphism; MAF > 0.2; heterozygosity rate < 0.05; SNP density with ~400 kb/SNP in each chromosome, we obtained 4857 high-quality core SNP loci. The characteristic statistics of the core SNP loci combination showed that the average call rate was nearly 100%; the average MAF was 0.34; and the average heterozygosity was 0.02. Using these core SNPs, more than 99% of the materials could be identified accurately and effectively. In addition, the identification results of core SNP loci showed extremely significant linear correlation with that of CottonSNP80K. Taken together, a core combination containing 4857 SNP loci for fingerprint identification of upland cotton varieties is constructed, which can accurately identify the purity and reality of modern upland cotton varieties.

Key words: DNA array, fingerprint, SNP, core loci, Upland cotton

图1

CottonSNP80K芯片SNP位点特征统计 横坐标代表被统计的SNP特征参数, 依次为位点检出率、最小等位基因频率、杂合率和相邻SNP之间的距离。纵坐标代表SNP的分布数目。"

表1

用于指纹图谱绘制的核心SNP位点染色体分布"

染色体
Chr.		 染色体长度
Chr. length
(Mb)		 标记数Number of SNPs 分布密度
Density
(kb/SNP)		 染色体
Chr.		 染色体长度
Chr. length
(Mb)		 标记数Number of SNPs 分布密度
Density
(kb/SNP)
A01 99.9 250 399.5 D01 61.5 153 401.7
A02 83.4 207 403.1 D02 67.3 169 398.1
A03 100.3 248 404.3 D03 46.7 118 395.7
A04 62.9 156 403.3 D04 51.5 128 402.0
A05 92.0 237 388.4 D05 61.9 156 397.0
A06 103.2 255 404.6 D06 64.3 164 392.0
A07 78.3 197 397.2 D07 55.3 138 400.8
A08 103.6 258 401.7 D08 65.9 164 401.8
A09 75.0 183 409.8 D09 51.0 128 398.4
A10 100.9 256 394.0 D10 63.4 156 406.2
A11 93.3 242 385.6 D11 66.1 169 391.1
A12 87.5 215 406.9 D12 59.1 150 394.1
A13 80.0 204 392.0 D13 60.5 156 388.0
Total 1160.2 2908 399.0 Total 774.4 1949 397.3

表2

用于品种指纹图谱分析的核心SNP位点特征统计"

类型
Type		 最大值
Max.		 最小值
Min.		 平均值
Mean
检出率 Call frequency 1.00 0.99 1.00
最小等位基因频率 MAF 0.50 0.20 0.34
杂合率 Heterozygosity 0.05 0.00 0.02
多态信息含量 PIC 0.50 0.32 0.44

图2

312份陆地棉品种遗传距离相关性分析横坐标代表利用芯片总位点计算的材料间遗传距离, 纵坐标代表利用核心位点计算的材料间遗传距离。"

表3

基于核心SNP位点的陆地棉近等基因系多态性检测"

材料
Accession		 多态位点(总位点/核心位点)
No. of polymorphic loci
(total loci/core loci)		 多态率(总位点/核心位点)
Polymorphic rate
(total loci/core loci)
新乡小吉和新乡小吉无绒无絮突变体
Xinxiangxiaoji linted-fuzzless vs. Xinxiangxiaoji lintless-fuzzless		 12529/1215 0.161/0.250
徐州142和徐州142无绒无絮突变体
Xuzhou-142 vs. Xuzhou-142 lintless-fuzzless		 14808/1488 0.190/0.306
7235和7235纤维突变体
7235 vs. 7235 mutant		 19451/2546 0.250/0.524
TM-1和SL1-7-1突变体
TM-1 vs. SL1-7-1		 20859/2188 0.268/0.450
TM-1和 MD-17突变体
TM-1 vs. MD-17		 23814/2367 0.306/0.487
TM-1和显性光子N1突变体
TM-1 vs. N1 (dominant naked seed)		 17678/2170 0.227/0.447
TM-1和隐性光子n2突变体
TM-1 vs. n2 (recessive naked seed)		 19996/2446 0.257/0.503
TM-1和im突变体
TM-1 vs. im (immature fiber)		 16333/3116 0.210/0.642

表4

芯片分型与SNP-PCR一致性分析"

SNP 染色体
Chr.		 位置
Position (bp)		 验证不一致数目
Number of mismatches		 引物
Primer (5'-3')
TM3086 A01 86363489 1 F: CTTGATTTTCAATTCAACCAAAAAACTCCG
R: AAAATACATGGACGCAACTATCTGATGCATAATTT
TM4824 A02 45647806 0 F: TTGAATGAGTCTCATTTTATCAATTGACCTTTATTA
R: TCATAAGGTAATTTGGCAAGTAATTTTGAAAAAGTC
TM7830 A03 89740533 1 F: AAGCCTATATTTTGCCAACTTACCTTGTGCAG
R: ATTAAGTTAAAAAGTGCCACATGTCACAAAACCAT
TM9574 A04 51844539 0 F: GGCCCTAATCTTAGGCATAGTTTACCCCAAATAA
R: CTTAGGGTAGACGGATGGAGGGATGAGA
TM19508 A07 21138995 0 F: TGCGAGGGATTAAGTTTAGCTTGGAAAGC
R: CCATTAGTTGACAATCTTCAAGAACCTTACGAAAA
TM27528 A08 63064554 0 F: GCTCCCATAATTTTCTTTCACTTCGAGACG
R: AAGAGAAAGGTGAAAATTTTAAGTACAAAAGGTTGA
TM27748 A08 65768181 0 F: GGCTTTTACTGATTTACATTTTGAACAATGTTAAAG
R: GGCTGAATGCTCCTTTACCTTATTTATGGGTT
TM33913 A10 4141534 2 F: GCTCCCTTAAACCCGCAACCTTAGTCA
R: AGAAAGTTGTTTCGGCTGGTATTTTGAGGTT
TM53506 D03 2560865 0 F: ATATTTTCTTTTTCTTTTTTCGATAAGATAGAGCAA
R: CCAAGGTTAGTTAACTAGAAAAACTGACCGAATCAA
TM58088 D05 33818451 0 F: CTCGTATGTCACAAGACCATCAAAATTTAAATGAA
R: TGTATCCCCATTTTGAACACCCACATG
TM69807 D08 63425972 0 F: TTTGTCAATTCTTACCAATTATACCTTCCTACCAAG
R: TATGTTTTGAAAATGCAACACCTATAGAAAAAGCAA
TM72841 D09 47573336 0 F: CAGGAATCATTGGGGTTGGACAAGC
R: TATGCTTGACTAAGGGATGCATTATCATACATTGTT
TM75830 D11 13710651 0 F: CGCGTCGTAATCGAGTTTCACCGA
R: GGTGTCCGGTCCGGTGTGCTTA
TM80004 D13 163793 0 F: GTGGCCACTGAAGAGCAAGCTTAATGA
R: ATGCATGTTCAAGCTTCTTGGTAATGTCATAATC

图3

基于SNP-PCR技术验证SNP位点芯片分型结果 M: DNA marker; 1: TM-1; 2: 7235; 3: J02-508(7-50); 4: 岱字棉16; 5: 鄂棉21; 6: 鄂棉23; 7: 国欣棉9; 8: 邯郸885; 9: 黑山棉1号; 10: 冀122; 11: 军棉1号; 12: 山农棉8号; 13: 山西W1; 14: 山西W8; 15: 斯字棉2B; 16: 泗棉3号; 17: 皖棉17; 18: 新陆早32; 19: 新陆早7号; 20: 新陆中26; 21: 新陆中35; 22: 豫棉15; 23: 中棉所12; 24: 中棉所41。"

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