欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2019, Vol. 45 ›› Issue (6): 872-878.doi: 10.3724/SP.J.1006.2019.83067

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于高通量测序开发玉米高效KASP分子标记

陆海燕1,周玲1,林峰1,王蕊2,王凤格2,赵涵1,*()   

  1. 1 江苏省农业科学院 / 江苏省农业生物学重点实验室, 江苏南京 210014
    2 北京市农林科学院玉米研究中心, 北京 100097
  • 收稿日期:2018-10-07 接受日期:2019-01-19 出版日期:2019-06-12 网络出版日期:2019-06-12
  • 通讯作者: 赵涵
  • 作者简介:E-mail: luhaiyan@jaas.ac.cn, Tel: 025-84391957
  • 基金资助:
    本研究由国家重点研发计划项目(2017YFD0101205, 2017YFD0102005);江苏省农业科技自主创新项目(CX(18)1001)

Development of efficient KASP molecular markers based on high throughput sequencing in maize

Hai-Yan LU1,Ling ZHOU1,Feng LIN1,Rui WANG2,Feng-Ge WANG2,Han ZHAO1,*()   

  1. 1 Provincial Key Laboratory of Agrobiology / Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China
    2 Maize Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
  • Received:2018-10-07 Accepted:2019-01-19 Published:2019-06-12 Published online:2019-06-12
  • Contact: Han ZHAO
  • Supported by:
    This study was supported by the National Key Research and Development Program(2017YFD0101205, 2017YFD0102005);the Jiangsu Agricultural Science and Technology Innovation Fund [CX(18)1001].(CX(18)1001)

RichHTML

108

PDF

1388

摘要：

SNP (Single Nucleotide Polymorphism)在基因组中数量多、分布广, 适用于大规模、自动化基因型检测。本研究利用205份不同来源的玉米自交系全基因组重测序数据鉴定出一系列多态性高的二态性SNP位点并开发出700个KASP分子标记。其中, 202个在46个玉米代表系中得到验证的KASP标记进一步用于系统进化树构建及群体结构分析。结果显示, 开发成功的KASP标记在染色体上分布均匀, 平均PIC为0.463, 平均MAF为0.451。基于KASP标记位点和总SNP位点的聚类分析结果高度吻合。KASP标记位点与总SNP位点的遗传距离相似性系数高达89.5%, 能成功区分玉米的杂种优势群。该KASP标记可在玉米种质资源分析、连锁群构建以及杂种优势群划分等方面发挥重要作用。

关键词: 玉米, 重测序, KASP标记, 种质资源

Abstract:

SNP (Single Nucleotide Polymorphism) which is abundant and dispersed widely in the genome is suitable for large-scale and automated genotyping. In this study, highly polymorphic bi-allelic SNP loci were screened and 700 KASP (Kompetitive Allele Specific PCR) molecular markers were developed based on resequencing data of 205 diverse maize inbred lines. Among them, 202 KASP markers validated by 46 representative lines were further used for phylogenetic tree construction and genetic structure analysis. The validated KASP markers distributed evenly on 10 chromosomes in maize with an average PIC of 0.463 and an average MAF of 0.451. The phylogenetic tree constructed by KASP markers is highly consistent with that by re-sequencing data. In addition, the genetic similarity coefficient evaluated between KASP loci and the total SNP loci achieved 89.5% which demonstrated the availability of KASP in heterotic group division. These findings suggest that 202 KASP markers play an important role in analysis of germplasm resource, construction of genetic map, and division of heterotic group in maize.

Key words: maize, resequencing, KASP markers, germplasm

表1

实验材料详细信息"

类群
Group		 205份供试材料
205Experimental cultivars		 用于KASP验证的46份材料
46 cultivars for KASP
样本
大小
Size		 占总样本比例
Percentage
(%)		 样本
大小
Size		 占总样本比例 Percentage
(%)		 代表自交系
Representative inbred lines
瑞德
Reid		 14 6.86 4 8.69 B73, A632, 郑32, H84
B73, A632, Zheng 32, H84
改良瑞德Improved Reid 33 16.09 9 19.57 郑58, 478, 5003, 黄C, 1205A, 综3, 铁7922, K22
Zheng 58, 478, 5003, Huang C, 1205A, Zong 3, Tie 7922, K22
热带
Tropic		 18 8.78 8 17.39 苏湾1611, 四路糯, CML162, DY206, CML52, Ki11, Ki3
Suwan 1611, Silunuo, CML162, DY206, CML52, Ki11, Ki3
PB 22 10.73 7 15.22 T877, 齐319, P138, 沈137, 沈135, Yu 87-1
T877, Qi 319, P138, Shen 137, Shen 135, Yu 87-1
兰卡斯特
Lancaster		 50 24.39 6 13.04 Mo17, OH43, LH51, LH61, LH54, 龙抗11
Mo17, OH43, LH51, LH61, LH54, Longkang 11
四平头
Sipingtou		 45 21.95 9 19.57 黄早四, 昌7-2, S22, Huangyesi 3, Wu 126, 444, K12
Huangzaosi, Chang 7-2, S22, Huangyesi 3, Wu 126, 444, K12
其他Other 23 11.22 3 6.52 F2, F7

图1

KASP标记基因分型图 a~d为编号不同的KASP标记的基因分型图; AA: 红色簇状是具有HEX型等位基因的品种; GG: 蓝色簇状是具有FAM型等位基因的品种; NTC: 是不含模板的空白对照。"

图2

202个SNP位点遗传多态性 A: 202个SNP位点在染色体上的物理位置, 右边数字是202个KASP标记的编号, 左边数字是202个KASP标记在染色体上物理位置, 单位为Mb; B: 202个SNP位点的最小等位基因频率分布(MAF)和多态信息含量分布(PIC)。"

图3

Neighbor-joining (N-J)树分析 A: 205份自交系基于202个位点的Neighbor-Joining (N-J)树分析, 红色线条代表46个实验材料; B: 205份自交系基于1,660,336个位点的Neighbor-Joining (N-J)树分析; Group 1~Group 7分别代表瑞德群、改良瑞德群、热带群、PB群、兰卡斯特群、四平头群、其他纬度种质。"

[1] 赵曦, 王荣焕, 于永涛, 王天宇, 黎裕 . 关联分析在玉米遗传学研究中的应用. 玉米科学, 2008,16(1):52-55.
Zhao X, Wang R H, Yu Y T, Wang T Y, Li Y . Application of association analysis in maize genetics. J Maize Sci, 2008,16(1):52-55 (in Chinese with English abstract).
[2] 简银巧 . 热带玉米全长泛转录组和基因组大小变异及应用. 中国农业科学院博士学位论文, 北京, 2017.
Jian Y Q . Variations in Pan-transcriptome and Genome Size in Tropical Maize (Zea mays L.) and Their Applications. PhD Dissertation of Chinese Academy of Agricultural Sciences, Beijing,China, 2017 (in Chinese with English abstract).
[3] 李向拓, 毛建昌, 吴权明 . 分子标记在玉米育种中的应用. 玉米科学, 2004,12(1):26-29.
Li X T, Mao J C, Wu Q M . Molecular markers and maize breeding. J Maize Sci, 2004,12(1):26-29 (in Chinese with English abstract).
[4] 卢柏山, 史亚兴, 宋伟, 徐丽, 赵久然 . 利用SNP标记划分甜玉米自交系的杂种优势类群. 玉米科学, 2015,23(1):58-62.
Lu B S, Shi Y X, Song W, Xu L, Zhao J R . Heterotic grouping of sweet corn inbred lines by SNP markers. J Maize Sci, 2015,23(1):58-62 (in Chinese with English abstract).
[5] 吴金凤, 宋伟, 王蕊, 田红丽, 李雪, 王风格, 赵久然, 蔚荣海 . 利用SNP标记对51份玉米自交系进行类群划分. 玉米科学, 2014,22(5):29-34.
Wu J F, Song W, Wang R, Tian H L, Li X, Wang F G, Zhao J R, Wei R H . Heterotic grouping of 51 maize inbred lines by SNP markers. J Maize Sci, 2014,22(5):29-34 (in Chinese with English abstract).
[6] Jiao Y, Zhao H, Ren L, Song W, Zeng B, Guo J, Wang B, Liu Z, Chen J, Li W, Zhang M, Xie S, Lai J . Genome-wide genetic changes during modern breeding of maize. Nat Genet, 2012,46:812-815.
doi: 10.1038/ng0914-1039 pmid: 22660547
[7] Sun S L, Zhou Y S, Chen J, Shi J P, Zhao H M, Zhao H N, Song W B, Zhang M, Cui Y, Dong X M, Liu H, Ma X X, Jiao Y P, Wang B, Wei X H, Stein J C, Glaubitz J C, Lu F, Yu G L, Liang C Z, Fengler K, Li B L, Rafalski A, Schnable P S, Ware D H, Buckler E S, Lai J S . Extensive intraspecific gene order and gene structural variations between Mo17 and other maize genomes. Nat Genet, 2018,50:1289-1295.
doi: 10.1038/s41588-018-0182-0 pmid: 30061735
[8] 宁洽, 刘文国, 杨伟光, 路明 . SNP标记在玉米研究上的应用进展. 玉米科学, 2017,25(1):57-61.
Ning Q, Liu W G, Yang W G, Lu M . Progress and application of SNP markers in maize. J Maize Sci, 2017,25(1):57-61 (in Chinese with English abstract).
[9] Gaudet M, Fara A G, Beritognolo I, Sabatti M . Allele-specific PCR in SNP genotyping. Method Mol Biol, 2009,578:415-424.
doi: 10.1007/978-1-60327-411-1_26
[10] Huang X Q, Roder M S . Development of SNP assays for genotyping the puroindoline b gene for grain hardness in wheat using pyrosequencing. J Agric Food Chem, 2005,53:2070-2075.
doi: 10.1021/jf047955b pmid: 15769137
[11] Tabone T, Mather D E, Hayden M J . Temperature switch PCR (TSP): robust assay design for reliable amplification and genotyping of SNPs. BMC Genomics, 2009,10:580.
doi: 10.1186/1471-2164-10-580 pmid: 2795770
[12] Rasheed A, Wen W, Gao F, Zhai S N, Jin H, Liu J D, Guo Q, Zhang Y J, Dreisigacker S, Xia X C, He Z H . Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theor Appl Genet, 2016,129:1843-1860.
doi: 10.1007/s00122-016-2743-x pmid: 27306516
[13] 赵久然, 李春辉, 宋伟, 王元东, 张如养, 王继东, 王凤格, 田红丽, 王蕊 . 基于SNP芯片揭示中国玉米育种种质的遗传多样性与群体遗传结构. 中国农业科学, 2018,51:626-634.
Zhao J R, Li C H, Song W, Wang Y D, Zhang R Y, Wang J D, Wang F G, Tian H L, Wang R . Genetic diversity and population structure of important Chinese maize breeding germplasm revealed by SNP-chips. Sci Agric Sin, 2018,51:626-634 (in Chinese with English abstract).
[14] 王文斌, 徐淑兔, 高杰, 张兴华, 郭东伟, 李向阳, 薛吉全 . 基于SNP标记的玉米自交系遗传多样性分析. 玉米科学, 2015,23(2):41-45.
Wang W B, Xu S T, Gao J, Zhang X H, Guo D W, Li X Y, Xue J Q . Analysis of genetic diversity of maize inbred lines based on SNP markers. J Maize Sci, 2015,23(2):41-45 (in Chinese with English abstract).
[15] Thomson M J . High-throughput SNP genotyping to accelerate crop improvement. Plant Breed Biotechnol, 2014,2:195-212.
doi: 10.9787/PBB.2014.2.3.195
[16] Wang S C, Wong D B, Forrest K, Allen A, Chao S M, Huang B E, Maccaferri M, Salvi S, Milner S G, Cattivelli L, Mastrangelo A M, Whan A, Stephen S, Barker G, Wieseke R, Plieske J, Lillemo M, Mather D, Appels R, Dolferus R, Brown-Guedira G, Korol A, Akhunova A R, Feuillet C, Salse J, Morgante M, Pozniak C, Luo M C, Dvorak J, Morell M, Dubcovsky J, Ganal M, Tuberosa R, Lawley C, Mikoulitch I, Cavanagh C, Edwards K J, Hayden M, Akhunov E . Characterization of polyploid wheat genomic diversity using a high-density 90,000 single nucleotide polymorphism array. Plant Biotechnol, 2014,12:787-796.
doi: 10.1111/pbi.12183 pmid: 4265271
[17] Semagn K, Babu R, Hearne S, Olsen M . Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Mol Breed, 2014,33:1-14.
[18] Kumpatla S P, Buyyarapu R, Abdurakhmonov I Y, Mammadov J A. Genomics-assisted plant breeding in the 21st century: technological advances and progress. In: Abdurakhmonov I ed. Plant Breeding. Rijeka, Croatia: InTech, 2012. pp 131-184.
[19] 吕远大, 李坦, 石丽, 张晓林, 赵涵 . 基于全基因组重测序信息开发玉米H99自交系特异分子标记. 作物学报, 2014,40:191-197.
Lyu Y D, Li T, Shi L, Zhang X L, Zhao H . Next-generation sequencing for molecular marker development in maize inbred H99. Acta Agron Sin, 2014,40:191-197 (in Chinese with English abstract).
[20] 周玲, 梁帅强, 林峰, 吕远大 . 玉米二态性InDel位点的鉴定和分子标记开发. 江苏农业学报, 2016,32:1223-1231.
Zhou L, Liang S Q, Lin F, Lyu Y D . Bi-allelic InDel loci detection and molecular marker development in maize. Jiangsu J Agric Sci, 2016,32:1223-1231 (in Chinese with English abstract).
[21] Lyu Y, Liu Y , Zhao H. mInDel: a high-throughput and efficient pipeline for genome-wide InDel marker development. BMC Genomics, 2016,17:290.
doi: 10.1186/s12864-016-2614-5 pmid: 4832496
[22] 周玲, 梁帅强, 吕远大, 张体付, 戴惠学, 赵松涛, 赵涵 . 中国黄淮海地区玉米杂种优势候选位点的鉴定. 玉米科学, 2017,25(1):15-23.
Zhou L, Liang S Q, Lyu Y D, Zhang T F, Dai H X, Zhao S T, Zhao H . Identification of candidate loci associated with maize heterosis in Huang-Huai-Hai region of China. J Maize Sci, 2017,25(1):15-23 (in Chinese with English abstract).
[23] Saitou N . The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 1987,4:406-425.
[24] 何中虎 ,阿韦斯·拉希德 ,卢家玲 ,夏先春. 基于KASP技术检测小麦功能基因的成套引物及其应用. CN105112546A[P]. 2015.
He Z H, Aves Rashid, Lu J L, Xia X C . Complete set of primers for detecting wheat functional genes and their application based on KASP technology. CN105112546A[P]. 2015 (in Chinese).
[25] 余四斌, 袁志阳, 孙文强, 熊银, 龚蓉 . 基于KASP技术用于水稻产量基因分型的引物组合及其应用. CN 106939349 A[P]. 2017.
Yu S B, Yuan Z Y, Sun W Q, Xiong Y, Gong R . Based primer composition and its application technique for rice yield based on KASP genotyping. CN 106939349 A[P]. 2017 (in Chinese).
[1] 杨扬, 常诗惠, 田红丽, 易红梅, 王璐, 任洁, 范亚明, 刘亚维, 王凤格, 赵久然. 不同生态区国审玉米品种的遗传多样性分析[J]. 作物学报, 2026, 52(5): 1352-1364.
[2] 杨欣雨, 崔文涛, 迪力尼格尔·阿力木, 汪凯翔, 吴鹏昊, 任姣姣. 玉米穗上叶片数全基因组关联分析和全基因组选择[J]. 作物学报, 2026, 52(5): 1573-1590.
[3] 韩亚鑫, 何冠华, 张小琼, 张登峰, 李永祥, 刘旭洋, 王天宇, 黎裕, 邹华文, 李春辉. 基于RNA-Seq和BSA-Seq联合分析挖掘玉米侧根密度基因资源[J]. 作物学报, 2026, 52(5): 1341-1352.
[4] 孙淑凤, 许振南, 黄嘉鑫, 翁建峰, 李新海. 玉米MAPK家族全基因组鉴定及其对拟轮枝镰孢菌感染的响应[J]. 作物学报, 2026, 52(5): 1291-1308.
[5] 姚术, 郭凯悦, 翟慧慧, 姚佳慧, 邓文琪, 闫玲, 黄驰, 高阳, 俞嫣然, 赵振邦, 李英慧, 王晓波, 李佳佳. 大豆苗期耐低铁综合评价及优异种质筛选[J]. 作物学报, 2026, 52(5): 1373-1387.
[6] 张宁宁, 滕雨菲, 任娜娜, 魏兴卓, 闫书豪, 樊可心, 王永宏, 陈文康, 张兴华, 朱万超, 徐淑兔, 薛吉全. 201份玉米自交系抗旱表型评价及可塑性分析[J]. 作物学报, 2026, 52(5): 1309-1325.
[7] 张鸿蓉, 王菲儿, 李盼, 仇海龙, 朱静, 赵连豪, 南运有, 何蔚, 樊志龙, 胡发龙, 柴强, 殷文. 减量20%灌水与25%有机肥替代化肥提高青贮玉米产量的光合特性[J]. 作物学报, 2026, 52(5): 1487-1500.
[8] 蔡宏玮, 于爱忠, 姜科强, 王鹏飞, 王玉珑, 霍建喆, 庞小能, 尹波, 尚永盼. 干旱灌区有机肥替代部分化肥促进甜玉米产量提升的关键机制[J]. 作物学报, 2026, 52(4): 1166-1180.
[9] 田红丽, 杨扬, 范亚明, 易红梅, 郭丹丹, 王凤格, 赵久然. 适于玉米品种鉴定的一套三等位变异SNP新型标记组合[J]. 作物学报, 2026, 52(4): 993-1005.
[10] 杨亚莉, 徐明睿, 马越飞, 海艺蕊, 刘凯栋, 刘万茂, 孙颖. 玉米根尖及整根响应缺铁的转录组比较研究[J]. 作物学报, 2026, 52(4): 1006-1021.
[11] 马亮, 马璐, 张舒钰, 章慧敏, 王仁明, 宋旭东, 张振良, 冒宇翔, 陆虎华, 陈国清, 郝德荣, 周广飞. 玉米苞叶数目转录组分析及候选基因鉴定[J]. 作物学报, 2026, 52(3): 790-801.
[12] 孟成, 王哲. 玉米ZmPFK基因家族全基因组鉴定及响应胁迫表达分析[J]. 作物学报, 2026, 52(3): 764-779.
[13] 李新浩, 邢梦柯, 周梓惠, 李思烨, 任昊, 王洪章, 赖华江. 外源褪黑素通过协调光反应与暗反应增强玉米苗期的耐热性[J]. 作物学报, 2026, 52(3): 839-856.
[14] 张超, 郭欢, 李忠玲, 岳淑宁, 赵娜. 基于BSA-seq技术定位玉米籽粒花青素关联基因[J]. 作物学报, 2026, 52(3): 780-789.
[15] 郭向阳, 涂亮, 王栋, 刘鹏飞, 王安贵, 易强, 任洪, 李刚, 祝云芳, 吴迅, 蒋喻林, 田丰, 陈泽辉. 热带Suwan种质在我国玉米种质改良中的创新与利用[J]. 作物学报, 2026, 52(3): 655-664.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2