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作物学报 ›› 2022, Vol. 48 ›› Issue (3): 553-564.doi: 10.3724/SP.J.1006.2022.11039

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用集群分离分析结合高密度芯片快速定位小麦成株期抗条锈病基因YrC271

刘丹1**(), 周彩娥1**, 王晓婷1, 吴启蒙1, 张旭1, 王琪琳1, 曾庆东2, 康振生2, 韩德俊1,*(), 吴建辉1,*()   

  1. 1西北农林科技大学农学院/旱区作物逆境生物学国家重点实验室, 陕西杨凌 712100
    2西北农林科技大学植物保护学院/旱区作物逆境生物学国家重点实验室, 陕西杨凌 712100
  • 收稿日期:2021-04-07 接受日期:2021-06-16 出版日期:2022-03-12 网络出版日期:2021-07-19
  • 通讯作者: 韩德俊,吴建辉
  • 作者简介:刘丹, E-mail: 17734508020@163.com第一联系人:**同等贡献
  • 基金资助:
    国家自然科学基金项目(31901494);国家自然科学基金项目(31901869);国家自然科学基金项目(31971890)

Rapid identification of adult plant wheat stripe rust resistance gene YrC271 using high-throughput SNP array-based bulked segregant analysis

LIU Dan1**(), ZHOU Cai-E1**, WANG Xiao-Ting1, WU Qi-Meng1, ZHANG Xu1, WANG Qi-Lin1, ZENG Qing-Dong2, KANG Zhen-Sheng2, HAN De-Jun1,*(), WU Jian-Hui1,*()   

  1. 1State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, China
    2State Key Laboratory of Crop Stress Biology for Arid Areas, Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi, China
  • Received:2021-04-07 Accepted:2021-06-16 Published:2022-03-12 Published online:2021-07-19
  • Contact: HAN De-Jun,WU Jian-Hui
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(31901494);National Natural Science Foundation of China(31901869);National Natural Science Foundation of China(31971890)

摘要:

由国际玉米小麦改良中心(CIMMYT)培育的春小麦高代选系C271对小麦条锈病保持抗性近40年。为明确C271的抗条锈病遗传组分, 利用感病品种晋麦79与C271杂交构建含有229个F2:3家系的遗传群体, 并于2019年在陕西杨凌和四川江油进行成株期病害调查。运用集群分离分析(BSA)结合高密度660K芯片策略在3B染色体短臂上快速挖掘出大量的与抗病关联的SNP, 利用等位基因特异的定量PCR标记(AQP)进行验证并作图, 成功检测到一个效应值较大的QTL, 可解释表型变异为22.7%~30.8%, 暂命名为YrC271, 位于标记AX-109001377AX-111087256之间, 约1.9 cM, 对应的物理距离1.9 Mb。利用已公布的小麦基因组信息对该区间进行比较基因组分析, 结果表明, 与中国春基因组相比, 不同材料间存在小片段的插入以及倒位现象, 但总体共线性良好。同时利用1484份小麦660K分型数据对该区间进行单倍型分析, 总体可分为5种区间单倍型, 其中C271所在的单倍型组的抗性优于其他组。虽然C271不含有Yr30Yr58连锁标记的阳性片段, 但从相对遗传位置、条锈病抗性表现以及育种系谱看, YrC271Yr30Yr58都很类似, 其关系需要进一步确认。对主效QTL定位来讲, 芯片结合BSA策略可快速锁定目标QTL区域, 再应用AQP技术既提高了作图效率, 也降低了标记分析的成本, 为高通量基因/QTL定位工作提供了借鉴。

关键词: 条锈病成株抗性, YrC271, SNP标记, 比较基因组分析, 单倍型分析

Abstract:

Wheat cultivar C271 registered as PI 210904 in the USDA National Small Grains Collection was developed from Punjab Pakistan in 1953 and it confers adult plant resistance (APR) to stripe rust both in the United States and China for many years. In the present study, we dissected the genetic basis of stripe rust resistance on 229 F2:3 populations produced by crossing Jinmai 79 and C271 in the fields of Yangling and Jiangyou. Bulked segregant analysis coupled with wheat 660K SNP array placed the majority of SNPs differences on chromosome arm 3BS. After using allele-specific quantitative PCR based genotyping assay (AQP) to confirm the SNPs, a linkage map was constructed and a major locus was detected across all environments based on IciMapping v4.1 software. The QTL, designated as YrC271, was flanked by SNP markers AX-109001377 and AX-111087256 with a genetic interval of 1.9 cM corresponding to a physical distance of 1.9 Mb in RefSeq v.1.0 (positions 6.1-8.0 Mb). Comparative genomics analysis was performed to detect the collinear genomic regions of different hexaploid wheat accessions (Triticum aestivum), T. dicoccoides, and T. turgidum. More than 340 SNPs in the physical region were extracted for haplotype analysis in a panel of over 1484 worldwide common wheat accessions, and five major haplotypes (Hap1, Hap2, Hap3, Hap4, and Hap5) were identified. And the favorable haplotype Hap1 was highly associated with stripe rust resistance. YrC271 appeared to be similar to YrC271 based on comparison of relative distance, stripe rust responses, and pedigree analyses, but allelism tests, cloning or precise phenotypic comparisons would be needed for confirmation. The YrC271 region provided the opportunity for further map-based cloning and haplotypes analysis enabled pyramiding favorable alleles into commercial cultivars by marker-assisted selection.

Key words: adult plant resistance to stripe rust, YrC271, SNP markers, comparative genomics analysis, haplotype analysis

图1

群体构建以及数据处理分析流程 A: 晋麦79和C271的田间抗性表现、群体构建和表型调查; B, C: 229个F2:3家系在杨凌和江油环境下的抗条锈病表型分布频率; D: 小麦660K SNP芯片差异SNP的染色体分布; E: 3B染色体上差异SNP物理位置及分布; F: 5A染色体上差异SNP物理位置及分布; G: YrC271的遗传图谱以及定位; H: 3BS染色体缺失系图谱。"

表1

晋麦79 × C271的F2:3群体的反应型(IT)和最大严重度(MDS)在多环境下的相关性分析"

环境
Environment
IT杨凌 2019
IT Yangling in 2019
IT江油 2019
IT Jiangyou in 2019
MDS杨凌 2019
MDS Yangling in 2019
MDS江油 2019
MDS Jiangyou in 2019
IT杨凌2019 IT Yangling in 2019 1
IT江油2019 IT Jiangyou in 2019 0.89** 1
MDS杨凌2019 MDS Yangling in 2019 0.86** 1
MDS江油2019MDS Jiangyou in 2019 0.85** 0.90** 1

表2

晋麦79 × C271的F2:3群体的反应型(IT)和最大严重度(MDS)方差分析"

变异来源
Source of variations
反应型IT 严重度MDS
自由度
DF
均方
Mean square
自由度
DF
均方
Mean square
基因型 Lines (L) 228 15.22** 228 1952.66**
重复 Replicates/environments 2 14.07 2 21,443.81
环境 Environments (E) 1 1.54 1 1360.89
基因型×环境 L × E 228 0.87 228 100.30
误差 Error 456 1.99 456 272.18
基因型方差 σ2g 3.31 420.12
广义遗传力 h2b 0.87 0.86

表3

多态性AQP标记的引物信息"

标记名称
Marker ID
引物序列
Primer sequence (5′-3′)
AX-110965644 A_FAM GAAGGTGACCAAGTTCATGCTGGGAGGCATAAAAAATTGCAGGCTG
B_HEX GAAGGTCGGAGTCAACGGATTGGGAGGCATAAAAAATTGCAGGCTA
C_reverse ATCGCTCCAGTTCTTGATGTGTCCA
AX-108828571 A_FAM GAAGGTGACCAAGTTCATGCTGCTTGTAGCAGACAGAATTACCA
B_HEX GAAGGTCGGAGTCAACGGATTGCTTGTAGCAGACAGAATTACCG
C_reverse CAGAACTCCAGTGCCCTTCT
AX-109001377 A_FAM GAAGGTGACCAAGTTCATGCTGGATACCATCCGCCCAAGT
B_HEX GAAGGTCGGAGTCAACGGATTGGATACCATCCGCCCAAGC
C_reverse TCGGGGTTCAGCCACACC
AX-109828842 A_FAM GAAGGTGACCAAGTTCATGCTGCATTCCCTGCCATCACG
B_HEX GAAGGTCGGAGTCAACGGATTGCATTCCCTGCCATCACA
C_reverse GCTTTTATGTGGCACCGGTG
AX-109925313 A_FAM GAAGGTGACCAAGTTCATGCTTTGCAGCGAAGAGTTTGGTT
B_HEX GAAGGTCGGAGTCAACGGATTTTGCAGCGAAGAGTTTGGTG
C_reverse CCGGCTGATCTGACCATCAT
AX-111087256 A_FAM GAAGGTGACCAAGTTCATGCTGAGAAATCAACTCACACAAATTT
B_HEX GAAGGTCGGAGTCAACGGATTGAGAAATCAACTCACACAAATTC
C_reverse TGTTTGATGTATGCGTGAGCTATT
AX-109966241 A_FAM GAAGGTGACCAAGTTCATGCTAGCATAAAAGGTGGCCCAAGCCATG
B_HEX GAAGGTCGGAGTCAACGGATTAGCATAAAAGGTGGCCCAAGCCATA
C_reverse TGTTGACTCGCAGATGTACTTTGCCTTTGCACAGTAATTTTTACC
AX-89750431 A_FAM GAAGGTGACCAAGTTCATGCTCTTTGCACAGTAATTTTTACC
B_HEX GAAGGTCGGAGTCAACGGATTCTTTGCACAGTAATTTTTACA
C_reverse CAGAAATCAGAGAGGAGAGTTC
AX-109997090 A_FAM GAAGGTGACCAAGTTCATGCTATGCTGAGGTTTCCGGTCAGAT
B_HEX GAAGGTCGGAGTCAACGGATTATGCTGAGGTTTCCGGTCAGAC
C_reverse TGACAAACCCGGGTCAAACA

表4

晋麦79 × C271的F2:3群体分别在2个环境下利用完备区间作图法进行成株期抗条锈QTL检测"

环境
Environment
标记区间
Marker interval
最大似然值
LOD
加性效应
Add
表型变异率
PVE (%)
反应型Infection type (IT)
杨凌2019 Yangling in 2019 AX-109001377-AX-111087256 15.9 -1.5 28.6
江油2019 Jiangyou in 2019 AX-109001377-AX-111087256 16.4 -1.5 29.9
平均 Mean AX-109001377-AX-111087256 17.2 -1.5 30.8
最大严重度Maximum disease severtiy (MDS)
杨凌2019 Yangling in 2019 AX-109001377-AX-111087256 12.2 -14.2 22.7
江油2019 Jiangyou in 2019 AX-109001377-AX-111087256 13.6 -15.5 25.0
平均Mean AX-109001377-AX-111087256 13.5 -14.8 24.8

图2

YrC271物理区间及其与不同倍性小麦品种基因组的共线性分析"

图3

YrC271区间单倍型分析(A)及抗条锈病表现(B) YL、TS、JY分别代表杨凌、天水和江油。"

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