新疆农业科学 ›› 2021, Vol. 58 ›› Issue (12): 2265-2273.DOI: 10.6048/j.issn.1001-4330.2021.12.014
• 植物保护·园艺特产·土壤肥料·节水灌溉·农业生态环境·农业装备工程与机械化 • 上一篇 下一篇
凌悦铭, 李寐华, 杨永, 杨文莉, 范蓉, 伊鸿平, 张红, 张学军
收稿日期:
2021-01-30
出版日期:
2021-12-20
发布日期:
2021-12-31
基金资助:
LING Yueming, LI Meihua, YANG Yong, YANG Wenli, FAN Rong, YI Hongping, ZHANG Hong, ZHANG Xuejun
Received:
2021-01-30
Online:
2021-12-20
Published:
2021-12-31
Supported by:
摘要: 目的 获得与甜瓜抗霜霉病基因连锁的分子标记或功能标记,用于甜瓜分子标记辅助选择育种。为进一步克隆甜瓜抗霜霉病主效基因奠定基础。方法 以野生高抗霜霉病资源DM-4和感病自交系DM-2为亲本,构建F2代分离群体,利用BSA-seq对甜瓜抗霜霉基因进行QTL定位,开发InDel分子标记,并在双亲及其F2群体单株进行筛选验证。结果 甜瓜抗霜霉病主效QTL位于第9号染色体。在候选区间开发了37个InDel分子标记,有9个PCR产物大小在抗、感双亲表现出差异,用F2单株验证,结合田间表型鉴定结果,引物InDel 15和InDel 20准确率分别为95 %和98 %,引物InDel 15和InDel 20在抗病亲本中扩增产物大小分别为251和349 bp,在感病亲本中分别为231和324 bp。结论 引物InDel 15和InDel 20可以作为分子标记进行抗霜霉病辅助选育,加快了甜瓜抗霜霉病育种速度。
中图分类号:
凌悦铭, 李寐华, 杨永, 杨文莉, 范蓉, 伊鸿平, 张红, 张学军. 基于BSA-Seq技术的甜瓜抗霜霉病InDel标记开发[J]. 新疆农业科学, 2021, 58(12): 2265-2273.
LING Yueming, LI Meihua, YANG Yong, YANG Wenli, FAN Rong, YI Hongping, ZHANG Hong, ZHANG Xuejun. Development of InDel Marker for Melon Resistance to Downy Mildew Based on BSA Resequencing[J]. Xinjiang Agricultural Sciences, 2021, 58(12): 2265-2273.
样本名 Sample | 原始数据量 Raw Base (bp) | 有效数据量 Clean Base (bp) | 碱基错误率 Error Rate(%) | Q20(%) | Q30(%) | GC含量 GC Content(%) |
---|---|---|---|---|---|---|
DM-4 | 5 230 484 100 | 5 205 287 100 | 0.01 | 97.74 | 94.94 | 37.39 |
DM-2 | 5 954 570 100 | 5 926 054 500 | 0.01 | 97.83 | 95.10 | 37.23 |
DM-R | 9 302 519 100 | 9 266 289 000 | 0.01 | 97.93 | 95.27 | 37.17 |
DM-S | 9 931 454 700 | 9 878 084 400 | 0.01 | 97.81 | 95.08 | 37.12 |
表1 测序数据质量情况汇总
Table 1 The quality of sequencing data
样本名 Sample | 原始数据量 Raw Base (bp) | 有效数据量 Clean Base (bp) | 碱基错误率 Error Rate(%) | Q20(%) | Q30(%) | GC含量 GC Content(%) |
---|---|---|---|---|---|---|
DM-4 | 5 230 484 100 | 5 205 287 100 | 0.01 | 97.74 | 94.94 | 37.39 |
DM-2 | 5 954 570 100 | 5 926 054 500 | 0.01 | 97.83 | 95.10 | 37.23 |
DM-R | 9 302 519 100 | 9 266 289 000 | 0.01 | 97.93 | 95.27 | 37.17 |
DM-S | 9 931 454 700 | 9 878 084 400 | 0.01 | 97.81 | 95.08 | 37.12 |
样本名 Sample | 比对序列数 Mapped reads | 有效序列数 Total reads | 比对率 Mapping rate(%) | 平均测序深度 Average depth(X) | 1X覆盖度 Coverage at least 1X(%) |
---|---|---|---|---|---|
DM-2 | 36 714 906 | 39 507 030 | 92.93 | 13.11 | 96.38 |
DM-4 | 32 024 597 | 34 701 914 | 92.28 | 11.57 | 96.37 |
DM-S | 61 510 228 | 65 853 896 | 93.40 | 21.29 | 97.75 |
DM-R | 57 254 273 | 61 775 260 | 92.68 | 19.85 | 97.70 |
表2 测序深度及覆盖度统计
Table 2 Sequencing depth and coverage
样本名 Sample | 比对序列数 Mapped reads | 有效序列数 Total reads | 比对率 Mapping rate(%) | 平均测序深度 Average depth(X) | 1X覆盖度 Coverage at least 1X(%) |
---|---|---|---|---|---|
DM-2 | 36 714 906 | 39 507 030 | 92.93 | 13.11 | 96.38 |
DM-4 | 32 024 597 | 34 701 914 | 92.28 | 11.57 | 96.37 |
DM-S | 61 510 228 | 65 853 896 | 93.40 | 21.29 | 97.75 |
DM-R | 57 254 273 | 61 775 260 | 92.68 | 19.85 | 97.70 |
图 2 Δ( SNP- Index)分布曲线 注:橙色曲线代表置信度为0.95的阈值线,绿色曲线代表置信度为0.99的阈值线
Fig. 2 Distribution curve of Δ( SNP-Index ) Note: Orange curve represents the confidence threshold line of 0.95, and green curve represents the confidence threshold line of 0.99
图 3 部分引物多态性筛选结果 注:M:Marker I;P1:以高抗霜霉病资源DM-4的DNA为模板进行PCR扩增;P2:以感病自交系DM-2的DNA为模板进行PCR扩增
Fig. 3 Polymorphism detected by InDel primers among F2 individual plants Note: M:Marker I; P1: PCR amplification in DM-4; P2: PCR amplification in DM-2
名称 Name | 正向引物序列5’- 3’ Forward primers sequence 5’ - 3’ | 反向引物5’- 3’ Reverse primers sequence 5’ - 3’ | 是否具有多态性 Polymorphism or not |
---|---|---|---|
InDel 1 | AGGTAAAGCCCCAGAGAGGA | CCTCCCGTGAAGTTTGGAGT | 否 |
InDel 2 | AGTGAATGTGGTCATTGTGCT | CCTCCCGTGAAGTTTGGAGT | 是 |
InDel 3 | ACACAGCACAACCCAGACAA | CTACGACCAACCCCCACATG | 是 |
InDel 4 | GCACTCTCCAGCCTTGAACT | GAGCAAAGGTACCGCTGTCT | 是 |
InDel 5 | TTTTCATTCGGATTCCTTGTTGT | AAGCTATGAGACCTACGCGC | 否 |
InDel 6 | TCAAAGTGACCCCATCCAGTG | TGTTTTGAGTTCAACGGGGGA | 否 |
InDel 7 | GTGACCCACGATAGGTACACC | TCAAGTTTGGGAGATTGGATCA | 否 |
InDel 8 | AGCATTCTCCCAGTTTCCCC | TGGGAACAAGAAAGTCAGGCA | 否 |
InDel 9 | ACACAATGAACCAAGATATTTGCCA | TGTTTGTAAGCCACTCGACT | 是 |
InDel 10 | ACGACACTAAATGACAGTCCGT | CCAAAATGGAAACCCATGCCC | 否 |
InDel 11 | GCAAAGTTGACCCTCTGTTCG | CAGCGTTCTAGGGGTTGGTT | 否 |
InDel 12 | TGTGAGAATGACAGCCCACA | CACATCTCTCAAGAGGCGGA | 否 |
InDel 13 | CCTCGCTCTATTTGCATTCACA | TTTATACGCGTACTAGTTCTCTCA | 否 |
InDel 14 | AAAGTCAGGATGGCCGAGTG | TCTCTTTCATCAGCGGCGTT | 是 |
InDel 15 | TCACAGCTCACAATCACAGGT | AGGGGAAGCTGGAGAAGACA | 是 |
InDel 16 | ACCTGCCGTCTATCGTGTTT | TGAGGTCGGTCCAAACAAGT | 否 |
InDel 17 | GCTGCCGTTTCGTTTTCAGT | ACGACCAAGAAACCCAGGAA | 否 |
InDel 18 | CCGTTGATTGCGCTAACACA | TGCCGCACGAGTATTCACTT | 是 |
InDel 19 | AGGAGTTTGGGTATGACACGAC | TTCTCCCTTTACCCGTTGCC | 是 |
InDel 20 | TGGGGTTGCTTGTGGATAGT | AGGCGAACACCTTTTGATTCA | 是 |
InDel 21 | GGAAGAGGAAGAGGCAGAGG | ACGAATCATTTCCTCCTCCGA | 否 |
InDel 22 | TGCTCTTGAGGCTAGGGCTA | TAGAGAGGTCCGAGCCCATC | 否 |
InDel 23 | TCATTGGCTAGAACTTCGACCA | CGAAAGTGATGGCCATGGGA | 否 |
InDel 24 | AGTGGTTGGTGGTGCATAGG | GCAACACTTTGATCCCCACA | 否 |
InDel 25 | ACCTCCAAGAAATGAATTCAGGA | TGAACGACGACGACCAATCT | 否 |
InDel 26 | CTTGCAAGGGCTAATGGTGC | ACGAATCAAAGGTGTAGCCA | 否 |
InDel 27 | ACAGTTTAGCTTCCATCCCTACA | TCGTTTAGGTCATTGTCCTCCA | 否 |
表3 InDel引物序列信息
Table 3 The information of InDel primers
名称 Name | 正向引物序列5’- 3’ Forward primers sequence 5’ - 3’ | 反向引物5’- 3’ Reverse primers sequence 5’ - 3’ | 是否具有多态性 Polymorphism or not |
---|---|---|---|
InDel 1 | AGGTAAAGCCCCAGAGAGGA | CCTCCCGTGAAGTTTGGAGT | 否 |
InDel 2 | AGTGAATGTGGTCATTGTGCT | CCTCCCGTGAAGTTTGGAGT | 是 |
InDel 3 | ACACAGCACAACCCAGACAA | CTACGACCAACCCCCACATG | 是 |
InDel 4 | GCACTCTCCAGCCTTGAACT | GAGCAAAGGTACCGCTGTCT | 是 |
InDel 5 | TTTTCATTCGGATTCCTTGTTGT | AAGCTATGAGACCTACGCGC | 否 |
InDel 6 | TCAAAGTGACCCCATCCAGTG | TGTTTTGAGTTCAACGGGGGA | 否 |
InDel 7 | GTGACCCACGATAGGTACACC | TCAAGTTTGGGAGATTGGATCA | 否 |
InDel 8 | AGCATTCTCCCAGTTTCCCC | TGGGAACAAGAAAGTCAGGCA | 否 |
InDel 9 | ACACAATGAACCAAGATATTTGCCA | TGTTTGTAAGCCACTCGACT | 是 |
InDel 10 | ACGACACTAAATGACAGTCCGT | CCAAAATGGAAACCCATGCCC | 否 |
InDel 11 | GCAAAGTTGACCCTCTGTTCG | CAGCGTTCTAGGGGTTGGTT | 否 |
InDel 12 | TGTGAGAATGACAGCCCACA | CACATCTCTCAAGAGGCGGA | 否 |
InDel 13 | CCTCGCTCTATTTGCATTCACA | TTTATACGCGTACTAGTTCTCTCA | 否 |
InDel 14 | AAAGTCAGGATGGCCGAGTG | TCTCTTTCATCAGCGGCGTT | 是 |
InDel 15 | TCACAGCTCACAATCACAGGT | AGGGGAAGCTGGAGAAGACA | 是 |
InDel 16 | ACCTGCCGTCTATCGTGTTT | TGAGGTCGGTCCAAACAAGT | 否 |
InDel 17 | GCTGCCGTTTCGTTTTCAGT | ACGACCAAGAAACCCAGGAA | 否 |
InDel 18 | CCGTTGATTGCGCTAACACA | TGCCGCACGAGTATTCACTT | 是 |
InDel 19 | AGGAGTTTGGGTATGACACGAC | TTCTCCCTTTACCCGTTGCC | 是 |
InDel 20 | TGGGGTTGCTTGTGGATAGT | AGGCGAACACCTTTTGATTCA | 是 |
InDel 21 | GGAAGAGGAAGAGGCAGAGG | ACGAATCATTTCCTCCTCCGA | 否 |
InDel 22 | TGCTCTTGAGGCTAGGGCTA | TAGAGAGGTCCGAGCCCATC | 否 |
InDel 23 | TCATTGGCTAGAACTTCGACCA | CGAAAGTGATGGCCATGGGA | 否 |
InDel 24 | AGTGGTTGGTGGTGCATAGG | GCAACACTTTGATCCCCACA | 否 |
InDel 25 | ACCTCCAAGAAATGAATTCAGGA | TGAACGACGACGACCAATCT | 否 |
InDel 26 | CTTGCAAGGGCTAATGGTGC | ACGAATCAAAGGTGTAGCCA | 否 |
InDel 27 | ACAGTTTAGCTTCCATCCCTACA | TCGTTTAGGTCATTGTCCTCCA | 否 |
名称 Name | 正向引物序列5’- 3’ Forward primers sequence 5’ - 3’ | 反向引物5’- 3’ Reverse primers sequence 5’ - 3’ | 是否具有多态性 Polymorphism or not |
---|---|---|---|
InDel 28 | TCATTGGCCGACACATGTCA | ACTCCAAAACTCCAAACCCCA | 否 |
InDel 29 | TGGTCCCTAACTAAATTACCCTCA | TCCCATAGCCGCCAACATTT | 否 |
InDel 30 | CTCAACTGGGTGTCGCTTCT | ATCGCAATGCCCCCAGTTAA | 否 |
InDel 31 | AACAGAGCTAACTTTGGCGC | TGTGAGAGTTTGGGCTTCCA | 否 |
InDel 32 | TCGAGCTAAAATGATTGTTGGCA | TCCTTCAAACATAAGGACACTCT | 否 |
InDel 33 | ACGAAACTCAATACTCGAGTGGA | TCACCACCTCCAATTCTTTGT | 否 |
InDel 34 | AAAAGCACCCCTCCCTTAGC | CGTACTCTAGACGTGTGCGG | 否 |
InDel 35 | TCTTTCACCCGTCAACTTAACT | TGTCCCACTTGAGTTGCATT | 否 |
InDel 36 | TGGTTGGGGAGCAAAATACA | AAACGTCCGTCGAAAATGCC | 否 |
InDel 37 | CGGCCGAATGGATGCTTTTT | TGCATTCGTTTTGGTTGGCA | 否 |
表3 续InDel引物序列信息
Table 3 The information of InDel primers
名称 Name | 正向引物序列5’- 3’ Forward primers sequence 5’ - 3’ | 反向引物5’- 3’ Reverse primers sequence 5’ - 3’ | 是否具有多态性 Polymorphism or not |
---|---|---|---|
InDel 28 | TCATTGGCCGACACATGTCA | ACTCCAAAACTCCAAACCCCA | 否 |
InDel 29 | TGGTCCCTAACTAAATTACCCTCA | TCCCATAGCCGCCAACATTT | 否 |
InDel 30 | CTCAACTGGGTGTCGCTTCT | ATCGCAATGCCCCCAGTTAA | 否 |
InDel 31 | AACAGAGCTAACTTTGGCGC | TGTGAGAGTTTGGGCTTCCA | 否 |
InDel 32 | TCGAGCTAAAATGATTGTTGGCA | TCCTTCAAACATAAGGACACTCT | 否 |
InDel 33 | ACGAAACTCAATACTCGAGTGGA | TCACCACCTCCAATTCTTTGT | 否 |
InDel 34 | AAAAGCACCCCTCCCTTAGC | CGTACTCTAGACGTGTGCGG | 否 |
InDel 35 | TCTTTCACCCGTCAACTTAACT | TGTCCCACTTGAGTTGCATT | 否 |
InDel 36 | TGGTTGGGGAGCAAAATACA | AAACGTCCGTCGAAAATGCC | 否 |
InDel 37 | CGGCCGAATGGATGCTTTTT | TGCATTCGTTTTGGTTGGCA | 否 |
图 4 引物InDel 15和InDel 20在双亲和F2单株中的PCR扩增结果 注:图2A为InDel 15引物扩增结果,其中11:抗霜霉病亲本DM-4,12:感霜霉病亲本DM-2,1~5:抗病单株,6~10:感病单株;图2B为InDel 20引物扩增结果,其中2:抗霜霉病亲本DM-4,1:感霜霉病亲本DM-2,3~7:抗病单株,8~12:感病单株;M均为:DL2000 DNA Marker
Fig.4 Result of amplification by primers InDel 15, InDel 20 among parents and F2 individual plants Note: Fig.2A: Result of amplification by primers InDel 15. 11: DM-4, 12: DM-2, 1~5: resistant individual, 6~10: susceptible individual. Fig.2B: Result of amplification by primers InDel 20. 2: DM-4, 1: DM-2, 3~7: resistant individual, 8~12: susceptible individual. M: DL2000 DNA Marker
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