基于BSA-Seq技术的甜瓜抗霜霉病InDel标记开发

doi:10.6048/j.issn.1001-4330.2021.12.014

摘要/Abstract

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。

关键词: 甜瓜; 霜霉病; InDel分子标记; BSA

Abstract:

【Objective】 o obtain molecular markers of melon downy mildew resistance genes, and improve the accuracy and scientific city of melon downy mildew resistance traits selection. 【Methods】 The F₂ generation segregation population was constructed by hybridization between wild high-resistant downy mildew resource DM-4 and susceptible inbred line DM-2BSA-seq was used to locate and regulate the candidate interval of downy mildew resistance gene in melon InDel molecular markers. parents and their F₂ individuals were used for screening and verification.【Results】 Genes regulating melon downy mildew resistance are located on 9th chromosome. 37 InDel molecular markers were developed in the candidate region, and 9 of them showed differences in the size of PCR products between resistant and susceptible parents. Further verification was carried out with F₂ plants. Combined with field phenotypic identification results, the accuracy rates of primer InDel 15 and InDel 20 were 95 % and 98 %, respectively. The amplified products of primer InDel 15 and InDel 20 in resistant parents were 251 bp and 349 bp, respectively, and 231 bp and 324 bp, respectively.【Conclusion】 Primers InDel 15 and InDel 20 can be used as molecular markers for downy mildew resistance breeding, which can accelerate the breeding speed of melon downy mildew resistance and lay the foundation for further cloning the main genes of melon downy mildew resistance.

Key words: Cucumis melo L.; downy mildew; In Del marker; BSA

中图分类号:

S652

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.

图/表 8

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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	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

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

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