基于重测序的长豇豆基因组InDel标记开发及应用

doi:10.16420/j.issn.0513-353x.2021-0347

摘要/Abstract

摘要：

选取3份长豇豆种质资源进行全基因组重测序,利用重测序数据挖掘InDel位点,根据这些位点开发分子标记,并验证这些标记的有效性和应用价值。经过严格筛选,获得981个易于凝胶电泳检测的长度大于30 bp且多态性高的InDel位点,挑选其中在基因组上均匀分布的165个设计引物并进行验证。用6份长豇豆材料对165对引物进行初筛,其中162对可获得PCR扩增产物,共扩增出673条条带;85对引物表现出多态性,多态性带333条（占总带数的49.48%）。利用85个多态性高的标记对173份长豇豆资源进行基因分型和遗传多样性分析,共检测到333个等位位点;多态性信息含量（PIC）为0.01 ~ 0.37,平均值为0.29;基因多样性指数变异范围为0.01 ~ 0.50,平均值为0.36,表明现有长豇豆资源遗传背景相对狭窄。通过聚类分析,173份长豇豆被分为2个类群,成功将不同类型长豇豆划分到不同亚群。另外,选用亲本间多态性标记用于6个杂交组合的F₁代真实性鉴定,鉴定出5个真实杂交种。本研究所开发的多态性 InDel 标记在长豇豆遗传多样性分析、杂交种纯度鉴定、遗传连锁图谱构建、基因定位和分子标记辅助选择育种等方面具有很好的应用前景。

关键词: 长豇豆, InDel标记, 遗传多样性, F₁代真实性鉴定

Abstract:

Based on resequencing data on three asparagus bean accessions,a set of InDel markers were developed and validated. Nine hundred and eight-one highly polymorphic InDel loci were screened out with insert or deletion bases longer than 30 bp suitable for PAGE gel electrophoresis detection. Among them,165 markers with uniform distribution across the genome were selected and checked in six asparagus bean inbreed lines. The results showed that 162 primer pairs yielded 673 amplification bands,and 85 primer pairs exhibited polymorphism yielded 333 polymorphic bands,accounted for 49.48% in the total amplification bands. These 85 polymorphic InDel markers were further used for genetic diversity analyzing in 173 asparagus bean accessions,and a total of 333 loci were generated. The polymorphism information content（PIC）value ranged from 0.01 to 0.37,with an average value of 0.29,while the gene diversity index value ranged from 0.01 to 0.50,with an average value of 0.36,indicating a relatively narrowed genetic diversity among these accessions. Population structure analysis revealed that there are two groups existed in these accessions. Further,these markers helped to identified five real hybrids from six hybrid combinations. Taken together,the polymorphic InDel markers developed in this study can be widely used for asparagus bean genetic analysis and molecular marker assisted selection breeding.

Key words: asparagus bean, InDel marker, genetic diversity analysis, F₁ identification

中图分类号:

S634.4

杨易, 黎庭耀, 李国景, 陈汉才, 沈卓, 周轩, 吴增祥, 吴新义, 张艳. 基于重测序的长豇豆基因组InDel标记开发及应用[J]. 园艺学报, 2022, 49(4): 778-790.

YANG Yi, LI Tingyao, LI Guojing, CHEN Hancai, SHEN Zhuo, ZHOU Xuan, WU Zengxiang, WU Xinyi, ZHANG Yan. Development and Application of Insertion-Deletion（InDel）Markers in Asparagus Bean Based on Whole Genome Re-sequencing Data[J]. Acta Horticulturae Sinica, 2022, 49(4): 778-790.

图/表 8

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编号No.	分类Type	资源数量Number	比例/%Ratio	命名Name
1	油白类White type	68	39.31	1-1,……1-68
2	油青类Green type	45	26.01	2-1,……2-45
3	紫豇豆Purple type	16	9.25	3-1,……3-16
4	特青类Dark green type	10	5.78	4-1,……4-10
5	纯白类Pure white type	14	8.09	5-1,……5-14
6	灰青类Gray green type	7	4.05	6-1,……6-7
7	无架豆类（矮生型）Shelf free type	4	2.31	7-1,……7-4
8	密豆类（籽粒密度高） Pod with high density grains type	9	5.20	8-1,……8-9

编号No.	分类Type	资源数量Number	比例/%Ratio	命名Name
1	油白类White type	68	39.31	1-1,……1-68
2	油青类Green type	45	26.01	2-1,……2-45
3	紫豇豆Purple type	16	9.25	3-1,……3-16
4	特青类Dark green type	10	5.78	4-1,……4-10
5	纯白类Pure white type	14	8.09	5-1,……5-14
6	灰青类Gray green type	7	4.05	6-1,……6-7
7	无架豆类（矮生型）Shelf free type	4	2.31	7-1,……7-4
8	密豆类（籽粒密度高） Pod with high density grains type	9	5.20	8-1,……8-9

染色体编号 Chromosome number	引物数 Number of primer	有效扩增引物数 Effective amplification primer	有效引物含量/% Effective amplification primer content	多态引物 Polymorphic primers number	多态引物含量/% Polymorphic primers content	扩增总条数 Total number of amplified band	多态条带数 Number of polymorphic band	多态条带比例/% Ratio
Vu1	17	17	100.00	7	41.18	94	35	37.23
Vu 2	13	13	100.00	6	46.15	60	24	40.00
Vu 3	22	22	100.00	7	31.82	95	19	20.00
Vu 4	19	19	100.00	13	68.42	76	47	61.84
Vu 5	14	14	100.00	10	71.43	48	37	77.08
Vu 6	17	16	94.12	10	58.82	54	38	70.37
Vu 7	9	8	88.89	5	55.56	27	22	81.48
Vu 8	11	10	90.91	4	36.36	45	14	31.11
Vu 9	16	16	100.00	10	62.50	66	43	65.15
Vu 10	16	16	100.00	9	56.25	61	42	68.85
Vu 11	11	11	100.00	4	36.36	47	12	25.53
总数Total	165	162	98.18	85	51.52	673	333	49.48

染色体编号 Chromosome number	引物数 Number of primer	有效扩增引物数 Effective amplification primer	有效引物含量/% Effective amplification primer content	多态引物 Polymorphic primers number	多态引物含量/% Polymorphic primers content	扩增总条数 Total number of amplified band	多态条带数 Number of polymorphic band	多态条带比例/% Ratio
Vu1	17	17	100.00	7	41.18	94	35	37.23
Vu 2	13	13	100.00	6	46.15	60	24	40.00
Vu 3	22	22	100.00	7	31.82	95	19	20.00
Vu 4	19	19	100.00	13	68.42	76	47	61.84
Vu 5	14	14	100.00	10	71.43	48	37	77.08
Vu 6	17	16	94.12	10	58.82	54	38	70.37
Vu 7	9	8	88.89	5	55.56	27	22	81.48
Vu 8	11	10	90.91	4	36.36	45	14	31.11
Vu 9	16	16	100.00	10	62.50	66	43	65.15
Vu 10	16	16	100.00	9	56.25	61	42	68.85
Vu 11	11	11	100.00	4	36.36	47	12	25.53
总数Total	165	162	98.18	85	51.52	673	333	49.48

上游引物 Forward primer	物理位置Physical location	引物序列（5′-3′） Forward primer sequence	下游引物 Reverse primer	物理位置 Physical location	引物序列（5′-3′） Reverse primer sequence
Vu1-4f	9 635 438	CTTGTGCATTGTTCTTGGTTCA	Vu1-4r	9 635 460	AGGGTAACTTGTTTTCAGCCAT
Vu1-5f	12 339 675	ATGGATAGGAGGGGAGGAAATT	Vu1-5r	12 339 697	TTTAGTCCCCTCTCCTCTACAC
Vu1-6f	14 580 618	TCGTTCTTTTACCATCTTCGCA	Vu1-6r	14 580 640	TTGAAGCGTGTTTAAATTACTGA
Vu1-10f	23 979 363	GAGTGATAGATCTTGGAGGTGC	Vu1-10r	23 979 385	CCTTCATAACCAGCTAAAAGGC
Vu1-12f	26 295 823	GCCCAACCGTGTTAAGTCTAAT	Vu1-12r	26 295 845	GAGGTTTCTAAGCACATGAGGT
Vu1-13f	28 949 546	ACATGCTGCATTCTCTACCATT	Vu1-13r	28 949 568	TCACGACGAGAACCATATTTGA
Vu1-17f	41 148 093	AGAAATTGGAGGAAAGGTGAGC	Vu1-17r	41 148 115	GACGATGATGGTGATGATGACA
Vu2-19f	18 161 417	AAGGTGGCGTGAATAATGTCTT	Vu2-19r	18 161 439	TTATCCAGAACTCCTTCAAGCC
Vu2-20f	19 179 823	ATCTTCATTGTCCATTCCTGCA	Vu2-20r	19 179 845	TTGGGTTGATCTTGTATGCCAA
Vu2-22f	22 321 069	CGTGAAGCTCAATCGAATCTTT	Vu2-22r	22 321 091	TCCAATCCATCATTCATTACGC
Vu2-25f	26 891 508	AAAGCATGTCATTAGCAGCATC	Vu2-25r	26 891 530	TGAAGAGCTTGAACGTGAGAAT
Vu2-28f	30 986 485	TTTCACGTTGGATCAAGCAAAC	Vu2-28r	30 986 507	TGTCACCAGAAAAGGCCATAAT
Vu2-29f	32 863 650	TTAGATTGGACAGTGAAGCTCG	Vu2-29r	32 863 672	TGATGCTGTTTGGATTGTGGG
Vu3-32f	5 016 424	TTCGTTTCAAACCTCCACTTCT	Vu3-32r	5 016 446	CTGTCTACCTGCAGTCGATTAG
Vu3-37f	23 098 591	CATGTCCACCCTGAAGCTATAG	Vu3-37r	23 098 613	AGAAAAGCAATACAAACGTGGC
Vu3-38f	25 801 706	CTGCTCTCAACCTTATCCCAAA	Vu3-38r	25 801 728	CTCTTGCAGTCCCTATTGTCAA
Vu3-44f	39 770 758	ATTTCAATAGTTCTGGCGAGCA	Vu3-44r	39 770 780	CACAAAGTGGCATATCTGGTTT
Vu3-45f	42 848 262	TATCCATGGGTTCAAATCGGAG	Vu3-45r	42 848 284	AGAACTGGCGAGAAGAAATGAT
Vu3-46f	43 685 697	CAGGCTTTGGTCATTGGATTTT	Vu3-46r	43 685 719	AAGAAAGAGACCTGAAGTGCTC
Vu3-52f	65 121 995	ATTGGGTGTGACATTCTTTACG	Vu3-52r	65 122 017	AACCTAATCGCACTGAACCGS
Vu4-53f	4 405 112	TGCAGCATTGAAGAATCTCCAA	Vu4-53r	4 405 134	GTGCAAGAATATGTGTTCCAGC
Vu4-54f	4 703 051	CTGGAATTGCAGTCATCTATCCT	Vu4-54r	4 703 074	AATTCCATTCCCGTGTTCTGAA
Vu4-55f	7 730 103	GGATAGCCAAAACCATAGTAGCT	Vu4-55r	7 730 126	CAAACTGTGGTGGTGAATATGA
Vu4-56f	9 241 355	GTGGTTATCTTGCTCTAGGTCC	Vu4-56r	9 241 377	TCTAAAGGGCACGATCAACATT
Vu4-57f	11 119 748	CTCATATATTTGTAGGGCCGGG	Vu4-57r	11 119 770	TGGGACCTTCAAGTGCAATTAA
Vu4-59f	14 278 136	AGGTCAAGTACATGTTGCATGA	Vu4-59r	14 278 158	GCACTGTTTTGGTTCTTAGCAT
Vu4-60f	15 050 272	GGGAGTCATTGCTGTTAGAGTT	Vu4-60r	15 050 294	GAAAAGGGTATGCATGAACAAC
Vu4-62f	16 066 798	ATTTTGGAGTGTATCAGTGGGG	Vu4-62r	16 066 820	ACTGAAAATCGCACTGGAAAAC
Vu4-63f	16 457 840	GGGTCCTTTTCTATTGCTCAGT	Vu4-63r	16 457 862	TCCTGAAACTGTCTTTCCCATC
Vu4-64f	18 682 066	GGGAACAAAAGATGCAAGTGAG	Vu4-64r	18 682 088	CAAAATTTGGGTTGTAGTCAGC
Vu4-65f	19 635 565	TTAACAAACCCATGAAACAGGC	Vu4-65r	19 635 587	GATATGGTTCTGTTGGTGGGAG
Vu4-70f	37 023 441	GGAGTTGAGACAGTAACATGGA	Vu4-70r	37 023 463	GGGTTTGATTCACTTTTGTCCC
Vu4-71f	40 172 227	TCACAACAGACAAACTCTACGT	Vu4-71r	40 172 249	GCATGCCACCATAATCACAAAT
Vu5-72f	879 582	GCCCCTCGATATTCTCAGATTC	Vu5-72r	879 604	AGTTTTGGCTAGCAGGTATTGT
Vu5-73f	2 307 937	TGTTCTTATGCATGGGTCAGTT	Vu5-73r	2 307 959	GCTCAAACAAGTGCAAGATCAT
Vu5-74f	4 191 243	CGAAGAAAGACAGTTGTTTGCA	Vu5-74r	4 191 265	CTGTTGATAGAGTGTGAGTCCC
Vu5-77f	19 695 657	CTTCACTGCTCTCCTTGCTATT	Vu5-77r	19 695 679	CTTGAAGTTGTGAGTGGACAGA
Vu5-78f	24 528 035	TCCCTGAGCATCCATTATTTCC	Vu5-78r	24 528 057	GTCATTCGGTCCATTTCACAAG
Vu5-80f	31 810 455	GTTGAAAGTGTATTGTTCTGCG	Vu5-80r	31 810 477	GGGACCACAGTAAAATCATGAC
Vu5-81f	34 174 933	ACATCGTGGTGCTCTTTCATTA	Vu5-81r	34 174 955	TGACTGGAAACCTTCTAACTGG
Vu5-82f	41 723 563	AGTTATTGGAGAACATGCCTCT	Vu5-82r	41 723 585	GGCGTTTAAGATTTAGTCGACA
Vu5-84f	46 562 084	CGGACAAAAGAAATAACCTGCA	Vu5-84r	46 562 106	TGTTCTCCAATAGTTCCTCACG
Vu5-85f	48 539 129	ACTTTCACCACTTACTCTCACT	Vu5-85r	48 539 151	CACATGTAAACAAGTAGTAGACGC
Vu6-87f	2 458 695	CCTACCCAGCCAATTTTGTCTA	Vu6-87r	2 458 717	CTGAATTCTCCAGGACTGATTC
Vu6-88f	4 157 381	CTGTACCAATCCACATGAACCT	Vu6-88r	4 157 403	CTGAATTGAACGAAGAAGTGCG
Vu6-90f	13 739 218	GCTTCCTAAGATTGCAACCTTG	Vu6-90r	13 739 240	AAACTCTAAGACCCAACCTGTC
Vu6-94f	19 440 325	AAGGTTTAGTTTAGGCTGTCCC	Vu6-94r	19 440 347	TGGAAAATGTGCACAACTAGGA
Vu6-95f	20 702 486	TTGTGTATTGGGTAAGTGCCAA	Vu6-95r	20 702 508	TCTTCCTCAGTCCCAATACTCA
Vu6-97f	23 626 647	ATTTTCTTCTTGGCTGCTGTTC	Vu6-97r	23 626 669	CTCTCTCCCACTCCTTTCAATG
Vu6-99f	28 111 300	CACTCCCAAGGAAGATGAGATC	Vu6-99r	28 111 322	GTTCTCAGGGGAAAAGGTGATT
Vu6-100f	30 559 787	ACATCTATCGCCAATCTCACAG	Vu6-100r	30 559 809	CAAATGAAGTGGGGAGGAAGAA
Vu6-101f	31 914 466	CACTTGAAGACGCTAAGAACAC	Vu6-101r	31 914 488	ACCTCCAACAAGTTCATGATCA
Vu6-102f	32 539 051	TATTGGAGAAAGGGCATAGCAG	Vu6-102r	32 539 073	CTTTTCACCGGTATCCCTTCTT
Vu7-105f	13 121 356	TCCTCTACCTTTCACCAAGCTA	Vu7-105r	13 121 378	ATGGTATGTCTTGTGATGGCAA
Vu7-106f	23 354 328	GATAGCTCACAACACAGACACT	Vu7-106r	23 354 350	TGTATCGGTACTCACATGCTTC
Vu7-108f	29 923 695	CACTTAGCATCTGATGGCCTTA	Vu7-108r	29 923 717	ACTGGGGTATTTCAAAGTGAGG
Vu7-109f	31 969 597	AAAGGGGTCACTCATACACATG	Vu7-109r	31 969 619	GACACCACTTTAACCCAAAACC
Vu7-110f	35 390 277	TGTGGTAGCTCAGTGTTAGTTG	Vu7-110r	35 390 299	AATATAACATAGGCCGGCTGAC
Vu8-113f	6 256 060	TGCGTGTAAGTGTCAGAGAAAT	Vu8-113r	6 256 082	AAGTGTGGTGAGGAAAGGTATG
Vu8-115f	10 801 610	TTGCTTAACCTGAGATGAACGT	Vu8-115r	10 801 632	ACGGGAGGTTATAGGCTATTTG
Vu8-116f	21 415 000	GACACAAGATCGAATCCAAGGA	Vu8-116r	21 415 022	GCACTGCATCTTTCACTTTCAT
Vu9-122f	1 063 922	CCTGGTACTTGGACTCAGATTT	Vu9-122r	1 063 944	CAGCTGTTGATGTTGAACCAAA
Vu9-123f	2 491 158	TTGTGTTAAAGGTGGTGCATTG	Vu9-123r	2 491 180	AGGGGTAAAGCAAGCAACATAT
Vu9-124f	4 327 558	GTCTCTATGGCGGTCAAGTTTA	Vu9-124r	4 327 580	CTTCATGATCTATCTGCCCCAG
Vu9-125f	5 918 421	GTATCGGATGCTGTTATTTGGG	Vu9-125r	5 918 443	GTGGAATTTCTATTTTGCCTGC
Vu9-126f	7 169 197	GATCATTGAACTCCCTTGGTCA	Vu9-126r	7 169 219	GTTAGCAACAGCAACCGATAAG
Vu9-127f	9 039 864	GCAGTGACCTCTCAATTCATCT	Vu9-127r	9 039 886	CCCTGCATCTCCATTTTGATTT
Vu9-129f	15 935 910	ACGATTCTACTGCTTAGCCATC	Vu9-129r	15 935 932	GCGAGAATTAAAGGAAACGTCA
Vu9-131f	22 071 671	CCCCGAATAAGTGTTCTAGGTG	Vu9-131r	22 071 693	CAAAACATGGCCATACATCTCG
Vu9-135f	33 519 683	CACTGCAACAATTATACGCCTT	Vu9-135r	33 519 705	TGTCACAGCTCCAAAGAATGAT
Vu9-136f	34 865 479	AAAGTTGTAGGCTAAGGTGGAC	Vu9-136r	34 865 501	CACAGATAACGAGGAGATAGGC
Vu9-138f	40 694 878	AAACCCACTCATGTACAAGACT	Vu9-138r	40 694 900	TGGTGTTCATCAAGATTTCACG
Vu10-141f	5 081 721	GCGATTGATTTAAGGCACAGTA	Vu10-141r	5 081 743	AAGTCAGGGGTTTTGATCAAGT
Vu10-142f	7 985 569	AGTCACAGTTTCAGTCACCAAA	Vu10-142r	7 985 591	ACGGAGATCAGTCACTATAACC
Vu10-144f	12 679 952	AGGGCTTCAATTTTCTCCTTCA	Vu10-144r	12 679 974	GAATTTGGAAGGCTGTTGTTGT
Vu10-145f	15 994 371	CCGGAAAACTCACAACATCATT	Vu10-145r	15 994 393	CCAACTTTTGAATGCTTGACCA
Vu10-146f	17 013 915	ACTGGTCTTCATTTTGTGCTGA	Vu10-146r	17 013 937	AGGCTTCATTGGGTAGTGTTAC
Vu10-147f	23 281 009	ACTTCATCAATGTCCACCTCTT	Vu10-147r	23 281 031	CGCAACGGATAGGAAATGGATA
Vu10-149f	28 197 383	TGTTTCTTGCGCTGACATAGTA	Vu10-149r	28 197 405	TTAGCAGTTGTCGAGTCTCTTC
Vu10-152f	36 129 524	AGTAAGCACAGTCTTAGAGCAA	Vu10-152r	36 129 546	TAGGCAATTCACAGTCTCTTGG
Vu10-154f	39 650 768	GACAGAAGAGGGGTTCAGAATC	Vu10-154r	39 650 790	AGTTCAAGTACATGCTCCAACA
Vu11-157f	10 620 552	CATTTCGCTCCCTGAAGTTTC	Vu11-157r	10 620 573	TGGTTTAGAAAGAGGTTACGTG
Vu11-160f	24 401 745	CCAACACGCTTTCATGATTCAA	Vu11-160r	24 401 767	GGTCTCGTGCAAAAGTAATGTC
Vu11-161f	28 839 447	AGTATCCAACAACCAACCATCA	Vu11-161r	28 839 469	GCGTGGAAATATGTATCGATCG
Vu11-162f	32 266 203	GGTCACGTCCAGAGATAATTACT	Vu11-162r	32 266 226	CACTTGCCAATATTCACAGCA