桃若干重要性状的KASP分子标记开发与应用

doi:10.3864/j.issn.0578-1752.2021.15.013

摘要/Abstract

摘要：

【目的】开发一系列高通量、低成本的桃重要性状竞争性等位基因特异PCR（Kompetitive Allele Specific PCR,KASP）标记,包括果皮有毛/无毛、果形扁平/圆形、果肉硬质/非硬质、DBF（Dominant Blood Flesh）红肉/非红肉、抗/感蚜等5对性状,加速桃优良品种培育,缩短桃育种年限。【方法】本研究在控制这些性状的候选基因及位点附近300 kb内,利用已有桃种质资源的基因组序列比对,开发KASP标记,对已知表型的桃种质材料进行基因分型验证,最终获得与目标性状紧密连锁的KASP标记。【结果】利用开发的5个性状的KASP分子标记对桃杂交分离群体和自然群体进行基因型检测,结果表明,标记鉴定结果与已知表型完全一致,准确率为100%。其中,杂交群体中果皮有毛/无毛性状分离比例为30﹕30,果形扁平/圆形分离比例为31﹕29,果实硬质/非硬质分离比例为27﹕26,抗蚜/感蚜分离比例为49﹕46,均符合孟德尔遗传1﹕1的分离定律。【结论】开发的KASP标记可高效检测桃果实外观、抗性、肉质等重要性状相关基因的等位变异,在基因型鉴定、亲本选配和杂种后代的分子标记辅助选择中有很好的应用前景。

关键词: 桃, KASP, 分子标记辅助选择, 育种

Abstract:

【Objective】 In order to improve breeding efficiency based on the marker-assisted selection in peach, a series of high-throughput and low-cost KASP markers for important traits (such as fruit skin fuzz, fruit shape, stony hard fruit texture, DBF blood-flesh and resistance to green peach aphids) of peach were developed. 【Method】 Based on the resequenced genome information of some peach cultivars, multiple sequence alignment was used to find specific SNP within 300 kb of both flanks of target genes or loci for related traits. KASP markers were developed according to the differences of SNPs, and the close link between KASP markers and the target characteristics was confirmed by genotyping.【Result】 The developed KASP molecular markers for five traits were utilized to detect the genotypes of peach hybrid populations and natural populations. The results showed that the markers identification was completely consistent with the known phenotypes, and the accuracy was 100%. In the hybrid populations of CN20 × 01-77-4, the segregation ratio of peach peel with fuzz/without fuzz, flat fruit/round fruit, and the segregation ratio of resistance to green peach aphids/susceptibility was 30﹕30, 31﹕29, and 49﹕46, respectively, which were in accordance with the 1﹕1 of Mendel’s first law. In population of CN20 × 10 xin 25-8, the high-throughput KASP molecular marker was used to detect genotypes of 53 offspring, and the results showed that genotypes of 27 were G/G appeared stony hard peach and 21 were T/C appeared DBF blood-flesh fruit, which were consistent with the phenotype identification results.【Conclusion】 The KASP marker could be efficiently used to detect allelic variations of genes related to important traits, such as peach fruit appearance, resistance, and fruit texture. It had a good application prospect in genotypic identification, parents apolegamy and hybrid progenies marker-assisted selection.

Key words: peach, KASP, marker-assisted selection, breeding

孟君仁,曾文芳,邓丽,潘磊,鲁振华,崔国朝,王志强,牛良. 桃若干重要性状的KASP分子标记开发与应用[J]. 中国农业科学, 2021, 54(15): 3295-3307.

MENG JunRen,ZENG WenFang,DENG Li,PAN Lei,LU ZhenHua,CUI GuoChao,WANG ZhiQiang,NIU Liang. Development and Application of KASP Molecular Markers of Some Important Traits for Peach[J]. Scientia Agricultura Sinica, 2021, 54(15): 3295-3307.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.15.013

https://www.chinaagrisci.com/CN/Y2021/V54/I15/3295

图/表 14

表1

本研究涉及的自然群体材料"

编号 Code	品种（系） Cultivar (line)	果皮茸毛 Peel fuzz	果实形状 Fruit shape	果肉颜色 Flesh color	果实肉质 Fruit flesh texture
1	中油蟠36-3 Zhongyoupan 36-3	无 Without fuzz	扁平 Flat fruit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
2	中油9号 Zhongyou 9	无 Without fuzz	圆形 Round fruit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
3	中油8号 Zhongyou 8	无 Without fuzz	圆形 Round fuit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
4	中油27号 Zhongyou 27	无 Without fuzz	圆形 Round fuit	黄 Yellow	非硬质（不溶质）Non-stony hard (Non-melting flesh)
5	中油20号 Zhongyou 20	无 Without fuzz	圆形 Round fuit	白 White	硬质 Stony hard
6	中油19号 Zhongyou 19	无 Without fuzz	圆形 Round fuit	黄 Yellow	硬质 Stony hard
7	中油18号 Zhongyou 18	无 Without fuzz	圆形 Round fuit	白 White	硬质 Stony hard
8	中桃红玉 Zhongtaohongyu	有 With fuzz	圆形 Round fuit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
9	中桃白玉 Zhongtaobaiyu	有 With fuzz	圆形 Round fuit	白 White	硬质 Stony hard
10	中桃5号 Zhongtao 5	有 With fuzz	圆形 Round fuit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
11	中桃10号 Zhongtao 10	有 With fuzz	圆形 Round fuit	黄 Yellow	硬质 Stony hard
12	中蟠2号 Zhongpan 2	有 With fuzz	扁平 Flat fruit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
13	中蟠1号 Zhongpan 1	有 With fuzz	扁平 Flat fruit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
14	中蟠102 Zhongpan 102	有 With fuzz	扁平Flat fruit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
15	早4号 Zao 4	无 Without fuzz	圆形 Round fuit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
16	双喜红 Shuangxihong	无 Without fuzz	圆形 Round fuit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
17	黄金蜜桃3号 Huangjinmi 3	有 With fuzz	圆形 Round fuit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
18	黄金蜜桃1号 Huangjinmi 1	有 With fuzz	圆形 Round fuit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
19	红芒果 Hongmangguo	无 Without fuzz	圆形 Round fuit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
20	春瑞 Chunrui	有 With fuzz	圆形 Round fuit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
21	春蜜 Chunmi	有 With fuzz	圆形 Round fuit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
22	春美 Chunmei	有 With fuzz	圆形 Round fuit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
23	春丽 Chunli	有 With fuzz	圆形 Round fuit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
24	NJC83	有 With fuzz	圆形 Round fuit	黄 Yellow	非硬质（不溶质）Non-stony hard (Non-melting flesh)
25	F4	有 With fuzz	圆形 Round fuit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
26	F3	无 Without fuzz	圆形 Round fuit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
27	F2	无 Without fuzz	圆形 Round fuit	黄 Yellow	非硬质（溶质）Non-stony hard (Melting flesh)
28	96-5-1	有 With fuzz	圆形 Round fuit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)
29	09南9-22 09nan9-22	有 With fuzz	圆形 Round fuit	白 White	硬质 Stony hard
30	05-1-135	无 Without fuzz	圆形 Round fuit	白 White	非硬质（溶质）Non-stony hard (Melting flesh)

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杂交组合 Cross	群体大小 Size of population	亲本基因型 Parental genotype
组合1：中油20号×01-77-4 Cross 1: Zhongyou 20×01-77-4	95	gg×Gg;ss×Ss;rr×Rr
组合2：中油20号×10新25-8 Cross 2: Zhongyou 20×10xin25-8	53	dfdf×DBFdf;hdhd×Hdhd

等位变异位点 Allelic variation sites	SNP在染色体上的位置 SNP location on chromosome	KASP引物及序列 KASP primer and sequence
果皮有毛/无毛位点 Peel with/without fuzz site	Chr05: 17571609	6-羧基荧光素 FAM: 5′-GATACTGACTTTCACAGCAACTTCGT-3′ 六氯-6-甲基荧光素 HEX: 5′-ATACTGACTTTCACAGCAACTTCGC-3′ 通用引物 Common: 5′-CTTACGACAACAAGAGTGTATGATCTGAT-3′
果形扁平/圆形位点 Fruit flat/round shape site	Chr06: 26924482	6-羧基荧光素 FAM: 5′-GTTTTACATTTGAGGAATTAAATTATAAGCATT-3′ 六氯-6-甲基荧光素 HEX: 5′-GTTTTACATTTGAGGAATTAAATTATAAGCATA-3′ 通用引物 Common: 5′-CCTCACTAAGAACAGGCAAGGACTT-3′
果肉硬质/非硬质位点 Fruit stony hard/non flesh site	Chr06: 14090769	6-羧基荧光素 FAM: 5′-ACTCTAGAAAGCATGTATTCACGACT-3′ 六氯-6-甲基荧光素 HEX: 5′-CTCTAGAAAGCATGTATTCACGACG-3′ 通用引物 Common: 5′-CCACTGAAGGTAATAAAGCCCCACTA-3′
DBF型果肉颜色红/非红位点 DBF flesh color red/non site	Chr05: 689647	6-羧基荧光素 FAM: 5′-GAGAAGGTGAACACAAGAAAGCCT-3′ 六氯-6-甲基荧光素 HEX: 5′-AGAAGGTGAACACAAGAAAGCCC-3′ 通用引物 Common: 5′-GAGGTGGGATAAGATACAGCTTCCAA-3′
寿星桃Rm3类型的抗蚜/感蚜位点 Rm3 Shouxing peach resistance to green peach aphid/susceptibility site	Chr06: 45741254	6-羧基荧光素 FAM: 5′-GGCTTTCCCAGGTAAAGTCCCT-3′ 六氯-6-甲基荧光素 HEX: 5′-GGCTTTCCCAGGTAAAGTCCCA-3′ 通用引物 Common: 5′-CAGCATGCCGTTGGCGCTTCTT-3′

群体 Population	表型 Phenotype	数量 No.	杂种后代/桃品种 Hybrids/Cultivar (line)	基因分型 Genotype	符合率 Coincidence rate (%)
组合1 Cross 1	有毛 With fuzz	30	51-34、51-36、51-37、51-38、51-39、51-52、51-55、51-56、51-57、51-59、51-62、51-63、51-65、51-66、51-69、51-72、51-74、51-78、51-80、51-84、51-87、51-89、51-92、51-93、51-95、51-96、52-1、52-2	Allele X and both alleles	100
组合1 Cross 1	无毛 Without fuzz	30	51-33、51-35、51-40、51-42、51-44、51-45、51-46、51-47、51-48、51-49、51-50、51-60、51-64、51-67、51-68、51-71、51-73、51-75、51-76、51-79、51-82、51-83、51-85、51-86、51-90、51-91、51-94、51-97、52-3	Allele Y	100
自然群体 Natural population	有毛 With fuzz	17	春美 Chunmei、黄金蜜桃3号 Huangjinmi 3、中蟠102 Zhongpan 102、黄金蜜桃1号 Huangjinmi 1、中桃10号Zhongtao 10、中蟠1号 Zhongpan 1、中蟠2号 Zhongpan 2、96-5-1、09南9-22 09nan9-22、中桃白玉 Zhongtaobaiyu、春瑞 Chunrui、NJC83、中桃5号 Zhongtao 5、F4、春丽 Chunli、春蜜 Chunmi、中桃红玉Zhongtaohongyu	Allele X and both alleles	100
自然群体 Natural population	无毛 Without fuzz	13	中油20号 Zhongyou 20、中油19号 Zhongyou 19、红芒果 Hongmangguo、中油蟠36-3 Zhongyoupan 36-3、中油8号 Zhongyou 8、中油27号 Zhongyou 27、双喜红 Shuangxihong、早4号 Zao 4、中油18号 Zhongyou 18、05-1-135、中油9号 Zhongyou 9、F2、F3	Allele Y	100

群体 Population	表型 Phenotype	数量 No.	杂种后代/桃品种 Hybrids/Cultivar (line)	基因分型 Genotype	符合率 Coincidence rate (%)
组合1 Cross 1	圆桃 Round	29	51-37、51-38、51-40、51-42、51-44、51-45、51-46、51-48、51-50、51-63、51-64、51-68、51-69、51-76、51-77、51-78、51-82、51-84、51-85、51-89、51-94、51-97、52-3、51-62、51-66、51-74、51-79、51-90、51-91	Allele X	100
组合1 Cross 1	蟠桃 Flat	31	51-33、51-34、51-35、51-36、51-39、51-47、51-49、51-52、51-54、51-55、51-56、51-57、51-59、51-60、51-65、51-67、51-71、51-72、51-73、51-75、51-80、51-81、51-83、51-86、51-87、51-92、51-93、51-95、51-96、52-1、52-2	Both alleles	100
自然群体 Natural population	圆桃 Round	27	中油20号 Zhongyou 20、中油19号 Zhongyou 19、春美Chunmei、黄金蜜桃3号 Huangjinmi 3、红芒果 Hongmangguo、霞脆 Xiacui、有名 Youming、黄金蜜桃1号 Huangjinmitao 1、中桃10号 Zhongtao 10、96-5-1、中油8号 Zhongyou 8、09南9-22 09nan9-22、中油27号 Zhongyou 27、双喜红 Shuanghongxi、白如玉 Bairuyu、春瑞 Chunrui、NJC83、早4号 Zao 4、中油18号 Zhongyou 18、05-1-135、中桃5号 Zhongtao 5、中油9号 Zhongyou 9、F1、F2、F3、春蜜Chunmi、中桃红玉 Zhongtaohongyu	Allele X	100
自然群体 Natural population	蟠桃 Flat	3	中油蟠36-3 Zhongyoupan 36-3、中蟠2号 Zhongpan 2、中蟠1号 Zhongpan 1	Both alleles	100

群体 Population	表型 Phenotype	数量 No.	杂种后代/桃品种 Hybrids/Cultivar (line)	基因分型 Genotype	符合率 Coincidence rate (%)
组合2 Cross 2	硬质桃 Stony hard	27	15-16、15-19、15-21、15-22、15-23、15-24、15-27、15-29、15-32、15-34、15-36、15-37、15-38、15-39、15-40、15-42、15-43、15-44、15-48、15-51、15-52、15-53、15-54、15-58、15-59、15-60、15-66	Allele Y	100
组合2 Cross 2	非硬质桃 Non-stony hard	26	15-15、15-17、15-18、15-20、15-25、15-26、15-28、15-30、15-31、15-33、15-35、15-41、15-45、15-46、15-47、15-49、15-50、15-55、15-56、15-57、15-61、15-62、15-63、15-64、15-65、15-67	Both alleles	100
自然群体 Natural population	硬质桃 Stony hard	5	09南9-22 09nan9-22、中桃10号 Zhongtao 10、中油19号 Zhongyou 19、中油18号 Zhongyou 18、白如玉 Bairuyu	Allele Y	100
自然群体 Natural population	非硬质桃 Non-stony hard	17	春瑞 Chunrui、中桃红玉 Zhongtaohongyu、双喜红 Shuangxihong、红不软 Hongburuan、96-5-1、春美 Chunmei、中蟠1号 Zhongpan 1、NJC83、中油9号 Zhongyou 9、中油8号 Zhongyou 8、05-135、黄金蜜桃3号 Huangjinmi 3、大久保 Dajiubao、中油20号 Zhongyou 20、中油蟠36-3 Zhongyoupan 36-3、中桃5号 Zhongtao 5、红芒果 Hongmangguo	Allele X and both alleles	100