野生二倍体草莓的SSR分子标记开发及核心种质的构建

doi:10.16420/j.issn.0513-353x.2022-0985

摘要/Abstract

摘要：

用MISA程序搜索森林草莓（Fragaria vesca L.）基因组获得106 829个SSR位点，比对森林草莓等8种二倍体草莓的基因组获得唯一且共同的SSR序列5 778个，其中单、二、三、四、五、六核苷酸类型以及复杂重复类型分别为3 461、1 223、999、57、7、5、26。在26个复杂重复类型中，有18个SSR位点在森林草莓、中国草莓（F. chinensis Lozinsk）、东北草莓（F. mandshurica Staudt）、黄毛草莓（F. nilgerrensis Schlecht.）、西藏草莓（F. nubicola Lindl.）、峨眉草莓（F. emeiensis Jia J. Lei）、五叶草莓（F. pentaphylla Lozinsk）和绿色草莓（F. viridis Duch.）等8个二倍体种的176份资源重测序数据中具有较高的覆盖度，通过检测获得了444个等位基因，平均有效等位基因数为7.899，Shannon信息指数在0.836 ~ 3.362之间，观察杂合度在0.006 ~ 0.477之间，期望杂合度在0.324 ~ 0.953之间，多态信息含量（PIC）介于0.315 ~ 0.951，平均值为0.756；利用选择的18对SSR标记构建野生二倍体草莓的核心种质，并利用t检验对核心种质进行评价，筛选出的43份材料保存了80%以上的遗传多样性，代表性较高，确定为野生二倍体草莓的核心种质。同时构建了每份材料的分子身份证并生成二维码。本研究中开发的多态性SSR位点以及筛选出的核心种质能够为今后野生草莓物种的鉴别、分子标记辅助育种等提供工具。

关键词: 草莓, 二倍体种, SSR, 多态性分析, 核心种质

Abstract:

Core collection can reduce the number of resources and maximize the genetic diversity of the population. The 106 829 SSR loci were obtained by searching the genome of Fragaria vesca L. with MISA program，and 5 778 unique and common SSR sequences were obtained by comparing the genome of eight diploid strawberries such as F. vesca L. Among the 26 complex repeats，18 SSR loci were found in F. vesca L，F. chinensis Lozinsk，F. mandshurica Staudt，F. nilgerrensis Schlecht.，F. nubicola Lindl.，F. emeiensis Jia J. Lei，F. pentaphylla Lozinsk and F. viridis Duch. Among them，the single，two，three，four，five，six nucleotide types and complex repeat types were 3 461，1 223，999，57，7，5，26，respectively. Among 26 complex repeat types，18 SSR loci have high coverage in 176 diploid strawberry re-sequencing data，which are used for construction of core collection. The results showed that 444 alleles were obtained from 176 accessions of wild diploid strawberry with 18 pairs of conservative sequence，the average number of effective alleles was 7.899，the Shannon's information index was 0.836-3.362，the observed heterozygosity was 0.006-0.477，the expected heterozygosity was 0.324-0.953，the PIC value was 0.315-0.951，and the average value was 0.756. The core collection of wild diploid strawberry was constructed with 18 pairs of SSR primers selected，and the core collection was evaluated by t-test. The fourty-three accessions were selected，which preserved more than 80% of genetic diversity and were highly representative. They were identified as the core collection of wild diploid strawberry，and at the same time，the molecular ID of two-dimensional code of core collection was constructed. The polymorphic SSR loci developed in this study can provide research tools for identification and molecular marker breeding of wild strawberry in the future.

Key words: strawberry, diploid species, SSR, polymorphic analysis, core collection

张咏琦, 王超, 许林林, 吴娥娇, 李天红, 赵密珍, 袁华招. 野生二倍体草莓的SSR分子标记开发及核心种质的构建[J]. 园艺学报, 2023, 50(11): 2365-2375.

ZHANG Yongqi, WANG Chao, XU Linlin, WU E’jiao, LI Tianhong, ZHAO Mizhen, YUAN Huazhao. Development of SSR Molecular Markers and Construction of Core Collection of Wild Diploid Strawberry[J]. Acta Horticulturae Sinica, 2023, 50(11): 2365-2375.

图/表 8

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位点 Loci	上游保守序列（5′-3′） Upstream conservative sequence	下游保守序列（5′-3′） Downstream conservative sequence
SSR1	TGCTAAGTAGAAGCCGCCA	AAAGCCATTTTACTACCTAGCC
SSR4	TTAACAAGCACACGACAAAG	CATGGGTCTTGCTTATAGCC
SSR6	TCCTCCATTATCTGCAGAAATC	TTCCAACCTCAAGCGTCT
SSR7	CAAAGAGGGTTTCATTCATG	CGAACCCATGCACAC
SSR9	AGACTTGTCTGGCCATTGTA	CACAGTACATCTCGCAAGGC
SSR10	GTGACTTGATGTTGCTGTAAGT	CATACTACTGGAGATGCTC
SSR11	GGTAATTTTCTTAAGTGACGCC	ATCGAACCAGCAGGCCT
SSR13	CACCAATTTCACCACATATC	TTCTGTGAGTCTTGAATGTTGC
SSR17	CCTCTCTTGTCCGGAACTT	CAGCAACTCCAAAGTTAAA
SSR18	ATGCAATGTGTGTAAACACA	CACGTGCGTTTGTCAAC
SSR19	TGTGCATTACTTGCTAATCC	CATCCTTTTGCTTTTGACCA
SSR20	AGGCCCATTTCACTTCTGTC	TCACCCAACCCGGAAA
SSR21	TATCTAGTAGTTGGAAAAGGA	TGTGGCATGGAAGTGG
SSR22	TGCATCATGCATTCAAT	GGAAGCTGCATTTCCATAGAA
SSR23	AATGTATATGTAGAATCC	GTCAATTGCCCATAGGTTAGC
SSR24	TGATCGAGTTCAATTGAGTCAC	GGATCGAGGACCTAATCCC
SSR25	CCCACACAAATTTAAAAGAGA	AGACTGAAATGGCCATGG
SSR26	GATTGCTCAAAGTGCTTTAC	GCCTATAATTGCTGTCATGCTAG

位点 Loci	上游保守序列（5′-3′） Upstream conservative sequence	下游保守序列（5′-3′） Downstream conservative sequence
SSR1	TGCTAAGTAGAAGCCGCCA	AAAGCCATTTTACTACCTAGCC
SSR4	TTAACAAGCACACGACAAAG	CATGGGTCTTGCTTATAGCC
SSR6	TCCTCCATTATCTGCAGAAATC	TTCCAACCTCAAGCGTCT
SSR7	CAAAGAGGGTTTCATTCATG	CGAACCCATGCACAC
SSR9	AGACTTGTCTGGCCATTGTA	CACAGTACATCTCGCAAGGC
SSR10	GTGACTTGATGTTGCTGTAAGT	CATACTACTGGAGATGCTC
SSR11	GGTAATTTTCTTAAGTGACGCC	ATCGAACCAGCAGGCCT
SSR13	CACCAATTTCACCACATATC	TTCTGTGAGTCTTGAATGTTGC
SSR17	CCTCTCTTGTCCGGAACTT	CAGCAACTCCAAAGTTAAA
SSR18	ATGCAATGTGTGTAAACACA	CACGTGCGTTTGTCAAC
SSR19	TGTGCATTACTTGCTAATCC	CATCCTTTTGCTTTTGACCA
SSR20	AGGCCCATTTCACTTCTGTC	TCACCCAACCCGGAAA
SSR21	TATCTAGTAGTTGGAAAAGGA	TGTGGCATGGAAGTGG
SSR22	TGCATCATGCATTCAAT	GGAAGCTGCATTTCCATAGAA
SSR23	AATGTATATGTAGAATCC	GTCAATTGCCCATAGGTTAGC
SSR24	TGATCGAGTTCAATTGAGTCAC	GGATCGAGGACCTAATCCC
SSR25	CCCACACAAATTTAAAAGAGA	AGACTGAAATGGCCATGG
SSR26	GATTGCTCAAAGTGCTTTAC	GCCTATAATTGCTGTCATGCTAG

位点Loci	N_a	N_e	I	H_o	H_e	PIC
SSR1	27.000	9.435	2.678	0.188	0.894	0.887
SSR4	13.000	1.479	0.836	0.006	0.324	0.315
SSR6	27.000	8.005	2.593	0.443	0.875	0.867
SSR7	24.000	5.728	2.378	0.188	0.825	0.816
SSR9	36.000	13.120	2.981	0.216	0.924	0.920
SSR10	29.000	10.574	2.704	0.352	0.905	0.898
SSR11	16.000	1.751	1.129	0.028	0.429	0.419
SSR13	7.000	1.687	0.911	0.051	0.407	0.387
SSR17	34.000	15.419	3.000	0.477	0.935	0.932
SSR18	18.000	2.448	1.472	0.119	0.591	0.569
SSR19	11.000	2.886	1.560	0.119	0.653	0.632
SSR20	49.000	21.158	3.362	0.432	0.953	0.951
SSR21	23.000	8.990	2.526	0.153	0.889	0.879
SSR22	21.000	4.063	2.015	0.222	0.754	0.739
SSR23	25.000	7.088	2.398	0.222	0.859	0.846
SSR24	30.000	14.681	2.934	0.307	0.932	0.928
SSR25	34.000	9.835	2.746	0.347	0.898	0.891
SSR26	20.000	3.827	2.003	0.199	0.739	0.726
平均Mean	24.667	7.899	2.235	0.226	0.766	0.756

位点Loci	N_a	N_e	I	H_o	H_e	PIC
SSR1	27.000	9.435	2.678	0.188	0.894	0.887
SSR4	13.000	1.479	0.836	0.006	0.324	0.315
SSR6	27.000	8.005	2.593	0.443	0.875	0.867
SSR7	24.000	5.728	2.378	0.188	0.825	0.816
SSR9	36.000	13.120	2.981	0.216	0.924	0.920
SSR10	29.000	10.574	2.704	0.352	0.905	0.898
SSR11	16.000	1.751	1.129	0.028	0.429	0.419
SSR13	7.000	1.687	0.911	0.051	0.407	0.387
SSR17	34.000	15.419	3.000	0.477	0.935	0.932
SSR18	18.000	2.448	1.472	0.119	0.591	0.569
SSR19	11.000	2.886	1.560	0.119	0.653	0.632
SSR20	49.000	21.158	3.362	0.432	0.953	0.951
SSR21	23.000	8.990	2.526	0.153	0.889	0.879
SSR22	21.000	4.063	2.015	0.222	0.754	0.739
SSR23	25.000	7.088	2.398	0.222	0.859	0.846
SSR24	30.000	14.681	2.934	0.307	0.932	0.928
SSR25	34.000	9.835	2.746	0.347	0.898	0.891
SSR26	20.000	3.827	2.003	0.199	0.739	0.726
平均Mean	24.667	7.899	2.235	0.226	0.766	0.756

类型 Type	种质数Number of germplasms	N	N_a	N_e	I	H_o	H_e	PIC
原始种质 Original collection	176.000	444	24.667	7.899	2.235	0.226	0.766	0.756
核心种质 Core collection	43.000 （24.43%）	366 （82.43%）	20.333 （82.43%）	10.497 （132.89%）	2.498 （111.77%）	0.260 （115.04%）	0.852 （111.23%）	0.842 （111.40%）
保留种质 Reservation collection	133.000 （75.57%）	370 （83.33%）	20.556 （83.33%）	6.804 （86.14%）	2.043 （91.41%）	0.216 （95.58%）	0.724 （94.52%）	0.713 （84.65%）
t1			0.142	0.177	0.255	0.494	0.125	0.349
t2			0.210	0.545	0.469	0.827	0.572	0.565