基于SSR荧光标记的79份桃种质遗传多样性分析

doi:10.3864/j.issn.0578-1752.2022.15.011

Abstract

Abstract:

【Objective】The aim of this study was to perform genotyping and genetic diversity analysis of 79 germplasms taken from National Fruit Germplasm Nanjing Peach Resource Nursery by using SSR fluorescent-labeled capillary electrophoresis technology, and to screen primers with high polymorphism, which could be used for identification of peach varieties and genetic relationship analysis and also be used for the establishment of molecular marker-assisted selection system.【Method】De novo sequencing was performed on the female parent Zhongyou No. 4, and SSR markers were developed in the whole genome with a physical distance of 1 Mb as the unit. 79 peach varieties in Nanjing were used as materials for PCR amplification, and the amplified products were detected by polyacrylamide gel electrophoresis and screened for markers with clear target bands and abundant polymorphisms. The 5' end of the screened markers were fluorescently modified, and the PCR products were sequenced on the ABI3730XL sequencer to realize the rescreening of the markers. Genmapper 4.0 software was used to count the sequencing results, and Data Formater 2.1 software converted the bp data obtained by statistics into the data format required by Power Marker v3.25. The selected primers were analyzed for polymorphism, and the fluorescent SSR primers with polymorphism information content (PIC) greater than 0.45 were selected as the core primers of 79 germplasm materials. Using Nei's as a parameter, the UPDM clustering method in NTSYSpc 2.1 software was used to analyze the genetic diversity among cultivars. The Structure v2.3.4 software based on Bayesian model was used to analyze the population genetic structure of 79 accessions. 【Result】Based on 79 germplasms, the SSR markers covering the whole gene were screened by polyacrylamide gel electrophoresis, and 207 pairs of primers with good polymorphism were screened out. The 207 pairs of primers were re-screened by SSR fluorescent-labeled capillary electrophoresis technology, and 26 pairs of core primers were finally screened, of which 5 pairs of core primers could completely distinguish 79 varieties. 26 pairs of SSR core primers amplified a total of 174 polymorphic genotypes in 79 peach germplasms, and each pair of primers were amplified 4-13 genotypes, with an average of 6.69 genotypes amplified per pair of primers. The polymorphism information content PIC of each pair of primers was above 0.45. Based on the locus information amplified by 207 pairs of SSR primers in 79 accessions, the genetic relationship map and population genetic structure map of 79 accessions were constructed. 207 pairs of primers divided 79 accessions into 7 groups based on genetic distance and 2 groups based on population structure. The genetic diversity of 79 cultivars was high, and the clustering results of some cultivars were consistent with the pedigree. 【Conclusion】The cluster analysis map and population genetic structure map of 79 materials were constructed, revealing the genetic relationship of 79 materials to a certain extent. The overall results showed that due to the complex genetic background of peach, the cultivars according to a single characteristic or determine the relationship between cultivars could not be classified. The screened 26 pairs of core primers could be used for genome-wide identification of linked traits, identification of peach germplasm resources, identification and protection of new varieties, and construction of molecular marker-assisted breeding systems.

Key words: peach, fluorescent labeling SSR, capillary electrophoresis, variety identification

WANG LuWei,SHEN ZhiJun,LI HeHuan,PAN Lei,NIU Liang,CUI GuoChao,ZENG WenFang,WANG ZhiQiang,LU ZhenHua. Analysis of Genetic Diversity of 79 Cultivars Based on SSR Fluorescence Markers for Peach[J].Scientia Agricultura Sinica, 2022, 55(15): 3002-3017.

Figures/Tables 10

Table 1

79 Cultivars of peach for SSR analysis"

编号 No.	种质材料 Germplasm	亲本 Parentage	来源 Source
1	雨花2号 Yuhua 2	63-9-22（西姆斯×菲利甫）自然实生 63-9-22 (SIMS×Philip) natural reality	中国江苏 Jiangsu, China
2	霞脆 Xiacui	雨花2号×77-1-6[(白花×橘早生)×朝霞] Yuhua 2×77-1-6 [(Baihua ×Tachibana Wase)×Zhaoxia]	中国江苏 Jiangsu, China
3	雨花1号 Yuhua 1	不详 Unknown	中国江苏 Jiangsu, China
4	瑞光18号 Ruiguang 18	丽格兰特×81-25-15（京玉×NJN76） Legrand×81-25-15 (Jingyu×NJN76）	中国北京 Beijing, China
5	瑞光19号 Ruiguang 19	丽格兰特×瑞光15号（京玉×NJN76） Legrand×Ruiguang No. 15 (Jingyu× NJN76）	中国北京 Beijing, China
6	早美 Zaomei	庆丰×朝霞 Qingfeng×Zhaoxia	中国北京 Beijing, China
7	红粉佳人 Pinklady	Rutgers Redleaf×白寿 Rutgers Redleaf×Baishou	中国江苏 Jiangsu, China
8	瑞光美玉 Ruiguangmeiyu	京玉×瑞光7号 Jingyu×Ruiguang 7	中国北京 Beijing, China
9	早花露 Zaohualu	雨花露自然实生 Yuhualu natural birth	中国江苏 Jiangsu, China
10	大久保 Okubo	白桃园中发现的实生后代 The postnatal offspring found in the white peach garden	日本 Japan
11	金陵黄露 Jilinghuanglu	99-8-3×Spring baby	中国江苏 Jiangsu, China
12	沪油002号 Huyou 002	瑞光3号×五月火 Ruiguang 3×May fire	中国上海 Shanghai, China
13	锦绣 Jinxiu	白花×云署1号 White flower×Yunshu No.1	中国上海 Shanghai, China
14	金童7号 Babygold 7	[Lemon Free×P.I.35201(云南桃实生 Yunnan peach seed）]×NJ196 (J.H.Hale×Goldfinch)	美国 US
15	沪油003号 Huyou 003	不详 Unknown	中国上海 Shanghai, China
16	玉霞蟠桃 Yuxiapantao	瑞蟠4号×瑞光18号 Ruipan 4×Ruiguang 18	中国江苏 Jiangsu, China
17	五月火 Mayfire	阿姆肯自然实生 Amken natural birth	美国 US
18	早凤玉 Zaofengyu	早凤桃芽变选育而成 Zaofeng peach was bred by bud mutation	中国北京 Beijing, China
19	沪021号 Hu 021	不详 Unknown	中国上海 Shanghai, China
20	日川白凤 Richuanbaifeng	白桃×橘早生 White peach×Tachibana Wase	日本 Japan
21	瑞光7号 Ruiguang 7	京玉×B7R2T129 Jingyu×B7R2T129	中国北京 Beijing, China
22	瑞光22号 Ruiguang 22	丽格兰特×82-48-12 Legrand×82-48-12	中国北京 Beijing, China
23	阿姆肯 Armking	Palomar×Springtime (春时 Chunshi)	美国 US
24	霞晖22号 Xiahui 22	朝晖×朝霞 Zhaohui×Zhaoxia	中国江苏 Jiangsu, China
25	瑞蟠3号 Ruipan 3	大久保×陈圃蟠桃 Okubo×Chenpu flat peach	中国北京 Beijing, China
26	早金露 Zaojinlu	不详 Unknown	未知 Unknown
27	瑞蟠5号 Ruipan 5	晚熟大蟠桃×油蟠桃 Late ripening flat peach×Youpantao	中国北京 Beijing, China
28	晚硕蜜 Wanshuomi	肥城桃 Feicheng peach	中国江苏 Jiangsu, China
29	早露蟠桃Zaolupantao	撒花红蟠桃×早香玉 Sahuahongpantao×Zaoxiangyu	中国北京 Beijing, China
30	霞晖8号 Xiahui 8	朝晖×瑞光18号 Zhaohui×Ruiguang 18	中国江苏 Jiangsu, China
31	晚白花 Wanbaihua	不详 Unknown	中国江苏 Jiangsu, China
32	连黄 Lian huang	早生黄金自然实生 Early Gold’s natural growth	中国辽宁 Liaoning, China
33	春花 Chun hua	北农2号×春蕾 Beinong 2×Chunlei	中国上海 Shanghai, China
34	黄露蟠桃Huanglupantao	不详 Unknown	中国浙江 Zhejiang, China
35	金童5号Babygold 5	PI35201（云南桃实生 Yunnan peach seed）×NJ196[NJ76925OP(J.H.Hale×Goldfinch)]	美国 US
36	砂子早生 Sunago Wase	偶发实生（神玉,久保?） Accidental birth (Shenyu, Jiubao?)	日本 Japan
37	早上海水蜜 Zaoshanghaishuimi	上海水蜜早熟变异 Early maturing variation of Chinese Cling	中国上海 Shanghai, China
38	金童9号 Babygold 9	PI35201 (云南桃实生 Yunnan peach seed)×PI43137	美国 US
39	紫金红2号 Zijinhong 2	霞光×早红宝石 Xiaguang × Early Ruby	中国江苏 Jiangsu, China
40	银花露 Yinhualu	白花×初香美 Baihua×Hatsukami	中国江苏 Jiangsu, China
41	弗雷德里克 Frederica	NJC95×D42-13W	美国 US
42	新白凤 Early Hakuho	不详 Unknown	中国江苏 Jiangsu, China
43	奉化玉露早 Fenghuayuluzao	不详 Unknown	未知 Unknown
44	霞晖4号 Xiahui 4	白花 Baihua	中国江苏 Jiangsu, China
45	早硕蜜 Zaoshuomi	白芒蟠桃×朝霞 Baimangpantao×Zhaoxia	中国江苏 Jiangsu, China
46	早魁蜜 Zaokuimi	晚蟠桃×124蟠桃 Wanpantao×124 pantao	中国江苏 Jiangsu, China
47	霞光 Xiaguang	理想 Fantasia	中国山西 Shanxi, China
48	瑞蟠4号 Ruipan 4	晚熟大蟠桃×扬州124蟠桃 Late ripening dapantao×Yangzhou 124 pantao	中国北京 Beijing, China
49	京玉 Jingyu	大久保×兴津油桃 Okubo×Okitsu	中国北京 Beijing, China
50	金霞蟠桃 Jinxiapantao	霞光×NF9260 Xiaguang×NF9260	中国江苏 Jiangsu, China
51	京红 Jinghong	不详 Unknown	中国北京 Beijing, China
52	银河 Galaxy	不详 Unknown	美国 US
53	瑞光28号Ruiguang 28	丽格兰特×瑞光2号（京玉×NJN76） Legrand×Ruiguang 2 (Jingyu×NJN76)	中国北京 Beijing, China
54	瑞蟠2号 Ruipan 2	晚熟大蟠桃×扬州124蟠桃 Late ripening dapantao×Yangzhou 124 pantao	中国北京 Beijing, China
55	金晖 Jinhui	早生黄金×罐桃5号 Early gold×Canned peach 5	中国江苏 Jiangsu, China
56	雨花露 Yuhualu	白花×早上海水蜜 Baihua×Zaoshanghaishuimi	中国江苏 Jiangsu, China
57	白花水蜜 Baihuashuimi	不详 Unknown	中国江苏 Jiangsu, China
58	丰黄 Fenghuang	早生黄金自然实生 Early Gold’s natural growth	中国辽宁 Liaoning, China
59	晖雨露 Huiyulu	朝晖×雨花露 Zhaohui×Yuhualu	中国江苏 Jiangsu, China
60	奉化玉露 Fenghuayulu	上海水蜜营养系变异 Variation of Chinese Cling nutrition system	中国上海 Shanghai, China
61	花玉露 Huayulu	玉露变异 Jade dew variation	中国浙江 Zhejiang, China
62	瑞光23号 Ruiguang 23	Fuzador×瑞光7号（京玉×B7R2T129）Fuzador×Ruiguang 7 (Jingyu× B7R2T129)	中国北京 Beijing, China
63	瑞蟠1号 Ruipan 1	大久保×陈圃蟠桃 Okubo×Chenpupantao	中国北京 Beijing, China
64	霞晖3号 Xiahui 3	白花 Baihua	中国江苏 Jiangsu, China
65	玉露蟠桃Yulupantao	地方品种 Local varieties	中国浙江 Zhejiang, China
66	霞晖1号 Xiahui 1	朝晖×朝霞（亲本均为白花） Zhaohui×Zhaoxia (both parents are Baihua)	中国江苏 Jiangsu, China
67	雨花3号Yuhua 3	不详 Unknown	中国江苏 Jiangsu, China
68	金陵锦桃 Jinlingjintao	红粉佳人实生后代 The offspring of Pink Lady	中国江苏 Jiangsu, China
69	源东白桃 Yuandongbaitao	砂子早生芽变 Bud mutation of Sunago Wase	中国浙江 Zhejiang, China
70	新白花 Xinbaihua	白花水蜜自然实生 Baihua honey naturally grows	中国江苏 Jiangsu, China
71	金童6号 Babygold 6	NJ13232(=NJ58127×OP)×NJ196	美国 US
72	紫金红3号 Zijinhong 3	W31×紫金红1号 W31×Zijinhong 1	中国江苏 Jiangsu, China
73	金山早红 Jinshanzaohong	早红宝石芽变 Bud mutation of Early Red Ruby	中国江苏 Jiangsu, China
74	早红港 Early Redhaven	不详 Unknown	未知 Unknown
75	霞晖5号 Xiahui 5	朝晖×63-17-1 Zhaohui×63-17-1	中国江苏 Jiangsu, China
76	南山甜桃 Nanshantiantao	地方品种 Local varieties	中国广东 Guangdong,China
77	锦香 Jinxiang	北农2号×60-27-7 Beinong 2×60-27-7	中国上海 Shanghai, China
78	佛尔蒂尼·莫蒂尼 Fertilia Morettini	Gialla Di Firenze×Fertilia I	意大利 Italy
79	京春 Jingchun	早生黄金自然实生 Early Gold’s natural growth	中国北京 Beijing, China

Table 1

Fig. 1

Fig. 2

Fig. 3

Table 2

Amplification results of 5 pairs of primers in 79 accessions"

种质材料编号 Germplasms No.	Pp01-3F-2356268	Pp01-64F-38037623	LG4-8F-21523456	LG3-37F-16205196	Pp01-19F-9009794
1	186\201	161	95\97	149	147\149
2	178\188	159\161	84\97	149	149
3	186\194	144	75\84	148\150	147\149
4	190	161	84\97	140\145	149
5	190\197	146	75\84	151	149
6	188\190	155\161	84\95	145\151	147\149
7	188\201	144	82\95	145\149	147\149
8	178\201	144	82\95	145\151	147
9	194	155\157	82	140\149	\
10	190	146	84	149	149
11	190	144\146	84\95	149\151	149
12	188\197	144	95\97	145\149	149
13	186\197	144	82\84	149	149
14	188\190	144	95	149	147\149
15	178\188	144	95	145\149	147
16	186\197	144	84\97	149\151	149
17	186	144	84	145\149	149
18	186\188	155\157	95	140\149	145\147
19	188	155\157	82\95	145\149	147
20	190\197	155\157	84	\	149
21	190\194.9	144	84	149\151	149
22	188\197	157\161	84\97	145\149	149
23	178\197	159\161	95\97	145\149	149
24	190	155\163	75\84	140\151	147\149
25	190\197	144	75\84	149	149
26	190\197	155\157	84\95	149\151	149
27	190\197	144	75\84	149	147\149
28	188	155\161	95\97	149	\
29	188\197	144	84	149	147\149
30	190	144	75	149\151	149
31	186\197	144	84\95	145\149	149
32	186	144	95\97	149	149
33	180	144	78\87	140\145	145
34	186\197	157	84	145\151	149
35	188	155\161	95\97	149	149
36	190	144	75\84	140\151	149
37	188\197	159	84	145\151	149
38	190	155\157	84\95	149\151	149
39	178\197	161	95\97	149	149
40	186\197	157\159	75\89	149	147\155
41	186\201	144	95\97	145\149	147
42	186\190	155\159	75\89	145\149	149
43	188	144	84\97	149	149
44	186\201	\	95\97	149	147\149
45	188\197	155\161	84	149	147\149
46	194	155\157	82	140\149	157
47	190	\	75\84	149	\
48	190\197	\	75\84	140\149	147\149
49	188\197	155\161	84\95	145\151	147\149
50	190\197	144	84	149\151	147\149
51	186\197	\	75	149	147\149
52	186\190	144	75\84	149\151	147\149
53	190\197	144	75\84	149	149
54	190\194	144	95\97	149\151	149
55	188	155\161	84	145\149	147\149
56	186\190	144	95	149	147\149
57	190\197	159\161	84\95	149	147\149
58	188	144	84	149\151	147\149
59	186	144	84\97	149\151	147\149
60	178\186	144	84/95	145	149
61	190\197	144\155	84	149\151	147\149
62	178\201	155\161	95	145	147\149
63	188	159\161	84	145\149	147
64	190\197	146	75\89	145\149	149\155
65	188\197	144	84\95	145	147\149
66	190\197	159\161	84\97	145\149	147\149
67	186\197	144	84	149	147\149
68	186\188	144	95	145\149	145\147
69	187\203	159\161	82	140	149\157
70	188\197	144	84\97	149	147\149
71	197\201	157	84\95	145\149	147
72	190\201	161	95\97	145\149	147\149
73	190\197	144	75\84	149	149
74	197	144\146	84\95	149	149
75	178\197	144	84	149\151	149
76	178\190	155\157	84	145\151	149
77	190\197	159\161	95	140\145	147\149
78	186	159\161	84	149	149
79	188	\	95\97	145\151	147\149

Table 2

Table 3

Information of 26 SSR core primers"

编号 No.	SSR位点 Code	重复单元 Motif	引物序列 Primer sequence (5'-3'）	退火温度 Temperature (℃)	片段长度 Size (bp)	等位基因数 Allele No	多态信息含量 PIC
1	Pp01-2356268	(TC)27	F-ACTGTGGAGAAATGTCACTAAATCC	53-56	187	10	0.79
1	Pp01-2356268	(TC)27	R-CGGCTGACTTCTGTGACTATGTATT
2	Pp01-4292817	(AG)16	F-AGAAATTAAAGACACGCCAAGAAAT	53-56	116	6	0.67
2	Pp01-4292817	(AG)16	R-TGACTCACACTTGTTTTTCTCTCTG
3	Pp01-6357400	(ATT)7	F-GGTTTACGGAGGTAAATTTTGTTTT	53-56	156	4	0.49
3	Pp01-6357400	(ATT)7	R-TCATTACCCTATAACCATCATCACA
4	Pp01-9009794	(AT)11	F-GGGTTCGCTCTTAACTTCATTTATT	53-56	145	8	0.47
			R-AACAAGCTGAACATAACCCACATAA
5	Pp01-12524641	(CTT)16	F-ACAAAAGAACTTGCACCCATATTT	53-56	106	7	0.58
			R-GTTGTTGGATCAAAGAAGAAGAAAA
6	Pp01-17577816	(TTA)12	F-TAAAATTGGAGTGGGAAAATCATTA	53-56	116	4	0.56
			R-CTCTTTTGATTGATAAAGAAACTGCT
7	Pp01-29068991	(GA)16	F-CTACCCAAGCCCATTAAATTTGG	53-56	150	13	0.75
			R-CGAAGTATGTGCATGTTGTCACTA
8	Pp01-32006223	(CT)11	F-AGGACAAAGAAAGAATAACCCAAAT	53-56	152	6	0.61
			R-CGTATTATATGTGTTGGTAAGTTGGC
9	Pp01-37824973	(GA)28	F-TACTGCGATCATATAATGGGTGAGT	53-56	158	5	0.48
			R-CTTCTCTTTCACAACAACATCCAC
10	Pp01-38545547	(TTA)16	F-CTTCAGGAGATTCAATTCGTTTTT	53-56	153	7	0.63
			R-TTATTAACAAGGTTGAGTTGAGTCG
11	Pp02-3112920	(TAT)5	F-ATTCATTGGTTGGTTCCAATACTTA	53-56	134	5	0.46
			R-TTATTCAATTGGATTGGATTTTGTT
12	Pp02-8084012	(AT)11	F-GATTCAGATTAAAGCATGTCCATTT	53-56	130	7	0.56
			R-CGGTGAGTAAAATACTTCATTCGTT
13	Pp02-26052271	(AT)6	F-GAATCGTTGAACATTTATATGGGAC	53-56	139	6	0.55
			R-AAAGGGAAACAAGAACTCAGGTAAT
14	Pp03-8152877	(CT)15	F-AGCATACTTGAAGAAAAATGCTCAC	53-56	126	8	0.67
			R-CTCAGCCAAAACCTATCAGAATTTA
15	Pp03-16205196	(AG)9	F-GGTTTCTTATGAGTGAGAATCCAAG	53-56	146	7	0.59
			R-GCTGAGGTGTGAATATCTGTTTCTT
16	Pp03-21358179	(TGACCA)4	F-ACCAAGTGTAAATGGTCAATTTTGT	53-56	132	4	0.5
			R-TCTGGATTGTACTGTTCACTATGGA
17	Pp04-7103540	(AT)11	F-CCACTATTCAAAAACCAGTTTGTCT	53-56	155	11	0.63
			R-ATAGATGGTTTGAATCATTGGAAAA
18	Pp04-19374257	(TTA)6	F-AATTCGTATTCCCCTTTTAACATTC	53-56	154	6	0.49
			R-CAAACTCAAACTAATCAACTGGAGG
19	Pp04-21523456	(TA)13	F-ATCCCTCAATGAAAGCTCTCTGTAT	53-56	97	8	0.7
			R-CAAAACTTGTGTCAAAAATATGGTG
20	Pp04-22537846	(CA)6	F-AAAGTTAACCCAACACACCTATCAA	53-56	125	5	0.47
			R-CTGTATTTTCAAACAGAAGATTGCC
21	Pp05-11324136	(GT)9	F-ATGATCAGTCAGAGGTCTCAAAGTT	55-58	105	5	0.52
			R-TTTTTCTTTCTCTTTTTGTCAAAGG
22	Pp05-17864845	(TA)12	F-AATCAAACAGAAAAGCGAACTGTAA	53-56	130	7	0.49
			R-TCGAGGATGATTTGTTTAGTACGTT
23	Pp06-374720	(CT)16	F-GAATAAGTCACACCCACATCACTAA	53-56	148	10	0.55
			R-TATGAAAGGGTGTAAGCTACCTCAA
24	Pp06-6343221	(AT)7	F-CCTTGAACCGGAAAATAATAGAAAT	53-56	120	5	0.68
			R-ATTCACGAGAATAGTGTCAGTCCTT
25	Pp06-8102398	(TA)11	F-GCCATTCACTCTACTAGAAGGACAC	53-56	149	5	0.63
			R-GCCCACATGCATCAATTAATATC
26	Pp07-21855211		F-AATTATGGAACCATACACACGTTTT	53-56	153	5	0.48
			R-AAAGGGTATTTTGGAGTGAGACAAT
	平均值 Mean					6.69	0.57

Table 3

Fig. 4

Table 4

Fig. 5

Fig. 6

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组别 Group	数量 No.	桃品种（系） Cultivar (line)
Ⅰ	14	1、40、34、77、2、66、44、64、19、33、10、13、67、69
Ⅱ	16	9、24、56、25、42、20、32、58、65、70、63、8、21、12、15、49
Ⅲ	17	11、62、31、39、45、60、59、57、18、36、75、38、61、28、46、51、79
Ⅳ	10	3、23、55、16、76、48、54、43、29、37
Ⅴ	14	4、5、53、14、35、74、52、72、71、73、78、7、27、68
Ⅵ	4	6、30、17、41
Ⅶ	4	22、26、47、50

Analysis of Genetic Diversity of 79 Cultivars Based on SSR Fluorescence Markers for Peach

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