甘薯近缘野生种ISBP分子标记的开发及其在遗传多样性分析和品种鉴定中的应用

doi:10.3969/j.issn.1004-1524.2023.03.01

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (3): 489-498.DOI: 10.3969/j.issn.1004-1524.2023.03.01

甘薯近缘野生种ISBP分子标记的开发及其在遗传多样性分析和品种鉴定中的应用

孟羽莎¹^,²(), 王寅¹^,², 赖齐贤¹^,², 刘雷¹^,², 项超³, 吴永华¹^,², 郑嫣然¹^,², 顾兴国¹^,², 方豪¹^,², 苗苗¹^,², 吴列洪³, 汤勇¹^,²^,^*()

1.浙江省农业科学院农村发展研究所,浙江杭州 310021
2.农业农村部创意农业重点实验室,浙江杭州 310021
3.浙江省农业科学院作物与核技术利用研究所,浙江杭州 310021

收稿日期:2021-12-15 出版日期:2023-03-25 发布日期:2023-04-07
作者简介:孟羽莎(1989—),女,河北保定人,博士,助理研究员,主要从事甘薯育种和分子生物学研究。E-mail: mengyusha200@163.com
通讯作者: ^*汤勇,E-mail:zaastangy@163.com
基金资助:
浙江省重点研发计划(2021C04028)

Assessment of genetic diversity and variety identification based on insertion site-based polymorphism (ISBP) markers developed in wild species related to sweet potato

MENG Yusha¹^,²(), WANG Yin¹^,², LAI Qixian¹^,², LIU Lei¹^,², XIANG Chao³, WU Yonghua¹^,², ZHENG Yanran¹^,², GU Xingguo¹^,², FANG Hao¹^,², MIAO Miao¹^,², WU Liehong³, TANG Yong¹^,²^,^*()

1. Institute of Rural Development, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2. Key Laboratory of Creative Agriculture, Ministry of Agriculture and Rural Affairs, Hangzhou 310021, China
3. Institute of Crops and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2021-12-15 Online:2023-03-25 Published:2023-04-07

摘要/Abstract

摘要：

从甘薯近缘野生种Ipomoea triloba和Ipomoea trifida的基因组测序序列中鉴定出2 360条长末端重复反转座子(long terminal repeat retrotransposons, LTR-RT)序列,在此基础上开发了100对基于插入位点的多态性(insertion-site-based polymorphisms,ISBP)引物,并利用4份甘薯种质资源对开发的ISBP引物进行多态性评估,发现24对ISBP引物具有较高的多态性,随后将其应用于浙江省收集的56份甘薯种质资源的遗传多样性分析和品种鉴定。结果显示, 24对ISBP引物在56份种质资源中共产生182条多态性条带,且每份种质资源都有独特的指纹信息。NTsys遗传分析结果显示,群体内不同种质资源之间的遗传距离从0.027 5至0.653 8,平均为0.373 2,引物多态性信息含量(polymorphic information content,PIC)平均为0.289 6,UPGMA和PCA结果显示,这些群体共被分为两个大群,分别包含11和45份种质资源。以上研究表明,该群体的遗传变异较小,不同种质资源之间的遗传背景狭窄。本研究开发的ISBP引物一方面增加了甘薯中逆转座子分子标记的数量,填补了ISBP标记在甘薯遗传分析中应用的空白,另一方面为甘薯品种资源鉴定、保护与利用提供了有力工具。

关键词: 甘薯, ISBP, LTR, 分子标记, 遗传多样性, 品种鉴定

Abstract:

Based on genome wide sequencing of wild species related to sweet potato Ipomoea triloba and Ipomoea trifida, 2 360 long terminal repeat retrotransposons (LTR-RT) sequences were identified. One hundred insertion-site-based polymorphisms (ISBP) primer pairs were developed and four sweet potato germplasm resources were used to evaluate the polymorphisms of ISBP primers. Twenty four pairs of ISBP primers were found to be highly polymorphic, which were then applied to genetic diversity analysis and variety identification of 56 sweet potato germplasm resources collected in Zhejiang province. The results showed that 24 pairs of ISBP primers generated 182 polymorphic bands in 56 germplasm resources, and each germplasm resource had unique fingerprint information. The genetic analysis results of NTsys, UPGMA and PCA showed that the genetic distance between different germplasm resources in the population ranged from 0.027 5 to 0.653 8, with an average of 0.373 2, the average of polymorphic information content (PIC) was 0.289 6, and these resources were divided into two populations containing 11 and 45 germplasm resources, respectively. Studies have shown that the genetic variation of this population is small and the genetic background among different germplasm resources is narrow. The ISBP primers developed in this study not only increased the number of transposon molecular markers, filled in the gap of application of ISBP markers in genetic analysis of sweet potato, but also provided a favorable tool for the identification, protection and utilization of sweet potato variety resources.

Key words: sweet potato, ISBP, LTR, molecular marker, genetic diversity, variety identification

中图分类号:

S531

孟羽莎, 王寅, 赖齐贤, 刘雷, 项超, 吴永华, 郑嫣然, 顾兴国, 方豪, 苗苗, 吴列洪, 汤勇. 甘薯近缘野生种ISBP分子标记的开发及其在遗传多样性分析和品种鉴定中的应用[J]. 浙江农业学报, 2023, 35(3): 489-498.

MENG Yusha, WANG Yin, LAI Qixian, LIU Lei, XIANG Chao, WU Yonghua, ZHENG Yanran, GU Xingguo, FANG Hao, MIAO Miao, WU Liehong, TANG Yong. Assessment of genetic diversity and variety identification based on insertion site-based polymorphism (ISBP) markers developed in wild species related to sweet potato[J]. Acta Agriculturae Zhejiangensis, 2023, 35(3): 489-498.

图/表 6

表1 LTR-RT分类结果

Table 1 LTR-RT classification summary

亚家族中LTR 的个数 LTR No. of subfamily	Ipomoea triloba				Ipomoea trifida
	Copia家族 Copia family		Gypsy家族 Gypsy family		Copia家族 Gypsy family		Gypsy家族 Gypsy family
		亚家族个数 No. of subfamily	LTR-RT 数量 LTR-RT No.	亚家族个数 No. of subfamily	LTR-RT 数量 LTR-RT No.	亚家族个数 No. of subfamily	LTR-RT 数量 LTR-RT No.	亚家族个数 No. of subfamily	LTR-RT 数量 LTR-RT No.
1	128	128	1 102	1 102	126	126	826	826
2	6	12	13	26	4	8	12	24
≥3	6	22	9	49	5	22	3	15
共计Total	149	162	1 124	1 177	135	156	841	865

图1 M,100 bp DNA ladder;1~16,分别对应17~32号引物。

Fig.1 Amplifications of partial ISBP primers M, 100 bp DNA ladder; 1-16, Respectively corresponding to primers of No.17-32.

图2 引物ISBP31在部分种质资源中扩增结果 M,100 bp DNA ladder;1~21,分别对应1~21份种质份资源编号。

Fig.2 Amplifications of partial sweet-potato varieties based on ISBP31 primer pairs M, 100 bp DNA ladder; 1-21, Respectively corresponding to 1-21 germplasm resources.

表2 二十四对ISBP引物在56份资源中的多态性特征

Table 2 The characters of the 24 ISBP primer pairs based on 56 sweet potato germplasm resources

引物名称 Primer name	Ne^*	H^*	I^*	PIC
IbISBP4	1.300 5	0.204 4	0.342 1	0.257 8
IbISBP14	1.563 0	0.335 8	0.503 4	0.326 4
IbISBP17	1.266 8	0.185 6	0.309 8	0.250 0
IbISBP18	1.490 8	0.288 4	0.433 7	0.281 5
IbISBP19	1.595 8	0.349 9	0.521 4	0.339 6
IbISBP26	1.620 2	0.356 5	0.527 8	0.319 2
IbISBP30	1.677 8	0.379 0	0.552 5	0.260 4
IbISBP31	1.448 1	0.286 3	0.445 0	0.314 5
IbISBP32	1.359 7	0.224 3	0.356 6	0.266 8
IbISBP57	1.557 2	0.323 0	0.479 4	0.318 4
IbISBP60	1.801 4	0.436 8	0.626 0	0.271 2
IbISBP67	1.604 5	0.345 7	0.511 6	0.288 7
IbISBP69	1.593 6	0.341 5	0.506 1	0.305 0
IbISBP70	1.619 2	0.345 3	0.508 9	0.294 4
IbISBP71	1.559 8	0.335 6	0.508 5	0.338 8
IbISBP72	1.670 8	0.377 7	0.555 0	0.326 6
IbISBP74	1.526 4	0.301 6	0.448 2	0.278 8
IbISBP79	1.582 3	0.320 5	0.459 9	0.200 8
IbISBP80	1.707 6	0.399 4	0.581 4	0.296 5
IbISBP107	1.523 4	0.314 5	0.476 9	0.314 5
IbISBP112	1.558 1	0.324 3	0.479 0	0.240 5
IbISBP120	1.251 4	0.172 6	0.283 8	0.233 7
IbISBP129	1.823 9	0.445 0	0.635 2	0.334 0
IbISBP135	1.531 9	0.314 8	0.474 2	0.291 2

图3 二十四对特异性引物对56份种质资源的聚类分析红色代表第一个大群,蓝色、紫色、青色和绿色分别代表第二个大群中不同的亚群。紫色圆形、青色的方形、青色的三角形、绿色的菱形和圆形分别代表来自淳安县、莲都区、建德市、缙云县和黄岩区且聚集在同一分支上的种质资源。

Fig.3 UPGMA analysis of 56 sweetpotato germplasm resources based on the 24 pairs of specific primers Red represents the first population, and bule, purple, cyan and green represent different subpopulations in the second population. Purple circle, cyan square, cyan triangle, green diamond and circle represent germplasm resources from Chun’an county, Liandu district, Jiande city, Jinyun county and Huangyan district, which are clustered in the same branch respectively.

图4 五十六份种质资源的主成分分析

Fig.4 PCA analysis of 56 sweetpotato germplasm resources

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甘薯近缘野生种ISBP分子标记的开发及其在遗传多样性分析和品种鉴定中的应用

Assessment of genetic diversity and variety identification based on insertion site-based polymorphism (ISBP) markers developed in wild species related to sweet potato

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