9个欧洲李品种自交不亲和S-RNase基因的克隆及序列分析

doi:10.6048/j.issn.1001-4330.2023.05.015

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (5): 1162-1169.DOI: 10.6048/j.issn.1001-4330.2023.05.015

• 园艺特产·贮藏保鲜加工·植物保护 • 上一篇下一篇

9个欧洲李品种自交不亲和S-RNase基因的克隆及序列分析

王亚铜¹^,²(), 玉米提·玉素甫², 耿文娟², 章世奎¹, 孙召展¹, 王绍鹏¹, 王尚栋¹, 樊国全¹()

1.新疆农业科学院轮台果树资源圃,新疆轮台 841600
2.新疆农业大学园艺学院,乌鲁木齐 830052

收稿日期:2022-09-16 出版日期:2023-05-20 发布日期:2023-05-22
通信作者: 樊国全(1980-),男,新疆焉耆人,副研究员,硕士,研究方向为果树种质资源,(E-mail)fgq2046@163.com
作者简介:王亚铜(1993-),男,宁夏固原人,研究实习员,硕士,研究方向为果树种质资源与分子育种,(E-mail)wyt012129@163.com
基金资助:
中央财政林草科技推广示范项目横向基金;新疆农业科学院青年科技骨干创新能力培训项目(xjnky-2020026);国家园艺种质新疆名特果树及砧木分库运行服务(NHGRC2022-NH13);新疆特有果树种质资源安全保存及普查收集资源鉴定评价与繁殖编目入圃(19221874)

Cloning and sequence analysis of self-incompatibility S-RNase gene from 9 European plum cultivars

WANG Yatong¹^,²(), Yumiti Yusufu², GENG Wenjuan², ZHANG Shikui¹, SUN Zhaozhan¹, WANG Shaopeng¹, WANG Shangdong¹, FAN Guoquan¹()

1. Luntai Fruit Tree Germplasm Resource Nursery, Xinjiang Academy of Agricultural Sciences, Luntai Xinjiang 841600, China
2. College of Horticulture, Xinjiang Agricultural University, Urumqi 830052, China

Received:2022-09-16 Published:2023-05-20 Online:2023-05-22
Supported by:
Fund project:Central Finance Horizontal Fund for Forestry and Grass Science and Technology Promotion Demonstration Project;Innovation Ability Training Project for Young Sci-Tech Backbone Talents Sponsored by Xinjiang Academy of Agricultural Sciences(xjnky-2020026);Operation Service of National Horticultural Germplasm Branch Bank of Xinjiang Famous Special Fruit Trees and Rootstocks(NHGRC2022-NH13);Safe Conservation of Unique Fruit Tree Germplasm Resources in Xinjiang, Identification and Evaluation of Resources Collected by Census and Breeding Cataloging in the Nursery(19221874)

摘要/Abstract

摘要：

【目的】鉴定9个欧洲李品种自交不亲和S-RNase基因型,为提高生产效率和高效育种提供理论依据。【方法】利用2对蔷薇科通用引物对9个欧洲李品种的叶片基因组DNA进行特异性PCR扩增,片段回收,将克隆测序的结果在GenBank数据库中进行同源性比对并分析序列。【结果】9个欧洲李品种均扩增出2条带,鉴定出9个样品中的8个欧洲李品种的S基因型,其中理查德早生和斯泰勒的S基因型相同为S1S9,法兰西和Silvia的S基因型相同为S1S5,乌孜干1号、总统、Decbrowice 和女神的S基因型分别为S1S11、S6SSJ、S1SSJ、SSAS11,塔城酸梅仅鉴定出具有一条S1-RNase等位基因,未能确定出其S基因型。基因频率分析发现S1-RNase基因出现频率最高,S6-RNase和SSA-RNase基因频率最低。欧洲李SSA、S6、S9、SSJ和S5亲缘关系较近,参试李亚科树种的S-RNase基因不能单独聚成一个亚类,而是彼此交错分布在李亚科各个分支中。【结论】欧洲李的S基因型是由2种不同的S等位基因组成,鉴定的9个样品中,有8个欧洲李品种得到完整的S基因型,其中S1-RNase基因出现频率最高,李亚科植物S基因的分化早于各个种形成之前,而S基因种内的进化发生在各自单独的系统中。

关键词: 欧洲李; 自交不亲和; S-RNase基因; 克隆

Abstract:

【Objective】 To study cloning and sequence analysis of self-incompatibility S-RNase gene from 9 European plum cultivars【Methods】 Leaf genomic DNA of 9 European Prunus varieties were amplified by specific PCR using two pairs of universal primers of Rosaceae, and the fragments were recovered.The cloned sequencing results were compared in GenBank database for homology and sequence analysis.【Result】 Two bands were amplified from 9 European plum cultivars and the S genotypes of 8 European plum cultivars in 9 tested samples were identified.The S genotypes of Richard Early and Stanley were S1S9, and French and Silvia were S1S5.The S genotypes of Uzigan 1, President, Decbrowice and Nmpress were S1S11, S6SSJ, S1SSJ and SSAS11, respectively.Only one S1-RNase allele was identified in Tacheng Suanmei, but the S genotype was not identified.Gene frequency analysis showed that the frequency of S1-RNase gene was the highest, and the frequency of S6-RNase and SSA-RNase gene was the lowest.The phylogenetic tree of Rosaceae showed that SSA, S6, S9, SSJ and S5 were closely related to each other.The S-RNase genes of the tested Prunus species could not cluster into a single subclass, but were interleaved with each other in each branch of Prunus.【Conclusion】 The S genotype of European plum is composed of two different S alleles.Of the nine samples identified, eight European plum cultivars yield the complete S genotype, in which S1-RNase gene appears the highest frequency.The S gene differentiation of Lyaceae plants is earlier than the formation of each species.Evolution within the S gene species takes place in separate systems.

Key words: European plum; self-incompatibility; S-RNase gene; clone

中图分类号:

S662.3

王亚铜, 玉米提·玉素甫, 耿文娟, 章世奎, 孙召展, 王绍鹏, 王尚栋, 樊国全. 9个欧洲李品种自交不亲和S-RNase基因的克隆及序列分析[J]. 新疆农业科学, 2023, 60(5): 1162-1169.

WANG Yatong, Yumiti Yusufu, GENG Wenjuan, ZHANG Shikui, SUN Zhaozhan, WANG Shaopeng, WANG Shangdong, FAN Guoquan. Cloning and sequence analysis of self-incompatibility S-RNase gene from 9 European plum cultivars[J]. Xinjiang Agricultural Sciences, 2023, 60(5): 1162-1169.

图/表 5

表1 选取的引物组合及其序列

Tab.1 Sequences of consensus primer combinations

序号 No.	引物组合 Primer combination	序列5'-3' Sequence5'-3'	文献Reference
1	Pru-C2	CTATGGCCAAGTAATTATTCAAACC	[21]
1	PruCE-R	TGTTTGTTCCATTCGCCTTCCC	[21]
2	EM-PC2consFD	TCACMATYCATGGCCTATGG	[22]
2	EM-PC3consRD	AWCTRCCRTGYTTGTTCCATTC	[22]

图1 Pru-C2/PruCE-R引物在9个欧洲李品种上的扩增结果注:M为2 000 bp的Marker,1.理查德早生;2.乌孜干1号;3.Silvia;4.法兰西;5.总统;6.Decbrowice;7.斯泰勒;8.女神;9.塔城酸梅

Fig.1 Pru-C2/PruCE-R Amplification results of primers on 9 European plum cultivars Note: M is the Marker of 2,000 bp, 1.Richard Early; 2.Uzigan 1; 3.Silvia; 4.French; 5.president plum; 6.Decbrowice; 7.Stanley; 8.Empress; 9.Tacheng Suanmei

图2 EM-PC2consFD/EM-PC3consRD引物在9个欧洲李品种上的扩增结果注:M为2 000 bp的Marker,10.理查德早生;11.乌孜干1号;12.Silvia;13.法兰西;14.总统;15.Decbrowice;16.斯泰勒;17.女神;18.塔城酸梅

Fig.2 EM-PC2consFD/EM-PC3consRD Amplification results of primers on 9 European plum cultivars Note: M is the Marker of 2,000 bp, 10.Richard Early; 11.Uzigan 1; 12.Silvia; 13.French; 14.president plum; 15.Decbrowice; 16.Stanley; 17.Empress; 18.Tacheng Suanmei

表2 13个基因片段在GenBank上的比对

Tab.2 Results of 13 gene fragments blasted in GenBank

序号 No.	片段大小 Fragment of length (bp)	GenBank上同源性最高的基因及登录号 The maximum identity gene and accesion No.in GenBank		同源性 Homology(%)
1	308	Prunus domestica S1,cds	MW407936.1	100
2	309	Prunus domestica S1,cds	MW407936.1	100
3	473	Prunus domestica SSA,cds	MN069629.1	99
4	653	Prunus domestica S9,cds	AM746948.1	99
5	659	Prunus domestica S11,cds	MW407941.1	99
6	662	Prunus domestica S11,cds	MW407941.1	99
7	330	Prunus domestica S1,cds	MW407936.1	99
8	331	Prunus domestica S1,cds	MW407936.1	100
9	482	Prunus domestica S6,cds	AM746947.1	99
10	736	Prunus domestica SSJ,cds	MN069637.1	99
11	828	Prunus domestica S5, cds	MW407935.1	99
12	830	Prunus domestica S5,cds	MW407935.1	99
13	678	Prunus domestica S16,cds	MW407946.1	96

图3 蔷薇科李亚科S-RNase基因系统进化树注:●表示研究中获得的基因;SSA:女神;S6:总统;S9:理查德早生,斯泰勒;SSJ:总统,Decbrowice;S5:法兰西,Silvia;S1:理查德早生,斯泰勒,法兰西,Silvia,乌孜干1号,Decbrowice,塔城酸梅;S11:乌孜干1号,女神

Fig.3 Phylogenetic tree of S-RNase genes in Lyaceae, Rosaceae Note: ●Represents the genes obtained in this study; SSA:Empress; S6: president plum; S9: Richard Early, Stanley; SSJ: president plum, Decbrowice; S5: French, Silvia; S1: Richard Early, Stanley, French, Silvia, Uzigan 1, Decbrowice, Tacheng Suanmei; S11: Uzigan 1, Empress

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9个欧洲李品种自交不亲和S-RNase基因的克隆及序列分析

Cloning and sequence analysis of self-incompatibility S-RNase gene from 9 European plum cultivars

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