一种鉴别栽培柿品种SNP标记的新方法

doi:10.13560/j.cnki.biotech.bull.1985.2023-0363

摘要/Abstract

摘要：

【目的】为了鉴别栽培柿品种间的遗传差异，开发一种基于核糖体DNA内转录间隔区（nrDNA internally transcribed spacer）序列简单、高效的SNP分子标记新方法，为种质的收集、利用及推广应用提供参考。【方法】以18种六倍体栽培柿品种叶片为试材，对其ITS区进行扩增测序，并分析序列差异，最后用Sau96 I限制性内切酶对151处杂合位点进行酶切验证。【结果】18份柿品种ITS长度均为730 bp，共存在6个杂合位点，分别在151、168、205、278、279和622 bp处。六倍体柿杂合位点处碱基峰图面积比例存在一定的规律，即C:T=2:1、C:T=1:1、C:T=1:2和A:G=1:1，利用出现的杂合位点及其峰图面积比例规律差异将18份栽培柿品种分成11类。【结论】151处位点的酶切结果验证了酶切产物浓度与峰图面积比例一致。这种基于nrDNA ITS序列SNP分子标记的新方法能将18份栽培柿品种分成11类。

关键词: 栽培柿, nrDNA ITS, SNP, 杂合位点, 遗传差异

Abstract:

【Objective】In order to identify the genetic differences among persimmon（Diospyros kaki）cultivars, a simple and efficient SNP molecular marker based on nrDNA internally transcribed spacer sequence was developed, which may provide reference for the collection, utilization and promotion of germplasm. 【Method】The leaves of 18 hexaploid cultivated persimmon cultivars were used as experimental materials, and the ITS regions of 18 cultivars were amplified and sequenced, and sequence differences were analyzed. Finally, Sau96 I restriction endonuclides were used to verify 151 heterozygous sites. 【Result】Sequencing results showed that ITS length of 18 persimmon varieties was 730 bp. Six heterozygous loci were found in the sequencing peak map, which were 151, 168, 205, 278, 279 and 622 bp. The proportion of base peak area of hexaploid persimmon heterozygous sites had special pattern, that is, C:T= 2:1, C:T= 1:1, C:T= 1:2 and A:G= 1:1. The 18 cultivated persimmon varieties could be divided into 11 categories according to the differences of heterozygous sites and the proportion of peak area. 【Conclusion】The results of enzyme digestion at 151 sites verified that the concentration of enzyme digestion products was consistent with the proportion of its peak map area. The results showed that the SNP molecular labeling method based on nrDNA ITS sequence could divide 18 cultivated persimmon varieties into 11 groups.

Key words: cultivated persimmon, nrDNA ITS, SNP, heterozygous site, genetic difference

梁晋军, 朱溯远, 张宇琴, 张鹏飞, 温鹏飞, 杨运良. 一种鉴别栽培柿品种SNP标记的新方法[J]. 生物技术通报, 2024, 40(1): 160-167.

LIANG Jin-jun, ZHU Su-yuan, ZHANG Yu-qin, ZHANG Peng-fei, WEN Peng-fei, YANG Yun-liang. A Novel SNP Marker for the Identification of Persimmon（Diospyros kaki）Cultivars[J]. Biotechnology Bulletin, 2024, 40(1): 160-167.

图/表 4

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编号Code	学名Scientific	倍性水平Ploidy level	种/品种名Species/Cultivar name	脱涩类型Astringent type	来源Source
1	D. kaki Thunb	2n=6X=90	骏河 Suruga	PCNA	日本Japan
2	D. kaki Thunb	2n=6X=90	晚御所 Oku-gosho	PCNA	日本Japan
3	D. kaki Thunb	2n=6X =90	太秋 Taishuu	PCNA	日本Japan
4	D. kaki Thunb	2n=6X =90	花御所 Hanagosho	PCNA	日本Japan
5	D. kaki Thunb	2n=6X =90	兴津20 Okitsu 20	PCNA	日本Japan
6	D. kaki Thunb	2n=6X =90	前川次郎Jirou	PCNA	日本Japan
7	D. kaki Thunb	2n=6X=90	伊豆 Yidou	PCNA	日本Japan
8	D. kaki Thunb	2n=6X=90	富有 Fuyuu	PCNA	日本Japan
9	D. kaki Thunb	2n=6X=90	早秋 Soshu	PCNA	日本Japan
10	D. kaki Thunb	2n=6X=90	阳丰 Youhou	PCNA	日本Japan
11	D. kaki Thunb	2n=6X=90	海库曼 Haikuman	PVNA	美国America
12	D. kaki Thunb	2n=6X=90	斤柿 Jinshi	PCA	日本Japan
13	D. kaki Thunb	2n=6X=90	法莲坊 Falianfang	PCA	日本Japan
14	D. kaki Thunb	2n=6X=90	火葫芦 Huohulu	PCA	中国山西Shanxi, China
15	D. kaki Thunb	2n=6X=90	小涩柿 Xiaoseshi	PCA	中国山西Shanxi, China
16	D. kaki Thunb	2n=6X=90	橘蜜柿 Jumishi	PCA	中国山西Shanxi, China
17	D. kaki Thunb	2n=6X=90	黑柿 Heishi	PCA	中国山西Shanxi, China
18	D. kaki Thunb	2n=6X=90	水化柿 Shuihuashi	PCA	中国山西Shanxi, China

编号Code	学名Scientific	倍性水平Ploidy level	种/品种名Species/Cultivar name	脱涩类型Astringent type	来源Source
1	D. kaki Thunb	2n=6X=90	骏河 Suruga	PCNA	日本Japan
2	D. kaki Thunb	2n=6X=90	晚御所 Oku-gosho	PCNA	日本Japan
3	D. kaki Thunb	2n=6X =90	太秋 Taishuu	PCNA	日本Japan
4	D. kaki Thunb	2n=6X =90	花御所 Hanagosho	PCNA	日本Japan
5	D. kaki Thunb	2n=6X =90	兴津20 Okitsu 20	PCNA	日本Japan
6	D. kaki Thunb	2n=6X =90	前川次郎Jirou	PCNA	日本Japan
7	D. kaki Thunb	2n=6X=90	伊豆 Yidou	PCNA	日本Japan
8	D. kaki Thunb	2n=6X=90	富有 Fuyuu	PCNA	日本Japan
9	D. kaki Thunb	2n=6X=90	早秋 Soshu	PCNA	日本Japan
10	D. kaki Thunb	2n=6X=90	阳丰 Youhou	PCNA	日本Japan
11	D. kaki Thunb	2n=6X=90	海库曼 Haikuman	PVNA	美国America
12	D. kaki Thunb	2n=6X=90	斤柿 Jinshi	PCA	日本Japan
13	D. kaki Thunb	2n=6X=90	法莲坊 Falianfang	PCA	日本Japan
14	D. kaki Thunb	2n=6X=90	火葫芦 Huohulu	PCA	中国山西Shanxi, China
15	D. kaki Thunb	2n=6X=90	小涩柿 Xiaoseshi	PCA	中国山西Shanxi, China
16	D. kaki Thunb	2n=6X=90	橘蜜柿 Jumishi	PCA	中国山西Shanxi, China
17	D. kaki Thunb	2n=6X=90	黑柿 Heishi	PCA	中国山西Shanxi, China
18	D. kaki Thunb	2n=6X=90	水化柿 Shuihuashi	PCA	中国山西Shanxi, China

类型 Type	品种编号 Cultivar No.	ITS变异位点Nucleotide of variable sites of ITS
类型 Type	品种编号 Cultivar No.	151	168	205	278	279	622
D1	14	T	T	C	T	T	G
D2	1, 2, 3, 4, 15, 16	C	T	C	T	T	G
D3	17	T	T	C	C	T	G
D4	18	T	T	C	T	Y₃	G
D5	12	T	T	C	Y₂	Y₃	G
D6	5	Y₁	Y₁	C	T	T	G
D7	13	Y₁	T	Y₁	T	Y₂	R
D8	6,7	Y₁	T	C	Y₃	T	G
D9	8,9	Y₂	T	C	Y₂	T	G
D10	10	Y₃	T	C	Y₁	T	G
D11	11	Y₃	T	C	Y₂	T	G

类型 Type	品种编号 Cultivar No.	ITS变异位点Nucleotide of variable sites of ITS
类型 Type	品种编号 Cultivar No.	151	168	205	278	279	622
D1	14	T	T	C	T	T	G
D2	1, 2, 3, 4, 15, 16	C	T	C	T	T	G
D3	17	T	T	C	C	T	G
D4	18	T	T	C	T	Y₃	G
D5	12	T	T	C	Y₂	Y₃	G
D6	5	Y₁	Y₁	C	T	T	G
D7	13	Y₁	T	Y₁	T	Y₂	R
D8	6,7	Y₁	T	C	Y₃	T	G
D9	8,9	Y₂	T	C	Y₂	T	G
D10	10	Y₃	T	C	Y₁	T	G
D11	11	Y₃	T	C	Y₂	T	G