利用Target SSR-seq技术鉴定温州蜜柑芽变材料

doi:10.3864/j.issn.0578-1752.2021.23.013

摘要/Abstract

摘要：

【目的】柑橘芽极易发生变异,用形态学的方法鉴定这些芽变材料,易受环境、栽培条件等因素影响,而利用深度测序技术开展柑橘芽变材料鉴定,能够获得变异位点的基因型;同时,建立的技术体系也有利于柑橘资源的精准鉴定、种质资源的遗传多样性研究和植物性品种权保护。【方法】本研究通过靶位点SSR测序技术对柑橘芽变材料开展鉴定。首先利用柑橘全基因组序列扫描发掘SSR,采用多态性较高的SSR位点用于柑橘芽变材料的区分,进一步利用多路复用PCR扩增构建SSR靶位点文库,并通过MiSeq深度测序方法对2份温州蜜柑芽变材料的SSR靶位点进行测序验证。再利用开发的SSR标记对22份优质柑橘种质资源进行遗传多样性分析。【结果】利用GMATA软件在克里曼丁红橘（Citrus clementina Hort. ex. Tanaka）参考基因组和温州蜜柑（Citrus unshiu Macf.）基因组中分别获得69 101个和80 193个SSR,其中以AT/TA为主的2基序SSR最多,3基序中以AAT最多,通过序列比对发掘出变异热点区域的高多态性SSR用于温州蜜柑芽变材料的检测,4对SSR引物能对22份柑橘材料进行准确区分。77对SSR靶位点的深度测序一次性对多个SSR位点进行基因分型,能准确鉴定出2份温州蜜柑芽变材料的SSR基因型,以及SSR在基因组上的准确位置。结合SSR以及SNP基因型,能够对2份温州蜜柑芽变材料进行有效区分。【结论】本研究开发了用于柑橘芽变材料区分的高效SSR位点发掘方法,结合靶位点多路扩增和Target SSR-seq的深度测序实现了柑橘芽变材料的高效区分。

关键词: 柑橘, 芽变, Target SSR-seq, 深度测序

Abstract:

【Objective】 Most citrus cultivars especially some seedless varieties were originated from bud sports mutation. The plant morphological traits of these bud sports were susceptible to environment and cultivation conditions, so discriminating the mutant lines from its parents have been still hard tasks although many molecular marker approaches were developed. As the use of deep sequencing technology could simultaneously genotype multiple loci of the mutation lines, it was already widely used to discriminate citrus bud sports in high efficiency. In addition, the technical approach could also benefit to accurate the identification of citrus genetic accessions, the genetic diversity research and the variety rights protection. 【Method】 In this study, the Target-SSR sequencing approach was adopted to discriminate citrus bud sport. The SSR loci were firstly discovered by scanning of the clementine mandarin (Citrus reticulata Blanco) reference genome and satsuma mandarin (Citrus unshiu Macf.) genome sequences, and those high polymorphic SSR loci were used to design primers to distinguish 22 citrus accessions which including some bud sports materials. Finally, multiplexed PCR production obtained through 18 runs PCR amplification with 77 pair primers on two bud sports materials were used to construct target SSR sequencing library, and sequenced with MiSeq apparatus to genotype SSR and SNP variation. In addition, the genetic diversity of 22 citrus accessions were also analyzed by using the developed SSR markers in this study. 【Result】 69 101 SSRs and 80 193 SSR were obtained respectively from clementine reference genome and satsuma genome by using GMATA software, among which AT/TA motif SSR were the most, and 3 motifs AAT rank the second. The high polymorphic SSR loci and its flanking sequences were extracted to design primers for bud sports discrimination and library construction. Four pairs of SSR primers could accurately distinguish 22 citrus germplasms. Target SSR-sequencing with MiSeq apparatus not only simultaneously genotype multiple SSR loci at one time but also accurately identified the variant loci of SSRs. Combining with the identified SSR and SNP genotypes, it could be effectively distinguished two satsuma bud sports. 【Conclusion】In this research, an efficient SSR loci discovering method was developed, combining with target SSR multiplex amplification and deep sequencing on MiSeq platform, which could effectively discriminate citrus bud sports. This study not only provided an important means to identify citrus buds sports, but also facilitated the protection of citrus varieties and the management of citrus germplasms genetic diversity in the future.

Key words: citrus, bud sports, Target SSR-seq, deep sequencing

胡冬梅,江东,李永平,彭磊,李冬云,朱延松,杨云光. 利用Target SSR-seq技术鉴定温州蜜柑芽变材料[J]. 中国农业科学, 2021, 54(23): 5083-5096.

HU DongMei,JIANG Dong,LI YongPing,PENG Lei,LI DongYun,ZHU YanSong,YANG YunGuang. Identification of Bud Sport Mutation of Satsuma Mandarin by Target SSR-seq Technology[J]. Scientia Agricultura Sinica, 2021, 54(23): 5083-5096.

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序号 Code	种质号 Germplasm number	采集地 Collection site	对应母本 Corresponding maternal parent	序号 Code	种质号 Germplasm number	采集地 Collection site	对应母本 Corresponding maternal parent
1	芽变1号 Bud sports No1.	华宁县 Huaning county	4	12	芽变5号 Bud sports No5.	华宁县 Huaning county	11
2	芽变2号 Bud sport No2.	华宁县 Huaning county	4	13	大分1号 Oita No1.	华宁县 Huaning county
3	芽变3号 Bud sport No3.	华宁县 Huaning county	9	14	HX10	华宁县 Huaning county
4	冰糖橙 Bingtang Orange	华宁县 Huaning county		15	MK11	华宁县 Huaning county
5	BJ2	华宁县 Huaning county		16	芽变6号 Bud sport No6.	新平县 Xinping county	4
6	SC3	华宁县 Huaning county		17	芽变7号 Bud sport No7.	新平县 Xinping county	4
7	芽变4号 Bud sport No4.	华宁县 Huaning county	不详（温州蜜柑） Unknown (Satsuma)	18	芽变8号 Bud sport No8.	新平县 Xinping county	4
8	宫本 Miyanoto Wase	华宁县 Huaning county		19	芽变9号 Bud sport No9.	新平县 Xinping county	4
9	大浦 Ooura Wase	华宁县 Huaning county		20	芽变10号 Bud Sport No10.	新平县 Xinping county	4
10	兴津 Okitsu Wase	华宁县 Huaning county		21	芽变11号 Bud sport No11.	新平县 Xinping county	4
11	日南1号 Nichihan No1.	华宁县 Huaning county		22	HG12	新平县 Xinping county

引物编号 Primer number	上游Forward (5′-3′)	下游Reward（5′-3′）
MK7	CTGGGGCTCACATAAATCGT	GATTTGTCCGCCAATCAAGT
MK9	GCATTGCAGCACTTTTGTCAT	GCATTGCAGCACTTTTGTCAT
MK19	GGGTTGGGAATGTGAATGAA	CCTAGGGGTGGGCATATTTT
MK17	CCCACCTCGTTGAATCTCTC	ACCAGTACCACTGCTTACTCTTTT

3.CHROM	3.POS	3.REF	3.ALT	7.ALT	3.DP	7.DP	3.AF	7.AF	3.SGACOV	7.SGACOV
SCAFFOLD_2	9464551	A	C		7955		.212		1688
SCAFFOLD_2	9464547	GAGAAGAAGAAGAAGA	GAGAAGAAGAAGA	GAGAAGAAGAAGA	7954	6366	.342	.397	2718	2528
SCAFFOLD_2	9464536	GAAA	GA	GA	7919	6328	.347	.409	2750	2590
SCAFFOLD_2	9464527	G	C	C	7914	6328	.366	.427	2894	2703
SCAFFOLD_2	9464521	A	G		7912		.213		1689
SCAFFOLD_6	23400577	GTATTATTATTATTATT	GTATTATTATTATTATTATTATT	GTATTATTATTATTATTATTATT	5603	5709	.606	.373	3396	2131
SCAFFOLD_6	23400577	GTATTATTATTATTATT	GTATTATTATTATTATTATTATT	T	5603	5709	.606	.227	3396	1298