Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (23): 5083-5096.doi: 10.3864/j.issn.0578-1752.2021.23.013

• HORTICULTURE • Previous Articles     Next Articles

Identification of Bud Sport Mutation of Satsuma Mandarin by Target SSR-seq Technology

HU DongMei1(),JIANG Dong2(),LI YongPing3,PENG Lei4(),LI DongYun5,ZHU YanSong2,YANG YunGuang1   

  1. 1Agricultural Technology Extension Station of Yuxi County, Yuxi 653100, Yunnan
    2Citrus Research Institute of Southwest University, Chongqing 400712
    3Yunnan Green Food Development Center, Kunming 650032
    4College of Landscape and Horticulture, Yunnan Agricultural University, Kunming 650201
    5Citrus Industry Development Office of Huaning County, Huaning 652800, Yunnan
  • Received:2021-02-22 Accepted:2021-05-20 Online:2021-12-01 Published:2021-12-06
  • Contact: Dong JIANG,Lei PENG E-mail:909625192@qq.com;jiangdong@cric.cn;1527873167@qq.com

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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

Table 1

22 citrus accessions used in this study"

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

Table 2

The four pairs of polymorphic SSR primers used to discriminate 22 citrus accessions"

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

Fig. 1

The SSR motifs and its proportion in citrus reference genome"

Fig. 2

The SSR motifs and its proportion of SSR in satsuma genome"

Fig. 3

SSR variation presented in some homologous sequences"

Fig. 4

The amplification band profiles by No.9 primer on 22 citrus accessions"

Fig. 5

The principal coordinates analysis graph of 22 citrus accessions based on SSR genotype 3, 9, 7, 8, 10, 11, and 12 on behalf early ripening accessions, and the others are medium and late ripening accessions; Diagram 13 said 14 samples, and so on"

Fig. 6

The amplification bands by No.9 (9#1) primer checked with agarose gel electrophoresis"

Table 3

The differentiation SSR and SNP variation on two citrus bud sports variants"

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

Fig. 7

SSR alleles variation of two satsuma bud sports aligned on scaffold_6"

Fig. 8

SSR allel variation of two satsuma bud sports aligned on scaffold_2"

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