基于转录组测序开发糜子SSR标记

doi:10.3864/j.issn.0578-1752.2020.10.002

中国农业科学 ›› 2020, Vol. 53 ›› Issue (10): 1940-1949.doi: 10.3864/j.issn.0578-1752.2020.10.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

基于转录组测序开发糜子SSR标记

陈小红¹,何杰丽²,石甜甜¹,邵欢欢¹,王海岗³,陈凌³,高志军⁴,王瑞云^1,³(),乔治军³()

¹ 山西农业大学农学院,山西太谷030801
² 山西农业大学文理学院,山西太谷030801
³ 山西省农业科学院农作物品种资源研究所/农业部黄土高原作物基因资源与种质创制重点实验室/杂粮种质资源发掘与遗传改良山西省重点实验室,太原030031
⁴ 内蒙古鄂尔多斯市农牧业科学研究院,内蒙古鄂尔多斯017200

收稿日期:2019-07-25 接受日期:2020-02-20 出版日期:2020-05-16 发布日期:2020-05-22
通讯作者: 王瑞云,乔治军
作者简介:陈小红,Tel：13466897634;E-mail：13466897634@163.com。|何杰丽,E-mail：15835437045@163.com。
基金资助:
现代农业产业技术体系建设专项(CARS-06-13.5-A16);国家自然科学基金(31271791);国家留学基金((2019)75号-201908140133);山西省自然科学基金(201901D11126);山西省重点研发计划(一般项目)(农业)(201803D221008-5);山西省回国留学人员科研资助项目(2016-066)

Developing SSR Markers of Proso Millet Based on Transcriptome Sequencing

CHEN XiaoHong¹,HE JieLi²,SHI TianTian¹,SHAO HuanHuan¹,WANG HaiGang³,CHEN Ling³,GAO ZhiJun⁴,WANG RuiYun^1,³(),QIAO ZhiJun³()

¹ College of Agronomy, Shanxi Agricultural University, Taigu 030801, Shanxi
² College of Arts and Sciences, Shanxi Agricultural University, Taigu 030801, Shanxi
³ Institute of Crop Germplasms Resources, Shanxi Academy of Agricultural Sciences/Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture/Shanxi Key Laboratory of Genetic Resources and Genetic Improvement of Minor Crops, Taiyuan 030031
⁴ Erdos Institute of Agriculture and Animal Husbandry, Erdos 017200, Inner Mongolia

Received:2019-07-25 Accepted:2020-02-20 Online:2020-05-16 Published:2020-05-22
Contact: RuiYun WANG,ZhiJun QIAO

摘要/Abstract

摘要：

【目的】以6份不同地理来源的糜子资源为试验材料,基于前期转录组测序获得的1 000对SSR引物,选出200对进行多态性检测,以期构建一批可以准确评估糜子种质遗传差异的分子标记。【方法】用Primer Premier 5.0软件设计引物,改良CTAB法提取DNA,PCR扩增DNA和聚丙烯酰胺凝胶电泳筛选引物多态性;用PowerMarker 3.25和PopGen 1.32计算遗传多样性参数。【结果】 200对引物中97对呈单态性,80对呈多态性。单碱基序列重复引物有20对,10对具多态性,其重复基元是A（50%）和T（50%）。二碱基序列重复引物有36对,15对具多态性,其碱基重复类型有7种（AG最多,TC、GC和GA次之,CA、TA和AC最少）。三碱基序列重复引物有144对,55对具多态性,其碱基重复类型有24种（GGC、GCG和GCC最多,GAA、GCT和CGC等次之,ACC、AGG、CAG、CGT、AAG、AAC、TCG、CGA、ATT、CAA和CCA最少）。就引物分辨率(Rp)值而言,1—3碱基序列重复引物分别为0.67—4.67（平均2.07）、1.33—4.33（平均2.73）和0.67—4.00（平均1.83）。基于Rp值分析SSR分布频次,发现80个标记分布在5个区间：0—1、1—2、2—3、3—4和4—5,分别包含17（21.25%）、36（45.00%）、11（13.75%）、14（17.50%）和2个（2.50%）。就$\overline{\text{Rp}}$值而言,1—3碱基序列重复引物分别为0.33—0.67（平均0.51）、0.40—0.78（平均0.59）和0.33—0.83（平均0.59）。单碱基序列重复标记共检测到22个等位变异,每个位点为2—3个（平均2.2000）,其中8个和2个位点分别具2和3个变异;二碱基序列重复标记共检测到38个等位变异,每个位点为2—3个（平均2.5333）,其中7个和8个位点分别具2和3个变异;三碱基重复标记共检测到136个等位变异,每个位点为2—3个（平均2.4727）,其中29个和26个位点分别具2和3个变异。就多态性信息含量而言,1—3碱基序列重复引物分别为0.3750—0.5355（平均0.4293）、0.2392—0.7438（平均0.4293）和0.2392—0.7438（平均0.3989）。就多样性指数而言,1—3碱基序列重复分别为0.6365—1.0776（平均0.7497）、0.5623—1.0986（平均0.8339）和0.5623—1.0889（平均0.8312）。【结论】基于转录组测序结果,检测200对SSR引物的多态性,发现177对（88.5%）可以扩增出完整条带,其中80对具多态性,多态率为40%。

关键词: 糜子（Panicum miliaceum L.）, SSR, 引物分辨率（Rp）, 多态性信息含量（PIC）, 多样性指数

Abstract:

【Objective】Based on the data of transcriptome sequencing, 200 pairs of SSR primers were detected using six proso millet accessions with different origin. The aim of this study is to construct a set of molecular markers for assessing the genetic diversity of proso millet germplasms.【Method】Primer Premier 5.0 software was used to design primers, DNA was extracted by modified CTAB method and amplified by PCR. Polyacrylamide gel electrophoresis was used to screen the polymorphism of primers. Genetic diversity parameters were calculated with PowerMarker 3.25 and PopGen 1.32.【Result】Ninety seven and eighty out of 200 pairs of primers were shown monomorphic and polymorphic, respectively. A total of 20 were mono-nucleotide SSRs, among them 10 were polymorphic with repeat motif A (50%) and T (50%). A total of 36 were di-nucleotide SSRs, among them 15 were polymorphic with seven types of repeat motif, AG class was the most frequent, followed by the TC, GC and GA, CA, TA and AC were the least. A total of 144 were tri-nucleotide SSRs, among them 55 were polymorphic with 24 types of repeat motif, GGC, GCG and GCC class were the most frequent, followed by the GAA, GCT, CGC, etc, ACC, AGG, CAG, CGT, AAG, AAC, TCG, CGA, ATT, CAA and CCA were the least. In terms of the mono-, di-, tri-nucleotide SSR, their Rp value ranged from 0.67 to 4.67 (mean 2.07), 1.33 to 4.33 (mean 2.73) and 0.67 to 4.00 (mean 1.83), respectively. Evaluating the distribution frequency based on Rp, 80 SSRs were divided into five intervals viz 0-1, 1-2, 2-3, 3-4 and 4-5, with 17 (21.25%), 36 (45.00%), 11 (13.75%), 14 (17.50%) and 2 (2.50%) markers, respectively. For the mean of the Rp value, the mono-, di-, tri-nucleotide SSR were 0.33 to 0.67 (average 0.51), 0.40 to 0.78 (average 0.59) and 0.33 to 0.83 (average 0.59), respectively. As for mono-nucleotide SSRs, a total of 22 allelic variations were detected with 2-3 alleles (average 2.2000) for each locus; 2 and 3 alleles were identified for 8 and 2 loci, respectively. In terms of di-nucleotide SSRs, a total of 38 allelic variations were generated with 2-3 alleles (average 2.5333) for each locus; 7 and 8 loci produced 2 and 3 alleles, respectively. As for tri-nucleotide SSRs, a total of 136 allelic variations were generated with 2-3 alleles (average 2.4727) for each locus; 29 and 26 loci produced 2 and 3 alleles, respectively. In terms of the polymorphism information content, the mono-, di-, tri-nucleotide SSR were 0.3750-0.5355, 0.2392-0.7438 and 0.2392-0.7438, respectively. In terms of the diversity index, the mono-, di-, tri-nucleotide SSR were 0.6365-1.0776 (average 0.7497), 0.5623-1.0986 (average 0.8339) and 0.5623-1.0889 (average 0.8312), respectively. 【Conclusion】Two hundreds pairs of SSR primers obtained from transcriptome sequencing were used to detect genetic diversity of 6 proso millet accessions. It was shown that 177 (88.5%) out of the 200 pairs primers amplified intact bands, and 80 SSRs appeared polymorphic with the polymorphic rate 40%.

Key words: proso millet (Panicum miliaceun L.), SSR, resolving power (Rp), polymorphism information content (PIC), diversity index

陈小红,何杰丽,石甜甜,邵欢欢,王海岗,陈凌,高志军,王瑞云,乔治军. 基于转录组测序开发糜子SSR标记[J]. 中国农业科学, 2020, 53(10): 1940-1949.

CHEN XiaoHong,HE JieLi,SHI TianTian,SHAO HuanHuan,WANG HaiGang,CHEN Ling,GAO ZhiJun,WANG RuiYun,QIAO ZhiJun. Developing SSR Markers of Proso Millet Based on Transcriptome Sequencing[J]. Scientia Agricultura Sinica, 2020, 53(10): 1940-1949.

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序号 No.	统一编号 Unicode	名称 Accession name	来源 Origin	生态栽培区 Ecotope
1	00000177	红糜子Hongmizi	黑龙江省宁安市 Ning’an, Heilongjiang	东北春糜子区 Northeast spring-sowing ecotope
2	00000750	白糜子Baimizi	新疆沙湾县 Shawan, Xinjiang	西北春夏糜子区 Northwest spring & summer-sowing ecotope
3	00006653	金黍Jinshu	海南省 Hainan	南方秋冬糜子区 Southern autumn & winter-sowing ecotope
4	00007238	大红糜子Dahongmizi	内蒙古巴彦淖尔 Bayanzhuoer, Inner Mongolia	北方春糜子区 Northern spring-sowing ecotope
5	00007478	白圪塔糜Baigetami	青海省湟中县 Huangzhong, Qinghai	西北春夏糜子区 Northwest spring & summer-sowing ecotope
6		红黍子Hongshuzi	河南省安阳市 Anyang, Henan	华北夏糜子区 Northern summer-sowing ecotope

基于转录组测序开发糜子SSR标记

Developing SSR Markers of Proso Millet Based on Transcriptome Sequencing

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