枇杷全基因组SSR标记开发及其多态性研究

doi:10.16420/j.issn.0513-353x.2020-0497

摘要/Abstract

摘要：

搜索枇杷全基因组范围内的SSR位点,分析其分布特点,同时使用10个枇杷品种的重测序数据对这些位点的基因型进行分析。结果显示共获得103 509个SSR位点,二核苷酸重复类型有73 604个,占71.1%,以AT/TA为主。三核苷酸重复类型占15.0%,以AAG/CTT为主。对10个枇杷样本进行重测序,分别获得32 ~ 206 Mb的reads,测序深度为12.9× ~ 81.3×。本研究中新开发2个脚本程序(Flank和SSRtype),对重测序数据Reads中包含的SSR位点长度进行了分析,确定基因型。‘早钟6号’的测序深度达到12.9×,能够覆盖80%的SSR位点,说明12×以上的测序深度能够满足SSR位点的分析。枇杷中大量SSR位点为多拷贝(占78.4%),为单个品种单个位点产生3条以上的条带,而在所有品种中呈现两拷贝的SSR位点有12 962个(占12.5%),这些位点产生的多态性条带分别为2 ~ 10条不等,多态性信息含量的中位数为0.269,大于0.5的位点有526个。从中挑选20对引物进行荧光PCR,结果显示这些引物的多态性与重测序分析结果一致。

关键词: 枇杷, SSR标记, 全基因组, 多态性, 重测序

Abstract:

In this study,we searched the SSR loci in the whole loquat genome and analyzed its distribution characteristics. The resequencing data of 10 cultivars were used to analyze the genotypes of these loci. The results showed that 103 509 SSR sites were obtained,of which 73 604 SSRs were dinucleotide repeat,accounting for 71.1% of total discovered SSRs. The main dinucleotide repeat motif was AT/TA. The trinucleotide repeat type of SSRs accounts for 15.0%,with AAG/CTT as the main type. Ten cultivars of loquat were resequenced,and 32-206 Mb reads were obtained with a depth of 12.9×-81.3×. Two new scripts(Flank and SSRtype)were developed to analyze the length of SSR sites embedded in the resequencing data reads. The sequencing depth of Zaozhong 6 loquat was 12.9×,covering 80% of SSR sites,indicating that a sequencing depth of more than 12 times was enough to the analysis of the SSR loci. A large number of SSR loci in loquat are multiple copies(accounting for 78.4%),producing more than three bands in a locus in a cultivar. There are 12 962 SSR loci with two copies in all cultivars(accounting for 12.5%). The polymorphic bands generated by these loci range from two to 10. The median of the polymorphism information content(PIC)is 0.269. The PIC value of 526 SSR sites was more than 0.5. Twenty pairs of primers were screened to perform fluorescent PCR. The results of polymorphism of these primers were consistent with the resequencing analysis.

Key words: loquat, SSR marker, whole genome, polymorphism, re-sequencing data

中图分类号:

S667.3

蒋爽, 张学英, 安海山, 徐芳杰, 章加应. 枇杷全基因组SSR标记开发及其多态性研究[J]. 园艺学报, 2021, 48(5): 1013-1022.

JIANG Shuang, ZHANG Xueying, AN Haishan, XU Fangjie, ZHANG Jiaying. Development and Analysis of Polymorphism of SSR Markers in the Whole Genome of Loquat[J]. Acta Horticulturae Sinica, 2021, 48(5): 1013-1022.

图/表 7

参考文献 26

[1]	Ai Ye, Chen Lu, Xie Taixiang, Chen Juan, Lan Siren, Peng Donghui. 2019. Construction of core collection of Cymbidium ensifoliumcultivars based on SSR fluorescent markers . Acta Horticulturae Sinica, 46(10):1999-2008. (in Chinese)
	艾叶, 陈璐, 谢泰祥, 陈娟, 兰思仁, 彭东辉. 2019. 基于SSR荧光标记构建建兰品种核心种质. 园艺学报, 46(10):1999-2008.
[2]	Fukuda S, Ishimoto K, Sato S, Terakami S, Hiehata N, Yamamoto T. 2016a. A high-density genetic linkage map of bronze loquat based on SSR and RAPD markers. Tree Genetics & Genomes, 12(4):79-92.
[3]	Fukuda S, Ishimoto K, Terakami S, Yamamoto T, Hiehata N. 2016b. Genetic mapping of the loquat canker resistance gene pse-cin loquat (Eriobotrya japonica). Scientia Horticulturae, 200:19-24. doi: 10.1016/j.scienta.2015.12.036 URL
[4]	Fukuda S, Nishitani C, Hiehata N, Tominaga Y, Yamamoto T. 2013. Genetic diversity of loquat accessions in Japan as assessed by SSR markers. Journal of the Japanese Society for Horticultural Science, 82(2):131-137. doi: 10.2503/jjshs1.82.131 URL
[5]	Gao Yuan, Wang Kun, Wang Dajiang, Liu Lijun, Li Lianwen, Piao Jicheng. 2019. Genetic diversity and genetic structure of Malus baccata and Malus prunifolia from China as revealed by fluorescent SSR markers . Acta Horticulturae Sinica, 46(7):1225-1237. (in Chinese)
	高源, 王昆, 王大江, 刘立军, 李连文, 朴继成. 2019. 中国山荆子和楸子种质资源遗传多样性和遗传结构的荧光SSR分析. 园艺学报, 46(7):1225-1237.
[6]	Gou Wei. 2016. The development and utilization of loquat EST-SSR markers based on transcriptome sequencing[Ph. D. Dissertation]. Chengdu:Sichuan Agricultural University. (in Chinese)
	芶维. 2016. 基于转录组测序枇杷EST-SSR标记的开发与利用[博士论文]. 成都:四川农业大学.
[7]	Guan Ling, Zhang Zhen, Wang Xin-wei, Xue Hua-bo, Liu Yan-hong, Wang San-hong, Qiao Yu-shan. 2011. Evaluation and application of the SSR loci in apple genome. China Agriculture Science, 44(21):4415-4428. (in Chinese)
	关玲, 章镇, 王新卫, 薛华柏, 刘艳红, 王三红, 乔玉山. 2011. 苹果基因组SSR位点分析与应用. 中国农业科学, 44(21):4415-4428.
[8]	Guo Jun, Zhu Jie, Xie Shangqian, Zhang Ye, Ye Beilei, Zheng Liyan, Ling Peng. 2020. Development of SSR molecular markers based on transcriptome and analysis of genetic relationship of germplasm resources in avocado. Acta Horticulturae Sinica, 47(8):1552-1564. (in Chinese)
	郭俊, 朱婕, 谢尚潜, 张叶, 叶蓓蕾, 郑丽燕, 凌鹏. 2020. 油梨转录组SSR分子标记开发与种质资源亲缘关系分析. 园艺学报, 47(8):1552-1564.
[9]	Gupta P, Varshney R. 2000. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica, 113(3):163-185. doi: 10.1023/A:1003910819967 URL
[10]	He Q, Li X W, Liang G L, Ji K, Guo Q G, Yuan W M, Zhou G Z, Chen K S, van de Weg W E, Gao Z S. 2011. Genetic diversity and identity of Chinese loquat cultivars/accessions(Eriobotrya japonica)using apple SSR markers. Plant Molecular Biology Reporter, 29(1):197-208. doi: 10.1007/s11105-010-0218-9 URL
[11]	Jiang S, An H, Xu F, Zhang X. 2020. Chromosome-level genome assembly and annotation of the loquat(Eriobotrya japonica) genome. GigaScience, 9(3):giaa015. doi: 10.1093/gigascience/giaa015 URL
[12]	Jiang Shuang, Luo Jun, Wang Xiao-qing, Shi Chun-hui. 2019. A study on screening efficiency of the polymorphism of SSR markers based on the re-sequencing data in Pyrus . Journal of Fruit Science, 36(2):265-274. (in Chinese)
	蒋爽, 骆军, 王晓庆, 施春晖. 2019. 基于基因组重测序数据高效筛选梨SSR标记多态性引物. 果树学报, 36(2):265-274.
[13]	Kalinowski S T, Taper M L, Marshall T C. 2007. Revising how the computer program cervus,accommodates genotyping error increases success in paternity assignment. Molecular Ecology, 16(5):1099-1106. doi: 10.1111/j.1365-294X.2007.03089.x URL
[14]	Li Yongtan, Zhang Jun, Huang Yali, Fan Jianmin, Zhang Yiwen, Zuo Lihui. 2020. Analysis of chloroplast genome ofPyrus betulaefolia . Acta Horticulturae Sinica, 2020, 47(6):1021-1032. (in Chinese)
	李泳潭, 张军, 黄亚丽, 范建敏, 张益文, 左力辉. 2020. 杜梨叶绿体基因组分析. 园艺学报, 47(6):1021-1032.
[15]	Liu Shuo, Liu Ning, Zhang Qiuping, Zhang Yuping, Zhang Yujun, Xu Ming, Ma Xiaoxue, Liu Weisheng. 2019. Genetic diversity of the apricot germplasms from North & Northeast China. Acta Horticulturae Sinica, 46(6):1045-1056. (in Chinese)
	刘硕, 刘宁, 章秋平, 张玉萍, 张玉君, 徐铭, 马小雪, 刘威生. 2019. 中国华北和东北地区杏种质资源遗传多样性分析. 园艺学报, 46(6):1045-1056.
[16]	Ouni R, Zborowska A, Sehic J, Choulak S, Hormazae J L, Garkava-Gustavsson L, Mars M. 2020. Genetic diversity and structure of Tunisian local pear germplasm as revealed by SSR markers. Horticultural Plant Journal, 6(2):61-70. doi: 10.1016/j.hpj.2020.03.003 URL
[17]	Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A. 1996. The comparison of RFLP,RAPD,AFLP and SSR(microsatellite)markers for germplasm analysis. Molecular Breeding, 2(3):225-238. doi: 10.1007/BF00564200 URL
[18]	Soriano J M, Romero C, Vilanova S, Llacer G, Badenes M L. 2005. Genetic diversity of loquat germplasm[Eriobotrya japonica (Thunb) Lindl] assessed by SSR markers. Genome, 48(1):108-114. pmid: 15729402
[19]	Sun Jun, Li Xiao-ying, Xu Hong-xia, Zhang Lin, Chen Jun-wei. 2018. Identification of white flesh loquat germplasms of Zhejiang province with MCID strategy using genic-SSR markers. Journal of Fruit Science, 35(5):539-547. (in Chinese)
	孙钧, 李晓颖, 徐红霞, 张林, 陈俊伟. 2018. 基于genic-SSR标记的MCID法鉴定浙江白沙枇杷地方种质资源. 果树学报, 35(5):539-547.
[20]	Toth G, Gaspari Z, Jurka J. 2000. Microsatellites in different eukaryotic genomes:survey and analysis. Genome Research, 10(7):967-981. doi: 10.1101/gr.10.7.967 URL
[21]	Varshney R K, Graner A, Sorrells M E. 2005. Genic microsatellite markers in plants:features and applications. Trends in Biotechnology, 23(1):48-55. doi: 10.1016/j.tibtech.2004.11.005 URL
[22]	Wen G, Dang J B, Xie Z Y, Wang J Y, Jiang P F, Guo Q G, Liang G L. 2020. Molecular karyotypes of loquat(Eriobotrya japonica)aneuploids can be detected by using SSR markers combined with quantitative PCR irrespective of heterozygosity. Plant Methods, 16(1):22. doi: 10.1186/s13007-020-00568-7 URL
[23]	Xiang Y, Huang C H, Hu Y, Wen J, Li S, Yi T, Chen H, Xiang J, Ma H. 2017. Evolution of Rosaceae fruit types based on nuclear phylogeny in the context of geological times and genome duplication. Molecular Biology Evolution, 34(2):262-281.
[24]	Xiong Yan, Zhang Jinzhu, Dong Jie, Wang Li, Gao Peng, Jiang Wan, Che Daidi. 2020. A review of reduced-representation genome sequencing technique and its applications in ornamental plants. Acta Horticulturae Sinica, 47(6):1194-1202. (in Chinese)
	熊燕, 张金柱, 董婕, 王力, 高鹏, 姜琬, 车代弟. 2020. 简化基因组测序技术在观赏植物中的应用研究进展. 园艺学报, 47(6):1194-1202.
[25]	Xue H, Zhang P, Shi T, Yang J, Wang L, Wang S, Su Y, Zhang H, Qiao Y, Li X. 2018. Genome-wide characterization of simple sequence repeats in Pyrus bretschneideri and their application in an analysis of genetic diversity in pear. BMC Genomics, 19(1):473. doi: 10.1186/s12864-018-4822-7 URL
[26]	Zheng Ting-ting, Wei Wei-lin, Yang Xiang-hui, Lin Shun-quan. 2015. Development of SSR molecular markers based on transcriptome sequencing of Eriobotrya japonica . Subtropical Plant Science, 44(4):274-278. (in Chinese)
	郑婷婷, 魏伟淋, 杨向晖, 林顺权. 2015. 基于枇杷转录组序列的SSR分子标记引物开发. 亚热带植物科学, 44(4):274-278.

标记 Marker	位置 Position	上游引物(5′-3′) F-primer	下游引物(5′-3′) R-primer
EjSSR1	LG06_22780531_22780756	GTGGCGGATACAAGATTTCA	CACCATTTGCCATTCTACCG
EjSSR2	LG07_28161826_28162057	GGCAAGTTTGTCACGCTATT	AAGAAAAGAAAGCGGTAATCGT
EjSSR3	LG02_12437057_12437286	TATGCAGCCATACTAATGTA	TTAGCGTAGACTCCAGGTGA
EjSSR4	LG15_2449146_2449395	ACAAGATTATGCAGCTACCTTAC	CCGCTAACGATGGAATGAAC
EjSSR5	LG17_10249126_10249370	GAGGGTCTGTTGGCTTGGAT	GAAATGTGGGCATCTTCTTC
EjSSR6	LG02_13068397_13068630	AATATGAGACGTACAAGCTG	TCTTTACTCGCACTGAATGT
EjSSR7	LG09_33605598_33605838	AAATGATCCCTCAACTATAACC	CATTGCTCTATACTCGCATC
EjSSR8	LG05_8474062_8474283	ATAAATAGCAGCAAGGGCTCCAT	GAAACCCCTACCTCAAATCCAA
EjSSR9	LG16_306060_306291	TTCGTACTCATTGAGTTTGGTAT	TGTTGATTAGAATACTGCCTCCC
EjSSR10	LG05_7308571_7308855	CCCATAATCCATCTAAGTACCAAA	GACCTTGAAGGACCTCCATACAC
EjSSR11	LG04_14003826_14004069	CTCGGAGGATGATAAATCAATTC	CCTATTCGACAAGGACGACAAT
EjSSR12	LG11_26083798_26084019	GCCTAAACTTGCCTAGATTCTGAC	ACGGAGCCTAAGCGGACTAA
EjSSR13	LG07_37064315_37064546	TTGGACGGGTTGAGGAACTTAG	CCCACGACTTTGTCTCGCTTCT
EjSSR14	LG17_32809624_32809843	TGCAGTGCCGAACTAAGCTACATA	ACCTGCTGTACCGCAACTTCTG
EjSSR15	LG06_27714710_27714930	ATTGGGAAGCCGTAGTTGGTGA	TCGCAGCTCTTTCTTCTCACTT
EjSSR16	LG01_38250590_38250807	AGTTGAGGCTTAGAAGGCAAGG	GAAAGAACTTTGAAAATTCCATCA
EjSSR17	LG12_5320901_5321127	GAGTGGGACTTCGCATTTGATA	GGAATAACTAACGCCCGCAAC
EjSSR18	LG03_45350948_45351163	CTCGTTATTGCGTTCAAGCATATT	TTTCTGTTTGGAGCTGGGATTT
EjSSR19	LG16_264901_265127	CCTTGCCCTGTAGTATTATGTGAT	ATCTGCGGATAAGGAGCACAAG
EjSSR20	LG14_23903168_23903387	TTTGCGTGATGATCGTTGTCCT	ACACTAAACGGGAGGATGTGGG

标记 Marker	位置 Position	上游引物(5′-3′) F-primer	下游引物(5′-3′) R-primer
EjSSR1	LG06_22780531_22780756	GTGGCGGATACAAGATTTCA	CACCATTTGCCATTCTACCG
EjSSR2	LG07_28161826_28162057	GGCAAGTTTGTCACGCTATT	AAGAAAAGAAAGCGGTAATCGT
EjSSR3	LG02_12437057_12437286	TATGCAGCCATACTAATGTA	TTAGCGTAGACTCCAGGTGA
EjSSR4	LG15_2449146_2449395	ACAAGATTATGCAGCTACCTTAC	CCGCTAACGATGGAATGAAC
EjSSR5	LG17_10249126_10249370	GAGGGTCTGTTGGCTTGGAT	GAAATGTGGGCATCTTCTTC
EjSSR6	LG02_13068397_13068630	AATATGAGACGTACAAGCTG	TCTTTACTCGCACTGAATGT
EjSSR7	LG09_33605598_33605838	AAATGATCCCTCAACTATAACC	CATTGCTCTATACTCGCATC
EjSSR8	LG05_8474062_8474283	ATAAATAGCAGCAAGGGCTCCAT	GAAACCCCTACCTCAAATCCAA
EjSSR9	LG16_306060_306291	TTCGTACTCATTGAGTTTGGTAT	TGTTGATTAGAATACTGCCTCCC
EjSSR10	LG05_7308571_7308855	CCCATAATCCATCTAAGTACCAAA	GACCTTGAAGGACCTCCATACAC
EjSSR11	LG04_14003826_14004069	CTCGGAGGATGATAAATCAATTC	CCTATTCGACAAGGACGACAAT
EjSSR12	LG11_26083798_26084019	GCCTAAACTTGCCTAGATTCTGAC	ACGGAGCCTAAGCGGACTAA
EjSSR13	LG07_37064315_37064546	TTGGACGGGTTGAGGAACTTAG	CCCACGACTTTGTCTCGCTTCT
EjSSR14	LG17_32809624_32809843	TGCAGTGCCGAACTAAGCTACATA	ACCTGCTGTACCGCAACTTCTG
EjSSR15	LG06_27714710_27714930	ATTGGGAAGCCGTAGTTGGTGA	TCGCAGCTCTTTCTTCTCACTT
EjSSR16	LG01_38250590_38250807	AGTTGAGGCTTAGAAGGCAAGG	GAAAGAACTTTGAAAATTCCATCA
EjSSR17	LG12_5320901_5321127	GAGTGGGACTTCGCATTTGATA	GGAATAACTAACGCCCGCAAC
EjSSR18	LG03_45350948_45351163	CTCGTTATTGCGTTCAAGCATATT	TTTCTGTTTGGAGCTGGGATTT
EjSSR19	LG16_264901_265127	CCTTGCCCTGTAGTATTATGTGAT	ATCTGCGGATAAGGAGCACAAG
EjSSR20	LG14_23903168_23903387	TTTGCGTGATGATCGTTGTCCT	ACACTAAACGGGAGGATGTGGG

品种 Cultivar	过滤后 reads数 Clean_reads No.	Q30/%	重测序数据登录号 Accession number in NGDC	测序深度 Sequencing depth	SSR序列匹配reads数 Mapped reads of SSR sequences	SSR序列平均匹配reads数 Average number of mapped reads for each SSR sequence	检测到的位点数 Detected loci
早钟6号 Zaozhong 6	32 739 024	92.35	CRR144065	12.9×	3 979 224	38.4	82 844
长崎早生 Changqizaosheng	35 296 384	92.02	CRR144066	13.9×	4 311 146	41.6	84 013
茂木 Maomu	37 259 455	92.30	CRR144067	14.7×	4 439 618	42.9	84 265
大五星 Dawuxing	53 101 602	90.54	CRR144068	20.9×	6 407 584	61.9	86 560
月光 Yueguang	53 088 745	91.36	CRR144069	20.9×	6 406 910	61.9	86 712
宁海白 Ninghaibai	34 771 714	91.50	CRR144070	13.7×	4 207 108	40.6	86 373
白玉 Baiyu	49 199 883	90.30	CRR144071	19.4×	5 915 743	57.2	86 267
二八东 Erbadong	37 032 139	92.03	CRR144072	14.6×	4 016 303	38.8	84 313
七星^* Sevenstar^*	158 575 610	93.41	CRR078405	62.6×	18 755 558	181.2	93 943
火炬^* Huoju^*	206 173 911	93.16	CRR056810	81.3×	25 660 354	247.9	90 345

品种 Cultivar	过滤后 reads数 Clean_reads No.	Q30/%	重测序数据登录号 Accession number in NGDC	测序深度 Sequencing depth	SSR序列匹配reads数 Mapped reads of SSR sequences	SSR序列平均匹配reads数 Average number of mapped reads for each SSR sequence	检测到的位点数 Detected loci
早钟6号 Zaozhong 6	32 739 024	92.35	CRR144065	12.9×	3 979 224	38.4	82 844
长崎早生 Changqizaosheng	35 296 384	92.02	CRR144066	13.9×	4 311 146	41.6	84 013
茂木 Maomu	37 259 455	92.30	CRR144067	14.7×	4 439 618	42.9	84 265
大五星 Dawuxing	53 101 602	90.54	CRR144068	20.9×	6 407 584	61.9	86 560
月光 Yueguang	53 088 745	91.36	CRR144069	20.9×	6 406 910	61.9	86 712
宁海白 Ninghaibai	34 771 714	91.50	CRR144070	13.7×	4 207 108	40.6	86 373
白玉 Baiyu	49 199 883	90.30	CRR144071	19.4×	5 915 743	57.2	86 267
二八东 Erbadong	37 032 139	92.03	CRR144072	14.6×	4 016 303	38.8	84 313
七星^* Sevenstar^*	158 575 610	93.41	CRR078405	62.6×	18 755 558	181.2	93 943
火炬^* Huoju^*	206 173 911	93.16	CRR056810	81.3×	25 660 354	247.9	90 345