青钱柳全基因组SSR位点分析及多态性引物开发

doi:10.12302/j.issn.1000-2006.202206001

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南京林业大学学报（自然科学版） ›› 2024, Vol. 48 ›› Issue (4): 67-75.doi: 10.12302/j.issn.1000-2006.202206001

所属专题：专题报道Ⅱ：乡村振兴视域下药用树种青钱柳培育研究

• 专题报道Ⅱ：乡村振兴视域下药用树种青钱柳培育研究（执行主编李维林、方升佐） • 上一篇下一篇

青钱柳全基因组SSR位点分析及多态性引物开发

刘莉¹^,²(), 瞿印权¹, 余延浩¹, 王倩¹, 洑香香¹^,^*()

1.南京林业大学林草学院,江苏南京 210037
2.上海市浦东新区林业站,上海 200120

收稿日期:2022-06-01 修回日期:2022-09-05 出版日期:2024-07-30 发布日期:2024-08-05
通讯作者: *(xxfu@njfu.edu.cn),洑香香,教授。
作者简介:
刘莉(943444013@qq.com)。
基金资助:
国家自然科学基金项目(31971642);中央财政林业科技推广示范项目(皖[2023]TG13号)

Analysis of SSR locus based on the whole genome sequences of Cyclocarya paliurus and the development of polymorphic primers

LIU Li¹^,²(), QU Yinquan¹, YU Yanhao¹, WANG Qian¹, FU Xiangxiang¹^,^*()

1. College of Forestry and Grassland, Nanjing Forestry University, Nanjing 210037, China
2. Shanghai Pudong Forestry Station, Shanghai 200120, China

Received:2022-06-01 Revised:2022-09-05 Online:2024-07-30 Published:2024-08-05

摘要/Abstract

摘要：

【目的】通过对青钱柳全基因组序列分析,开发基因组SSR分子标记;尝试构建19个青钱柳优良药用无性系的DNA分子身份证,为后续种质资源评价、遗传多样性和种质鉴定提供技术支撑。【方法】利用MISA(microsatellite identification tool)软件对青钱柳全基因组进行SSR位点搜寻、筛选、识别及富集分析,采用Primer 3.0进行SSR引物设计;用重复性和稳定性高的SSR标记构建青钱柳无性系的识别系统。【结果】①从全基因组中共检测出89 741个SSR位点,SSR位点的发生频率为62.07%。②基因组SSR位点中单核苷酸重复单元比例最高,占总SSR位点的62.67%;六核苷酸重复单元比例最低,占0.15%;SSR位点的重复基序大多以(A/T)n为主。③单核苷酸和二核苷酸重复类型的SSR位点基序重复次数集中在6~16次;随重复次数增加,各SSR位点重复类型出现频率均呈下降趋势。④基因组SSR序列长度介于10~476 bp,不同类型重复单元的SSR序列长度存在变异性;随着重复次数的增加,SSR序列出现的频率整体呈下降趋势。⑤利用Primer 3.0成功设计出78 285对SSR引物;合成的377对中有75对引物可扩增出多态性条带;用5对单碱基重复的多态性SSR引物分析19个药用无性系,构建出无性系的二维码DNA分子身份证。【结论】青钱柳基因组SSR位点出现频率高,位点种类丰富,可为种质资源评价及指纹图谱的构建提供丰富的候选分子标记。

关键词: 青钱柳, 简单重复序列(SSR), 全基因组, DNA分子身份证

Abstract:

【Objective】Genomic simple repeat sequence (SSR) loci were analyzed by screening the whole genome of Cyclocarya paliurus. DNA molecular ID cards of 19 excellent medicinal clones of C. paliurus were constructed based on the newly-developed SSR primers. These genomic SSR markers could support further research, such as the evaluation of the germplasm resource, analysis of genetic diversity, and identification of cultivars/clones.【Method】The SSR loci were screened along with the whole genome of C. paliurus and were enriched and analyzed using MISA software. Subsequently, SSR primers were designed using Primer 3.0. Furthermore, a system for identifying clones of C. paliurus was constructed based on selected SSR markers with high reproducibility and stability.【Result】(1) We detected 89 741 SSR loci from the whole genome, with an occurrence frequency of 62.07%. (2) Among all SSR loci, the proportion of SSRs with a mononucleotide motif was the highest (62.67%) and a hexa-nucleotide repeat was the lowest (0.15%). Most of the repeated motifs in the SSR loci were dominated by (A/T)n. (3) The repeat number of mono-nucleotide and di-nucleotide motifs ranged from 6 to 16. With the increase in the repeat number, the frequencies of various SSR repetition types displayed a downward trend. (4) The length of the SSR sequences varied from 10 to 476 bp, and this length variation existed in different repetitive motifs. Additionally, the frequency of SSR occurrence tended to decrease as the repeat number increased. (5) We successfully designed 78 285 pairs of SSR primers using Primer 3.0. A total of 377 primer pairs were randomly synthesized for amplifying polymorphic SSR fragments, among which 75 pairs primers were successful. Moreover, quick response code DNA molecular ID cards for 19 medical-use clones of C. paliurus were constructed by five pairs of polymorphic SSR primers with a mono-nucleotide motif.【Conclusion】The frequency of genomic SSR loci was high, and there was variability in the type of SSR loci. Simple repeat sequences developed from the whole genome of C. paliurus could be effective candidate molecular markers with further applications in germplasm resource evaluation and fingerprint construction for multi-use clones of C. paliurus.

Key words: Cyclocarya paliurus, simple sequence repeat (SSR), whole genome, DNA molecular ID card

中图分类号:

S722
Q756

刘莉,瞿印权,余延浩,等. 青钱柳全基因组SSR位点分析及多态性引物开发[J]. 南京林业大学学报（自然科学版）, 2024, 48(4): 67-75.

LIU Li, QU Yinquan, YU Yanhao, WANG Qian, FU Xiangxiang. Analysis of SSR locus based on the whole genome sequences of Cyclocarya paliurus and the development of polymorphic primers[J].Journal of Nanjing Forestry University (Natural Science Edition), 2024, 48(4): 67-75.DOI: 10.12302/j.issn.1000-2006.202206001.

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SSR类型 SSR type	数量 number	重复次数 repeat time	平均长度/bp average length	百分比/% percentage	重复单元类型 motif type
SSR类型 SSR type	数量 number	重复次数 repeat time	平均长度/bp average length	百分比/% percentage	数量 number		主要基序 main motif
单核苷酸 mono-nucleotide	56 242	10~73	12.61	62.67	4	A/T、C/G
二核苷酸 di-nucleotide	27 291	6~86	19.78	30.41	12	AC/GT、AG/CT、AT/AT、CG/CG
三核苷酸 tri-nucleotide	5 014	5~32	18.68	5.59	59	AAT/ATT、AAG/CTT、ACG/CGT
四核苷酸 tetra-nucleotide	712	5~10	21.67	0.79	101	AAAG/CTTT、AAAT/ATTT、ACAT/ATGT
五核苷酸 penta-nucleotide	351	5~9	26.62	0.39	90	AGATG/ATCTC、AACTC/AGTTG AAAAG/CTTTT、 AAAAT/ATTTT
六核苷酸 hexa-nucleotide	131	5~17	33.02	0.15	88	AAAAAG/CTTTTT、AAAAAT/ATTTTT、 AGAGGG/CCCTCT
总计 total	89 741			100.00	354

引物来源 primer source	引物数量 number of primers	多态性引物数 number of polymorphic primers	有效引物数 number of valid primers	有效扩增率/% effective amplification rate	多态性比例/% polymor- phism ratio
单核苷酸 mono-nucleotide	142	10	75	52.82	7.04
二核苷酸 di-nucleotide	104	8	86	90.38	7.69
三核苷酸 tri-nucleotide	131	57	119	90.84	43.51
总计 total	377	75	280	74.27	19.89

引物名称 primer	引物序列(5'-3') primer sequence (5'-3')	重复基元 repeat unit	预计产物长度/bp expected length of production	实际产物长度/bp actual length of production	扩增总条带数 bands amplified	多态性条带数 polymorphic bands	多态性比率/% polymorphic ratio
CpSSR-6	F: AAGCTTGGCTTTTGCATGAT	(A)12	124	126~129	3	1	33.3
	R: AACTCCAGTAGCAGGGCTCA
CpSSR-10	F: AAGTCCCTGGTGATGGTGAG	(A)10	141	136~234	4	3	75.0
	R: GTCCGCTGAGTTCTTGAAGG
CpSSR-43	F: AAGGAGCGCAGGTATTCAGA	(A)15	191	143~296	5	4	80.0
	R: GCACAGGCAACTCAATCTCA
CpSSR-46	F: GCTTGGATTGCTCACAGTCA	(A)11	222	223~269	3	2	66.7
	R: AAAGCCGCTTGACTACGAAA
CpSSR-139	F: AAGGAGCGCAGGTATTCAGA	(T)10	191	118~192	5	4	80.0
	R: GCACAGGCAACTCAATCTCA
合计total					20	14	67.0
均值mean					4	2.8

引物名称 primer	荧光基团 fluorescent group	多态性片段编码/bp code of polymorphism fragments
		A 192
		B 192/190
CpSSR-139	FAM	C 192/118
		D 192/190/143
		E 192/190/187
		A 147/144
		B 144
CpSSR-10	ROX	C 144/136
		D234/147/144
		E234/144
		A 239/192/190
		B 296/192
CpSSR-43	FAM	C 192/190
		D 192
		E 296/192/190
		F192/190/143
CpSSR-46	TAMRA	A 223
		B 226/223
		C 226
		D 229/226/223
CpSSR-6	TAMRA	A 129/127/126
		B 127/126

青钱柳全基因组SSR位点分析及多态性引物开发

Analysis of SSR locus based on the whole genome sequences of Cyclocarya paliurus and the development of polymorphic primers

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