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

doi:10.12302/j.issn.1000-2006.202206001

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2024, Vol. 48 ›› Issue (4): 67-75.doi: 10.12302/j.issn.1000-2006.202206001

Special Issue: 专题报道Ⅱ：乡村振兴视域下药用树种青钱柳培育研究

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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
Contact: FU Xiangxiang E-mail:943444013@qq.com;xxfu@njfu.edu.cn

Abstract

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

CLC Number:

S722
Q756

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, 2024, 48(4): 67-75.

Figures/Tables 7

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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

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

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[8]	FANG Shengzuo. A review on the development history and the resource silviculture of Cyclocarya paliurus industry [J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2022, 46(6): 115-126.
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