Designing of microsatellite primers for Quercus glauca and Q. glaucoides (Fagaceae) based on RAD-seq data

doi:10.12302/j.issn.1000-2006.202212002

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2024, Vol. 48 ›› Issue (6): 62-70.doi: 10.12302/j.issn.1000-2006.202212002

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Designing of microsatellite primers for Quercus glauca and Q. glaucoides (Fagaceae) based on RAD-seq data

OUYANG Zeyi¹^,²(), LI Zhihui¹, MOU Honglin¹, JIANG Xiaolong¹, CHENG Yong³, WU Jiyou³^,^*()

1. College of Forestry, Central South University of Forestry and Technology, Changsha 410116, China
2. Hunan Botanical Garden, Changsha 410116, China
3. Hunan Academy of Forestry, Changsha 410004, China

Received:2022-12-01 Revised:2023-01-10 Online:2024-11-30 Published:2024-12-10
Contact: WU Jiyou E-mail:455281453@qq.com;805610788@qq.com

Abstract

Abstract:

【Objective】Quercus glauca and Q. glaucoides are valuable and dominant species in the subtropical evergreen broadleaf forests of East Asia. They represent typical geographical vicarious species with significant ecological and economic importance. Therefore, the development of SSR primers for these two species can facilitate the analysis of genetic patterns and genetic diversity for the management and resource development of evergreen broadleaf forests as well as provide a reference for the development of microsatellite markers across species.【Method】This study developed SSR primers based on RADseq data from three Q. glauca and three Q. glaucoides individuals, respectively. The sequencing data were filtered and extracted using the process_radtags model in Stacks 2.0b software and SciRoKo 3.4 software sequentially. SSR primers were designed using Primer premier V6.0 software.【Result】The sequences were clustered using pyRAD 3.0.66, identifying a total of 217 SSR loci, 35% (76) of which were polymorphic in both species. Twenty-eight SSR primer pairs were designed and validated in two Q. glauca populations and two Q. glaucoides populations (48 individuals in total) through nested polymerase chain reaction (PCR) amplification. The 28 SSR primer pairs are distributed across ten chromosomes of the Q. glauca genome and successfully amplified in Q. glauca and Q. glaucoides individuals, with an amplification rate of 90.7%. The SSR genotyping analysis detected a total of 176 alleles, with the number of alleles per primer ranging from 3 to 13, and an average of 6.29. The expected and observed heterozygosity of the primers ranged from 0.223 to 0.886 and from 0.159 to 0.830, respectively.【Conclusion】The universal microsatellite primers developed in this study using RADseq data for Q. glauca and Q. glaucoides provide a basis for further population genetics studies of these species. In addition, this study demonstrates that RADseq data can be employed to rapidly, efficiently, and be used to cost-effectively develop universal microsatellite primers for closely related species.

Key words: Quercus glauca, Quercus glaucoides, microsatellite, RAD-seq

CLC Number:

S795

OUYANG Zeyi, LI Zhihui, MOU Honglin, JIANG Xiaolong, CHENG Yong, WU Jiyou. Designing of microsatellite primers for Quercus glauca and Q. glaucoides (Fagaceae) based on RAD-seq data[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2024, 48(6): 62-70.

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类型 type	样本编号 sample ID	采集地 location of collection	东经/(°) longitude (E)	北纬/(°) latitude (N)	海拔/m elevation
	gDM16207_5	浙江省遂昌县	118.85	28.38	718
	gDM18375	江西省吉安市	114.20	26.52	406
简化基因组测序RADseq	gDM18581	西藏自治区察隅县	97.08	28.56	1 645
	sDM11817	云南省晋宁县	102.77	24.70	2 004
	sDM17689	云南省巧家县	102.92	27.28	2 189
	sDM17825	云南省广南县	104.97	23.77	1 715
	gDM20391-gDM20402	江西省铅山县	117.75	27.89	658
	gDM21167-gDM21178	日本埼玉县	139.42	35.98	35
扩增验证PCR	sDM12688-sDM12693 sDM13035-sDM13040	云南省元谋县	101.95	25.72	2 093
	sDM10272-sDM 10275 sDM10280-sDM10287	广西壮族自治区隆林县	105.19	24.65	1 497

样本编号 sample ID	序列数 number of sequences	过滤后序列数 number of filter sequences	SSR聚类位点数 number of SSR Clustering Loci	一致性位点数 number of consistent loci	杂合度 heterozygosity	测序错误率/% sequencing error rate
gDM16207_5	9 391 215	8 073 849	20 415	1 808	0.006 656	0.070 44
gDM18375	9 095 105	8 382 814	23 195	1 808	0.007 887	0.080 43
gDM18581	9 140 314	8 397 947	26 498	1 808	0.006 120	0.067 51
sDM11817	9 515 559	8 166 681	28 418	1 808	0.007 409	0.069 65
sDM17689	9 364 847	8 376 899	30 229	1 808	0.006 690	0.079 68
sDM17825	8 466 497	7 689 333	33 170	1 808	0.007 648	0.075 63

引物号 primer code	正向序列(5'—3') forward sequence	反向引物序列(5'—3') forward sequence	NCBI序列号 accession number
GS2	TTGATGAACTGCGTACTCC	AAGCGAACCCAATTCCAC	MZ170721
GS3	ATGATTTACCGGATCTGGAG	CATGATTCAGTGCCATCTG	MZ170722
GS6	CTTGTGCCGACATTCTTG	ACCACCAGCCTGAAATTC	MZ170723
GS15	ATTTCTCTTCTGGGTCGGA	GCTGACCTTTCTTGACTTTG	MZ170724
GS19	GGTGAGTAGACGGAGGTT	CCTTCACTTCTTCTTCATCCT	MZ170725
GS28	CACCAAGTGTGGCCTTTC	TGAAGAGCAGAAATGGATGG	MZ170726
GS32	TCTGGTATAGAATCCAACGA	GCCCTTCATACTTCTTTGTT	MZ170727
GS33	GGTTCTGGTTCTGGTTCTG	CGCTACAAATTCCTTGCTTT	MZ170728
GS35	CTCATCTCCTACCTCATTCTT	TCCCTTTGCTGACTTTGT	MZ170729
GS74	TTCCTACTTCCTAGTGAGAG	ATGAGGTCAGGCTCCTTA	MZ170730
GS83	CACAATCACAATCACCAACA	TCCTTGTCTTCCTCCTCTT	MZ170731
GS84	TGGCTTCTTCTTCCTATATGAG	GCGTATGCTGGTAAGTGAT	MZ170732
GS88	TCGGCACGGATCGTAATG	CAACCCATGAAGCTGAACT	MZ170733
GS105	CGCTTTCGCTTTGAAATTG	CTACACAAATTGCGTTTCAG	MZ170734
GS117	AGGATCTGGATCAGTAAGC	AACGTAGCACAGTAACATG	MZ170735
GS119	ATCCGGTCATGTACTTCATT	GCAACCAAACGGAGAGTA	MZ170736
GS122	CGCCATTTGGTTGTAATCT	GCTGTTAGATGATCCGTTAG	MZ170737
GS150	GGGATGAAACAAATTAGGAC	CCAATCGTTATTTAGCAAGG	MZ170738
GS160	CCTGTCTTCTTCTTCTTCTTC	ACTAGGAGATGATGATATACCC	MZ170739
GS165	CGGGTTTGGTCCAACAAC	CGCACTGAGCTTCCCTTT	MZ170740
GS169	GGCTGGCTTCCAAATCTT	AATATGGAATGGACCACTCA	MZ170741
GS171	TTCCTCTAGTCTCTTCAGTCT	TGAGATACATACTTGGCTTACC	MZ170742
GS172	CTCTCAGCTACAACAACAA	AGACCCATTCTCACTTCAA	MZ170743
GS180	CCAAAGAACTGGAGCTTTATG	CTTTCACCATGTGTCCCTC	MZ170744
GS185	TAGGTGGCGGTCTAGGTT	AGCTTTGCACAGAGTCTC	MZ170745
GS186	TCTGTGTTATGGACTCTGAG	ACAACGTAAGACGGTAAGA	MZ170746
GS209	TTCAGTGAAATATCACCTGC	CCAAGAAATCATCCTCTTCTC	MZ170747
GS216	AAGTGAGACTCGGTTCAA	GGCTTCCCTCTAAGTTTCT	MZ170721

引物号 primer code	重复基序 repeat motifs	序列长度/bp sequences length	个体数 sample size	N_Aa	N_Ab	期望杂合度 expected heterozygosity	观测杂合度 observed heterozygosity
GS2	TGT	138~156	47	5	7	0.739	0.319
GS3	GTT	137~155	48	2	5	0.471	0.333
GS6	CT	134~160	42	4	13	0.862	0.714
GS15	CCA	125~134	44	3	4	0.553	0.341
GS19	TGGTTT	114~126	47	3	3	0.594	0.617
GS28	CT	133~153	46	5	11	0.841	0.783
GS32	AACAAA	109~133	39	2	5	0.642	0.410
GS33	GGTTCT	121~133	48	3	3	0.484	0.750
GS35	AG	121~143	47	4	12	0.873	0.830
GS74	GA	124~128	33	3	3	0.432	0.212
GS83	AG	108~130	46	5	12	0.886	0.739
GS84	CA	129~133	44	2	3	0.223	0.159
GS88	TGT	121~127	40	3	3	0.405	0.250
GS105	AG	115~129	41	5	8	0.808	0.463
GS117	AACAC	121~141	33	2	5	0.730	0.636
GS119	TTTGC	117~127	43	2	3	0.577	0.326
GS122	AG	130~136	42	2	4	0.584	0.500
GS150	ATAAA	139~154	46	2	4	0.664	0.283
GS160	TTC	119~138	43	4	8	0.810	0.465
GS165	GAA	120~129	40	3	4	0.669	0.475
GS169	GTGA	145~153	46	3	3	0.591	0.326
GS171	CT	105~121	45	3	10	0.836	0.733
GS172	AAG	134~152	34	3	7	0.755	0.382
GS180	AGA	108~132	43	5	9	0.740	0.442
GS185	TC	146~148	48	2	6	0.736	0.396
GS186	GA	146~168	48	5	9	0.795	0.792
GS209	GA	124~144	48	4	7	0.678	0.667
GS216	AGT	124~136	47	4	5	0.701	0.404

Designing of microsatellite primers for Quercus glauca and Q. glaucoides (Fagaceae) based on RAD-seq data

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