Genome-wide Development of SSR Primers and Genetic Diversity Analysis in Blueberry

doi:10.13560/j.cnki.biotech.bull.1985.2025-1404

Abstract

Abstract:

Objective To address the challenges posed by morphologically ambiguous blueberry cultivars and the lack of a precise identification system, this study aimed to mine genome-wide SSR loci, analyze their characteristics, and develop novel SSR markers, thereby providing a solid foundation for accurate authentication of blueberry germplasm and assessment of genetic diversity. Method SSR loci within the 'Draper' genome were mined and analyzed using Krait software, and primers were designed with Primer 3.0. Preliminary screening of SSR marker primers was performed through bioinformatics analysis. PCR amplification was conducted using a gradually expanded blueberry germplasm collection. A combination of non-denaturing polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis was employed to screen for highly polymorphic SSR markers. Subsequently, genetic diversity analysis of blueberry germplasm was carried out using the finally selected highly polymorphic primers. Result A total of 888 906 SSR loci were identified in the 'Draper' genome. Dinucleotide repeat motifs were the dominant type, accounting for 87.80% of the total, followed by trinucleotide repeats, which represented 6.34%. Following bioinformatic primer design and preliminary screening, 103 pairs of SSR marker primers were obtained. Subsequent multi-round electrophoretic screening yielded 17 pairs of highly polymorphic SSR markers. These were used to analyze the genetic diversity of 64 blueberry germplasm accessions. A total of 204 alleles were detected. The average effective number of alleles (Ne) was 6.165, the average Shannon’s information index (I) was 1.956, and the average polymorphism information content (PIC) was 0.985, indicating that the developed SSR markers are highly polymorphic. UPGMA cluster analysis showed that the 64 blueberry accessions could be grouped into three distinct clusters at a Nei’s genetic distance of 0.000 8. Conclusion This study characterized the abundance and motif distribution of SSR loci in the blueberry genome. Seventeen highly polymorphic SSR primer pairs were developed, which proved effective for distinguishing blueberry germplasm accessions. Furthermore, the genetic diversity among blueberry cultivars was found to be high.

Key words: Vaccinium spp., whole-genome, SSR molecular marker, SSR primer, genetic diversity

LIU Si-wen, WANG Bo-wei, XING Jin-jie, WANG Jing, LI Ya-dong, SUN Hai-yue. Genome-wide Development of SSR Primers and Genetic Diversity Analysis in Blueberry[J]. Biotechnology Bulletin, 2026, 42(11): 134-144.

Figures/Tables 11

References 40

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编号 Code	品种名 Cultivar name	编号 Code	品种名 Cultivar name	编号 Code	品种名 Cultivar name	编号 Code	品种名 Cultivar name
1	Draper	17	蓝塔	33	努伊	49	Emil
2	利宝	18	泽西	34	伯灵顿	50	L25
3	甜心	19	雷格西	35	艾玛	51	1502
4	Biloxi	20	博尼法西	36	AF1	52	H5
5	X13	21	赫伯特	37	AF2	53	Y42
6	北春	22	布里吉塔	38	AF3	54	F6
7	North sky	23	托柔	39	Small fruit	55	L29
8	North blue	24	日升	40	Jgdrzej	56	122
9	北陆	25	奥林匹克	41	HL2	57	L11
10	慧蓝	26	早蓝	42	HL3	58	49
11	圣云	27	都克	43	HL4	59	O2
12	蓝金	28	晚蓝	44	HL5	60	L45
13	普特	29	艾玛蓝	45	HL9	61	YOU
14	蓝丰	30	瑞卡	46	贝尔	62	DS01
15	H7	31	考林	47	格瑞萨	63	DS04
16	齐伯瓦	32	Record	48	北卫	64	D15

编号 Code	品种名 Cultivar name	编号 Code	品种名 Cultivar name	编号 Code	品种名 Cultivar name	编号 Code	品种名 Cultivar name
1	Draper	17	蓝塔	33	努伊	49	Emil
2	利宝	18	泽西	34	伯灵顿	50	L25
3	甜心	19	雷格西	35	艾玛	51	1502
4	Biloxi	20	博尼法西	36	AF1	52	H5
5	X13	21	赫伯特	37	AF2	53	Y42
6	北春	22	布里吉塔	38	AF3	54	F6
7	North sky	23	托柔	39	Small fruit	55	L29
8	North blue	24	日升	40	Jgdrzej	56	122
9	北陆	25	奥林匹克	41	HL2	57	L11
10	慧蓝	26	早蓝	42	HL3	58	49
11	圣云	27	都克	43	HL4	59	O2
12	蓝金	28	晚蓝	44	HL5	60	L45
13	普特	29	艾玛蓝	45	HL9	61	YOU
14	蓝丰	30	瑞卡	46	贝尔	62	DS01
15	H7	31	考林	47	格瑞萨	63	DS04
16	齐伯瓦	32	Record	48	北卫	64	D15

名称 Name	正向引物序列 Forward primer sequences （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）	退火温度 Tm （℃）	预期产物 Expected size （bp）	荧光类型 Fluorescent types
spSSR03	CTGTGTCGACCTCTCCAACG	TGAGATCGGTGGCTTTCTCG	57	242	FAM
spSSR04	CTTGGCGATCTCCTCCTTCC	TCAAGGAACAACAGGCCTCC	55	320	HEX
spSSR05	GCTTCCGGTACCTCTCTTCG	TCATCCCGCACAAGAGTTCC	57	283	HEX
spSSR08	ATCTGAAGTCTCCAACGGCG	GCTAACCCGAGTCTGGTTCC	55	262	HEX
spSSR09	TGTGGCTGGAGATGTTGACG	CAAAATGCGATTCCTCCTCCC	57	236	FAM
spSSR11	AAAGGGGACAAGCAACTCCC	CACCACTTCCGGGATAGTGC	57	252	FAM
spSSR12	ACGACGTGAGAAACCACTCC	CTATCCCTCAACCGCGATGG	57	262	HEX
spSSR13	GAGAGAGAGCCAGTCCTTGC	ACGACGAACCAGACGAAACC	57	194	FAM
spSSR14	ACGACGAACCAGACGAAACC	GAGAGAGAGCCAGTCCTTGC	59	209	FAM
spSSR15	TGAACCAGACGAAACCCACC	GAGAGAGAGCCAGTCCTTGC	59	178	FAM
spSSR16	CAAGTCGACAACATGCTCCG	TTTACAAGCACATACGCGCG	59	218	FAM
spSSR18	CTTGGCGATCTCCTCCTTCC	TCAAGGAACAACAGGCCTCC	59	332	HEX
spSSR19	TTCCAAAACCACCAAACGCC	AGAGCAATGTGGTAGCCAGG	55	326	HEX
spSSR20	GGCTTACAGCTCCTCCATGG	GGTGATGAGAGAGAGCCAGG	59	175	FAM
spSSR21	CTATCCCTCAACCGCGATGG	ACGACGTGAGAAACCACTCC	59	268	HEX
spSSR22	TGTGCATCCAAACCTTGAGC	TGTGGTGGTGGTTATGGAGG	59	238	FAM
spSSR23	GAGGCGGTAGTACATGTGGG	CTCTCAACTCCCCTTCAGCC	57	340	HEX

名称 Name	正向引物序列 Forward primer sequences （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）	退火温度 Tm （℃）	预期产物 Expected size （bp）	荧光类型 Fluorescent types
spSSR03	CTGTGTCGACCTCTCCAACG	TGAGATCGGTGGCTTTCTCG	57	242	FAM
spSSR04	CTTGGCGATCTCCTCCTTCC	TCAAGGAACAACAGGCCTCC	55	320	HEX
spSSR05	GCTTCCGGTACCTCTCTTCG	TCATCCCGCACAAGAGTTCC	57	283	HEX
spSSR08	ATCTGAAGTCTCCAACGGCG	GCTAACCCGAGTCTGGTTCC	55	262	HEX
spSSR09	TGTGGCTGGAGATGTTGACG	CAAAATGCGATTCCTCCTCCC	57	236	FAM
spSSR11	AAAGGGGACAAGCAACTCCC	CACCACTTCCGGGATAGTGC	57	252	FAM
spSSR12	ACGACGTGAGAAACCACTCC	CTATCCCTCAACCGCGATGG	57	262	HEX
spSSR13	GAGAGAGAGCCAGTCCTTGC	ACGACGAACCAGACGAAACC	57	194	FAM
spSSR14	ACGACGAACCAGACGAAACC	GAGAGAGAGCCAGTCCTTGC	59	209	FAM
spSSR15	TGAACCAGACGAAACCCACC	GAGAGAGAGCCAGTCCTTGC	59	178	FAM
spSSR16	CAAGTCGACAACATGCTCCG	TTTACAAGCACATACGCGCG	59	218	FAM
spSSR18	CTTGGCGATCTCCTCCTTCC	TCAAGGAACAACAGGCCTCC	59	332	HEX
spSSR19	TTCCAAAACCACCAAACGCC	AGAGCAATGTGGTAGCCAGG	55	326	HEX
spSSR20	GGCTTACAGCTCCTCCATGG	GGTGATGAGAGAGAGCCAGG	59	175	FAM
spSSR21	CTATCCCTCAACCGCGATGG	ACGACGTGAGAAACCACTCC	59	268	HEX
spSSR22	TGTGCATCCAAACCTTGAGC	TGTGGTGGTGGTTATGGAGG	59	238	FAM
spSSR23	GAGGCGGTAGTACATGTGGG	CTCTCAACTCCCCTTCAGCC	57	340	HEX

名称 Name	等位基因数Na	有效等位基因数Ne	Shannon’s信息指数I	观测杂合度Ho	期望杂合度He	无偏期望杂合度uHe	多态性信息量PIC
spSSR03	6	3.626	1.405	0.714	0.724	0.730	0.983
spSSR04	16	10.431	2.520	0.750	0.904	0.911	0.990
spSSR05	5	2.602	1.083	0.500	0.616	0.623	0.978
spSSR08	9	4.958	1.786	0.583	0.798	0.807	0.986
spSSR09	9	3.292	1.475	1.000	0.696	0.702	0.973
spSSR11	10	4.698	1.789	1.000	0.787	0.794	0.982
spSSR12	9	4.898	1.735	1.000	0.796	0.803	0.984
spSSR13	17	5.737	2.154	1.000	0.826	0.832	0.988
spSSR14	15	7.008	2.188	1.000	0.857	0.864	0.990
spSSR15	19	10.139	2.558	0.800	0.901	0.908	0.990
spSSR16	14	9.806	2.383	0.667	0.898	0.905	0.989
spSSR18	16	10.711	2.542	0.200	0.907	0.914	0.990
spSSR19	13	6.122	2.099	1.000	0.837	0.844	0.985
spSSR20	13	5.306	1.993	1.000	0.812	0.818	0.980
spSSR21	11	3.924	1.631	1.000	0.745	0.751	0.981
spSSR22	10	5.760	1.913	1.000	0.826	0.833	0.983
spSSR23	12	5.796	1.992	1.000	0.827	0.834	0.983
平均Mean	12	6.165	1.956	0.836	0.809	0.816	0.985