橄榄转录组SSR信息分析及分子标记开发与应用

doi:10.16420/j.issn.0513-353x.2023-0355

摘要/Abstract

摘要：

选取不同品种（系）橄榄成熟果进行转录组测序，结果获得296 314条序列，应用MISA软件进行SSR位点分析，获得86 084个SSR位点，分布在70 686条序列中，SSR在所检测序列中出现频率为23.86%，其中54 735条序列含有两个及以上的SSR位点，占比达68.4%；橄榄转录组中SSR序列以复合型核苷酸、单核苷酸、二核苷酸重复类型为主，三者占SSR总数的88.88%；单核苷酸、二核苷酸重复类型中优势基元分别为A/T、AG/CT/TC/GA。以6个不同橄榄品系（种）为研究对象，对随机挑选的99对SSR引物进行有效性与多态性筛选，最终开发了53个有效性SSR分子标记，12个多态性SSR分子标记。利用开发的12个多态性EST-SSR标记对59份橄榄种质进行多态性评价与群体结构分析，结果共检测到48个多态性位点，香农多样性指数平均0.876，多态信息含量平均0.426。利用混群模型分组、UPGMA聚类和PCA对59份橄榄种质进行群体结构分析，混群模型分析结果与UPGMA聚类结果、主成分分析结果有类群上的交叉，但类群数有所不同，混群模型将其划分为3个类群；UPGMA聚类分析在遗传相似系数为0.68处划分为2大种群，与PCA方法分类结果相同。研究结果认为开发的12对SSR标记遗传多样性丰富、有效，为橄榄种质资源鉴别提供可靠的基础工具。

关键词: 橄榄, 转录组, SSR, 多态性引物, 遗传多样性, 群体结构

Abstract:

The mature fruits of different varieties（lines）of Canarium album L. were selected for transcriptome sequencing，and 296 314 sequences were obtained. MISA software was used to analyze SSR loci，and 86 084 SSR loci were obtained，distributed in 70 686 sequences. The frequency of SSR in the detected sequences was 23.86%，of which 54 735 sequences contained two or more SSR loci，accounting for 68.4%. The SSR sequences in the Chinese olive transcriptome were mainly composed of compound nucleotide，single nucleotide and dinucleotide repeats，accounting for 88.88% of the total SSRs. The dominant motifs in mononucleotide and dinucleotide repeat types were A/T and AG/CT/TC/GA，respectively. The effectiveness and polymorphism of 99 pairs of SSR primers randomly selected from six different Chinese olive lines（species）were screened. Finally，53 effective SSR molecular markers and 12 polymorphic SSR molecular markers were developed. The polymorphism evaluation and population structure analysis of 59 Chinese olive germplasm were carried out by using 12 polymorphic EST-SSR markers. A total of 48 polymorphic loci were detected. The average Shannon diversity index was 0.876，and the average polymorphic information content was 0.426. The population structure of 59 Chinese olive germplasms was analyzed by three methods of mixed group model grouping，UPGMA clustering and PCA. The results of mixed group model analysis were crossed with those of UPGMA clustering and principal component analysis，but the number of groups was different. The mixed group model was divided into three groups. UPGMA cluster analysis was divided into two populations at the genetic similarity coefficient of 0.68，which was the same as the PCA method. The results of the study concluded that the 12 pairs of SSR markers developed were genetically diverse and valid，and provided a reliable basic tool for Chinese olive germplasm resource identification.

Key words: Canarium album, transcriptome, SSR, polymorphic primer, genetic diversity, population structure

谢倩, 张诗艳, 江来, 丁明月, 刘玲玲, 吴如健, 陈清西. 橄榄转录组SSR信息分析及分子标记开发与应用[J]. 园艺学报, 2023, 50(11): 2350-2364.

XIE Qian, ZHANG Shiyan, JIANG Lai, DING Mingyue, LIU Lingling, WU Rujian, CHEN Qingxi. Analysis of Canarium album Transcriptome SSR Information and Molecular Marker Development and Application[J]. Acta Horticulturae Sinica, 2023, 50(11): 2350-2364.

图/表 11

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种质来源 Germplasm source	编号 Number	代号 Code	种质名称 Germplasm name
福建福州闽侯 Minhou，Fuzhou，Fujian	1	MA1	福榄1号 Fulan 1
	2	MA2	惠圆 Huiyuan
	3	MA3	青皮长营 Qingpi Changying
	4	MA4	大长营 Dachangying
	5	MA5	黄皮长营 Huangpi Changying
	6	MA6	长营 Changying
	7	MA7	檀头 Tantou
	8	MA8	甜榄1号 Tianlan 1
	9	MA9	自来圆1号 Zilaiyuan 1
	10	MA10	自来圆2号 Zilaiyuan 2
福建福州闽清 Minqing，Fuzhou，Fujian	11	MA11	福榄2号 Fulan 2
	12	MA12	灵峰Lingfeng
	13	MA13	闽清1号 Minqing 1
	14	MA14	清榄1号 Qinglan 1
福建福州晋安 Jin’an，Fuzhou，Fujian	15	MA15	实生1 Shisheng 1
	16	MA16	实生2 Shisheng 2
	17	MA17	实生3 Shisheng 3
	18	MA18	实生4 Shisheng 4
福建莆田 Putian，Fujian	19	MB1	庄边1号 Zhuangbian 1
福建泉州南安 Nan’an，Quanzhou，Fujian	20	MC1	南安1号 Nan’an 1
	21	MC2	南安2号 Nan’an 2
福建漳州云霄 Yunxiao，Zhangzhou，Fujian	22	ME1	穗橄榄 Suiganlan
福建漳州漳浦 Zhangpu，Zhangzhou，Fujian	23	ME2	漳浦11号Zhangpu 11
	24	ME3	漳浦7号 Zhangpu 7
福建龙岩永定 Yongding，Longyan，Fujian	25	MF1	永定1号Yongding 1
	26	MF2	永定2号Yongding 2
福建龙岩长汀 Changting，Longyan，Fujian	27	MF3	长汀1号Changting 1
福建南平延平 Yanping，Nanping，Fujian	28	MH1	埔头1号 Putou 1
	29	MH2	剑州榄 Jianzhoulan
福建宁德福安 Fu’an，Ningde，Fujian	30	MJ1	葡萄橄榄 Grape Chinese Olive
	31	MJ2	四季榄1号 Sijilan 1
	32	MJ3	四季榄2号 Sijilan 2
	33	MJ4	子弹橄榄 Bullet Chinese Olive
	34	MJ5	子阳1号 Ziyang 1
广东茂名电白 Dianbai，Maoming，Guangdong	35	YK1	青皮榄 Green-Rind Chinese Olive
广东茂名高州 Gaozhou，Maoming，Guangdong	36	YK2	高州1号 Gaozhou 1
	37	YK3	高州2号 Gaozhou 2
	38	YK4	香榄 Xianglan
广东潮州饶平 Raoping，Chaozhou，Guangdong	39	YU1	棱尖 Lengjian
广东揭阳揭西 Jiexi，Jieyang，Guangdong	40	YV1	大白榄 Dabailan
	41	YV2	东山长穗 Dongshan Changsui
	42	YV3	三棱榄 Sanlenglan
广西钦州浦北 Pubei，Qinzhou，Guangxi	43	GN1	猪腰榄 Zhuyaolan
四川泸州合江 Hejiang，Luzhou，Sichuan	44	CE1	大梭子Dasuozi
	45	CE2	丁香榄 Dingxianglan
	46	CE3	青果Chinese White Olive
	47	CE4	三白圆 Sanbaiyuan
浙江温州平阳 Pingyang，Wenzhou，Zhejiang	48	ZC1	平阳1号 Pingyang 1
	49	ZC2	平阳2号 Pingyang 2
	50	ZC3	平阳3号 Pingyang 3
	51	ZC4	平阳4号 Pingyang 4
	52	ZC5	平阳5号 Pingyang 5
	53	ZC6	平阳6号 Pingyang 6
	54	ZC7	平阳7号 Pingyang 7
	55	ZC8	平阳8号 Pingyang 8
浙江温州瑞安 Rui’an，Wenzhou，Zhejiang	56	ZC9	瑞安1号 Rui’an 1
	57	ZC10	瑞安2号Rui’an 2
	58	ZC11	瑞安3号Rui’an 3
	59	ZC12	瑞安4号Rui’an 4

种质来源 Germplasm source	编号 Number	代号 Code	种质名称 Germplasm name
福建福州闽侯 Minhou，Fuzhou，Fujian	1	MA1	福榄1号 Fulan 1
	2	MA2	惠圆 Huiyuan
	3	MA3	青皮长营 Qingpi Changying
	4	MA4	大长营 Dachangying
	5	MA5	黄皮长营 Huangpi Changying
	6	MA6	长营 Changying
	7	MA7	檀头 Tantou
	8	MA8	甜榄1号 Tianlan 1
	9	MA9	自来圆1号 Zilaiyuan 1
	10	MA10	自来圆2号 Zilaiyuan 2
福建福州闽清 Minqing，Fuzhou，Fujian	11	MA11	福榄2号 Fulan 2
	12	MA12	灵峰Lingfeng
	13	MA13	闽清1号 Minqing 1
	14	MA14	清榄1号 Qinglan 1
福建福州晋安 Jin’an，Fuzhou，Fujian	15	MA15	实生1 Shisheng 1
	16	MA16	实生2 Shisheng 2
	17	MA17	实生3 Shisheng 3
	18	MA18	实生4 Shisheng 4
福建莆田 Putian，Fujian	19	MB1	庄边1号 Zhuangbian 1
福建泉州南安 Nan’an，Quanzhou，Fujian	20	MC1	南安1号 Nan’an 1
	21	MC2	南安2号 Nan’an 2
福建漳州云霄 Yunxiao，Zhangzhou，Fujian	22	ME1	穗橄榄 Suiganlan
福建漳州漳浦 Zhangpu，Zhangzhou，Fujian	23	ME2	漳浦11号Zhangpu 11
	24	ME3	漳浦7号 Zhangpu 7
福建龙岩永定 Yongding，Longyan，Fujian	25	MF1	永定1号Yongding 1
	26	MF2	永定2号Yongding 2
福建龙岩长汀 Changting，Longyan，Fujian	27	MF3	长汀1号Changting 1
福建南平延平 Yanping，Nanping，Fujian	28	MH1	埔头1号 Putou 1
	29	MH2	剑州榄 Jianzhoulan
福建宁德福安 Fu’an，Ningde，Fujian	30	MJ1	葡萄橄榄 Grape Chinese Olive
	31	MJ2	四季榄1号 Sijilan 1
	32	MJ3	四季榄2号 Sijilan 2
	33	MJ4	子弹橄榄 Bullet Chinese Olive
	34	MJ5	子阳1号 Ziyang 1
广东茂名电白 Dianbai，Maoming，Guangdong	35	YK1	青皮榄 Green-Rind Chinese Olive
广东茂名高州 Gaozhou，Maoming，Guangdong	36	YK2	高州1号 Gaozhou 1
	37	YK3	高州2号 Gaozhou 2
	38	YK4	香榄 Xianglan
广东潮州饶平 Raoping，Chaozhou，Guangdong	39	YU1	棱尖 Lengjian
广东揭阳揭西 Jiexi，Jieyang，Guangdong	40	YV1	大白榄 Dabailan
	41	YV2	东山长穗 Dongshan Changsui
	42	YV3	三棱榄 Sanlenglan
广西钦州浦北 Pubei，Qinzhou，Guangxi	43	GN1	猪腰榄 Zhuyaolan
四川泸州合江 Hejiang，Luzhou，Sichuan	44	CE1	大梭子Dasuozi
	45	CE2	丁香榄 Dingxianglan
	46	CE3	青果Chinese White Olive
	47	CE4	三白圆 Sanbaiyuan
浙江温州平阳 Pingyang，Wenzhou，Zhejiang	48	ZC1	平阳1号 Pingyang 1
	49	ZC2	平阳2号 Pingyang 2
	50	ZC3	平阳3号 Pingyang 3
	51	ZC4	平阳4号 Pingyang 4
	52	ZC5	平阳5号 Pingyang 5
	53	ZC6	平阳6号 Pingyang 6
	54	ZC7	平阳7号 Pingyang 7
	55	ZC8	平阳8号 Pingyang 8
浙江温州瑞安 Rui’an，Wenzhou，Zhejiang	56	ZC9	瑞安1号 Rui’an 1
	57	ZC10	瑞安2号Rui’an 2
	58	ZC11	瑞安3号Rui’an 3
	59	ZC12	瑞安4号Rui’an 4

重复类型 Repeat types	序列数量 Number of sequences	位点数量 Number of loci	占全部SSR/% Proportion of all SSRs	EST中出现频率/% Frequency in EST	平均距离/ kb Average distance	平均长度/ bp Average length
单核苷酸Single nucleotide	25 262	27 390	31.82	8.53	12.59	10.34
二核苷酸Dinucleotide	9 370	9 742	11.32	3.16	35.39	13.97
三核苷酸Trinucleotide	7 773	8 114	9.43	2.62	42.48	16.88
四核苷酸Tetranucleotide	954	961	1.12	0.32	358.71	21.91
五核苷酸Pentanucleotide	269	269	0.31	0.09	1 281.50	26.43
六核苷酸Hexanucleotide	234	234	0.27	0.08	1 473.17	37.10
复合型核苷酸Compound nucleotides	36 135	39 374	45.74	12.19	8.76	24.45

重复类型 Repeat types	序列数量 Number of sequences	位点数量 Number of loci	占全部SSR/% Proportion of all SSRs	EST中出现频率/% Frequency in EST	平均距离/ kb Average distance	平均长度/ bp Average length
单核苷酸Single nucleotide	25 262	27 390	31.82	8.53	12.59	10.34
二核苷酸Dinucleotide	9 370	9 742	11.32	3.16	35.39	13.97
三核苷酸Trinucleotide	7 773	8 114	9.43	2.62	42.48	16.88
四核苷酸Tetranucleotide	954	961	1.12	0.32	358.71	21.91
五核苷酸Pentanucleotide	269	269	0.31	0.09	1 281.50	26.43
六核苷酸Hexanucleotide	234	234	0.27	0.08	1 473.17	37.10
复合型核苷酸Compound nucleotides	36 135	39 374	45.74	12.19	8.76	24.45

引物代号 Primer code	正向引物序列 Forward primer sequence	反向引物序列 Reverse primer sequence	产物大小/bp Product size	退火温度（正/反）/℃ Annealing temperature (F/R)	重复基元 Repeat primitive	等位位点数 Number of alleles
1	CTCCAAGGGGCAAGACAGAG	TCACTGTCGCCGTCGTTAAA	174 ~ 178	60.04/59.97	(A)10；(GA)6	2
8	GCCAACAGGACGATTCTCCA	AGCTAGGAGGTGAGACAGGG	119 ~ 125	60.04/60.03	(A)10；(TT)6(T)13*	2
15	AGCTTCCCCACACATGTCAG	ACTGGTATGCCCTTCACGTG	221 ~ 226	59.96/60.04	(TA)6	2
38	TCATGCTCCTCCTCTCTGCT	CAGTGCACTTGTTCAGCCAC	291 ~ 294	60.03/59.97	(AAA)5(A)17(AA)8	3
49	GGAGACTCAAGGTCACTGCC	TTTGCTCTCCAGTGACGGTC	290 ~ 305	60.04/59.97	(GAGA)5(GA)10*	2
53	AATCTCCCAAGCAGATGGCC	ATCAGGCCACATCACTTGGG	147 ~ 153	60.11/60.03	(A)10；(TT)8(TTT)5(T)17	2
59	CTCCCACCCACAACCACTAC	GCCGCAAATCTCGCAGAAAT	156 ~ 159	59.96/59.90	(TGGAGA)13	2
63	ACACCACCAAGCTTGTCACA	ACATTCCAGTCAGCAGCTCC	205 ~ 215	60.04/60.04	(A)10；(TT)6(T)12*	3
64	GGCAGATACGGGGCTCTTAC	CCATTCTCGTCCGCTGATGA	293 ~ 326	59.97/59.90	(CCT)6；(T)12(TT)6*	6
69	TCCGGGTATTGGAAGGCAAC	GGCTCTCTGCAAGGCGATAT	180 ~ 183	60.04/59.97	(AGG)5；(AA)6(A)12ttcatctag aagg(A)10gtattagaataacaaaaagctgagctagccaatcctagatgaagatgtgtacagaatagtgtgtga(T)12(TT)6	2
72	GGTCCAAAGAATCCGGGTGT	TGCTTTGATGGCAACGTGTG	209 ~ 216	59.96/59.97	(T)10	3
77	GCCCCTTCCTGTTCTTCCTC	GGAACCGGAGTTTGGGCTAA	177 ~ 194	60.04/59.96	(CT)7	2