基于SLAF-seq技术的石斛兰SNP标记开发及亲缘关系分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1386

摘要/Abstract

摘要：

利用SNP标记技术对收集的石斛兰种质资源进行亲缘关系分析，为新品种选育亲本选择提供理论依据。以60份石斛兰种质资源为对象，用特异性位点扩增片段测序技术（SLAF-seq）对种质进行SNP标记开发及亲缘关系分析。通过对各样品的测序数据进行统计，共获得157.34 Mb Clean Reads数据，各样本Reads数据在576 195-5 359 710 之间。样本平均测序质量值Q30为93.85%，平均GC含量为40.16%。通过测序数据分析，共获得1 337 217个SLAF标签，标签的平均测序深度为9.63×，其中具多态性的SLAF标签有1 049 638个。共开发得到11 248 186个群体SNP标记，每个样品的SNP 标记数目介于694 015-6 367 379之间，SNP标记完整度为2.71%-24.89%，杂合率为1.13%-5.74%。对群体SNP 过滤，共获得31 499个高度一致性的有效SNP 标记。利用筛选获得的SNP标记构建系统发育树，60份石斛兰种质资源被分为3个亚群，这3个亚群分别包含材料为 Q1：3份，Q2：21份，Q3: 36份。种质聚类结果与形态学分类结果基本一致。利用SLAF-seq技术能高效、精确地开发出适用于石斛兰种质遗传分析的SNP标记，开发的SNP标记可为石斛兰育种、遗传图谱构建、品种鉴定以及农艺性状的关联分析等研究提供分子基础。

关键词: 石斛兰, SLAF-seq, SLAF标签, SNP标记, 群体结构, 发育树, 亲缘关系

Abstract:

The genetic relationship of the collected Dendrobium germplasm resources was analyzed by SNP marker technology to provide theoretical basis for the selection of new varieties breeding parents. Total 60 Dendrobium germplasm resources were used for SNP marker development and genetic relationship analysis using specific-locus amplified fragment sequencing technology（SLAF-seq）. The sequencing data of each sample were statistically analyzed, and 157.34 Mb Clean Reads data were obtained. The Reads data of each sample ranged from 576 195 to 5 359 710. The average sequencing quality value（Q30）and GC content of samples was 93.85% and 40.16%. Through sequencing data analysis, a total of 1 337 217 SLAF tags and 1 049 638 polymorphic SLAF tags were obtained. The average sequencing depth of the tags was 9.63×. A total of 11 248 186 population SNP markers were developed. The number of SNP markers in each sample ranged from 694 015 to 6 367 379. The integrity ratio was 2.71% to 24.89%, and the hetloci ratio was 1.13% to 5.74%. The population SNPs were filtered, and a total of 31 499 highly consistent and effective SNP markers were obtained. The phylogenetic tree was constructed by using the SNP markers obtained. The 60 Dendrobium germplasm resources were divided into 3 subgroups. These three subgroups contained germplasm resources: Q1（3）, Q2（21）, and Q3（36）. The results of germplasm clustering were basically consistent with the morphological classification. Using SLAF-seq technology can efficiently and accurately develop SNP markers suitable for the genetic analysis of Dendrobium. The SNP markers developed may provide molecular basis for Dendrobium breeding, genetic map construction, variety identification and association analysis of agronomic traits.

Key words: Dendrobium, SLAF-seq, SLAF tags, SNP marker, population structure, phylogenetic tree, genetic relationship

崔学强, 黄昌艳, 邓杰玲, 李先民, 李秀玲, 张自斌. 基于SLAF-seq技术的石斛兰SNP标记开发及亲缘关系分析[J]. 生物技术通报, 2023, 39(6): 141-148.

CUI Xue-qiang, HUANG Chang-yan, DENG Jie-ling, LI Xian-min, LI Xiu-ling, ZHANG Zi-bin. SNP Markers Development and Genetic Relationship Analysis of Dendrobium Germplasms Using SLAF-seq Technology[J]. Biotechnology Bulletin, 2023, 39(6): 141-148.

图/表 5

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种质编号 Germplasm No.	总读数 Total reads	GC含量 GC content/%	Q30含量 Q30 content/%	种质编号 Germplasm No.	总读数 Total reads	GC含量 GC content/%	Q30含量 Q30 content/%
1	2 206 424	40.80	94.43	32	5 359 710	39.13	92.60
2	3 392 758	39.52	94.06	33	1 927 034	39.04	91.41
3	1 685 725	40.68	94.30	34	1 001 860	40.88	89.71
4	2 397 802	41.36	93.61	35	1 738 186	38.96	91.29
5	2 407 877	40.88	94.25	36	1 570 013	38.62	90.59
6	3 816 221	37.99	93.65	37	2 817 541	42.10	94.61
7	1 236 847	45.65	92.95	38	2 262 024	41.10	94.49
8	2 603 097	40.01	94.46	39	1 506 192	40.21	94.35
9	2 266 706	40.64	94.09	40	1 658 896	39.49	94.57
10	2 741 663	39.42	93.77	41	2 398 814	41.12	94.63
11	2 888 849	39.57	94.72	42	2 010 374	39.09	94.46
12	5 033 204	39.52	94.34	43	2 253 253	38.95	94.61
13	1 177 834	39.60	93.56	44	2 443 737	39.60	94.03
14	1 558 521	39.04	93.06	45	3 295 369	39.07	94.86
15	3 350 246	42.91	94.67	46	1 067 643	38.75	94.67
16	2 508 628	39.29	93.40	47	2 170 007	39.61	94.40
17	3 008 190	39.45	93.23	48	1 954 914	39.23	94.37
18	2 892 775	42.48	94.54	49	2 394 534	39.06	94.48
19	3 799 474	41.30	94.54	50	2 899 967	39.27	94.52
20	3 265 332	41.98	94.56	51	2 199 124	46.38	94.72
21	2 223 263	38.82	93.44	52	3 270 085	40.08	94.66
22	2 536 748	40.32	94.25	53	1 303 118	39.41	94.51
23	3 663 842	40.96	94.71	54	3 465 111	38.97	94.13
24	576 195	42.98	93.34	55	2 955 467	39.29	94.76
25	5 218 665	39.01	93.00	56	4 757 901	39.73	94.79
26	2 318 866	39.08	93.03	57	4 802 384	40.06	94.62
27	2 752 538	41.06	94.50	58	2 339 558	40.16	94.63
28	1 709 658	38.85	90.70	59	2 787 875	38.80	94.67
29	3 965 547	38.44	94.48	60	1 857 241	42.29	93.80
30	1 831 691	38.73	91.14	SD	7 293 431	42.86	94.33
31	3 836 959	40.67	94.49

种质编号 Germplasm No.	总读数 Total reads	GC含量 GC content/%	Q30含量 Q30 content/%	种质编号 Germplasm No.	总读数 Total reads	GC含量 GC content/%	Q30含量 Q30 content/%
1	2 206 424	40.80	94.43	32	5 359 710	39.13	92.60
2	3 392 758	39.52	94.06	33	1 927 034	39.04	91.41
3	1 685 725	40.68	94.30	34	1 001 860	40.88	89.71
4	2 397 802	41.36	93.61	35	1 738 186	38.96	91.29
5	2 407 877	40.88	94.25	36	1 570 013	38.62	90.59
6	3 816 221	37.99	93.65	37	2 817 541	42.10	94.61
7	1 236 847	45.65	92.95	38	2 262 024	41.10	94.49
8	2 603 097	40.01	94.46	39	1 506 192	40.21	94.35
9	2 266 706	40.64	94.09	40	1 658 896	39.49	94.57
10	2 741 663	39.42	93.77	41	2 398 814	41.12	94.63
11	2 888 849	39.57	94.72	42	2 010 374	39.09	94.46
12	5 033 204	39.52	94.34	43	2 253 253	38.95	94.61
13	1 177 834	39.60	93.56	44	2 443 737	39.60	94.03
14	1 558 521	39.04	93.06	45	3 295 369	39.07	94.86
15	3 350 246	42.91	94.67	46	1 067 643	38.75	94.67
16	2 508 628	39.29	93.40	47	2 170 007	39.61	94.40
17	3 008 190	39.45	93.23	48	1 954 914	39.23	94.37
18	2 892 775	42.48	94.54	49	2 394 534	39.06	94.48
19	3 799 474	41.30	94.54	50	2 899 967	39.27	94.52
20	3 265 332	41.98	94.56	51	2 199 124	46.38	94.72
21	2 223 263	38.82	93.44	52	3 270 085	40.08	94.66
22	2 536 748	40.32	94.25	53	1 303 118	39.41	94.51
23	3 663 842	40.96	94.71	54	3 465 111	38.97	94.13
24	576 195	42.98	93.34	55	2 955 467	39.29	94.76
25	5 218 665	39.01	93.00	56	4 757 901	39.73	94.79
26	2 318 866	39.08	93.03	57	4 802 384	40.06	94.62
27	2 752 538	41.06	94.50	58	2 339 558	40.16	94.63
28	1 709 658	38.85	90.70	59	2 787 875	38.80	94.67
29	3 965 547	38.44	94.48	60	1 857 241	42.29	93.80
30	1 831 691	38.73	91.14	SD	7 293 431	42.86	94.33
31	3 836 959	40.67	94.49