基于BSA-seq结合连锁分析发掘大豆荚粒性状QTLs及候选基因

doi:10.13304/j.nykjdb.2022.0089

摘要/Abstract

摘要：

豆荚和籽粒是大豆重要的产量形成器官，同时，荚粒性状作为大豆重要外观品质，还直接影响其商品性。鉴于此，基于大豆重组自交系群体（recombinant inbred line， RIL）在多种环境条件下的荚粒性状（荚长、荚宽、荚重、粒长、粒宽和粒重），筛选极端家系构建2个混池——大荚大粒池（large pool，LP）和小荚小粒池（small pool，SP）；经混合群体分离测序（bulked segregant analysis sequencing， BSA-seq）寻找控制荚粒性状的遗传位点，同时结合前期该群体连锁定位QTLs以及RIL亲本荚粒转录组数据，发掘多环境、多方法、多性状共定位区间以及候选基因。结果表明，构建的LP与SP混池在不同环境下的荚粒性状存在显著差异，BSA-seq结果与Williams82参考基因组的平均比对效率在98%以上，10×基因组覆盖率在94%以上；基于此，在置信度0.95时，经SNP-index、Euclidean distance（ED）和G-statistic算法分别获得27、41和51个关联区域，在置信度0.99时，分别获得5、15和16个关联区域，位于11条染色体（1、3、4、6、7、12、13、15、17、19和20号）；经与前期连锁定位QTLs比较发现，在7、19和20号染色体上存在多环境、多方法、多性状共定位遗传位点，且20号染色体SNP标记ss715637629及19号染色体SNP标记ss715635705、ss715635755、ss715635844等在连锁定位与BSA-seq同时被检测到；进一步分析3种BSA-seq算法共关联区域内的候选基因，发现40个基因存在外显子非同义突变，其中15个基因在RIL群体亲本C813和KN7豆荚和籽粒表达量较高，并有3个基因随豆荚发育进程表达量增加。以上结果为开展大豆荚粒性状分子遗传改良以及遗传基础解析奠定了重要基础。

关键词: 大豆, 荚粒性状, BSA-seq, 连锁定位, QTLs, 候选基因

Abstract:

Pod and seed are important organs for yield formation in soybean， while pod and seed traits are also important characteristics of soybean appearance quality， which directly influence the commercial values of soybean product. In view of this， a recombinant inbred line （RIL） population was evaluated for the pod and seed related traits （pod length， pod width， pod weight， seed length， seed width and seed weight） under different environments， and the bulked segregant analysis sequencing （BSA-seq） was conducted to identify the genetic loci for pod and seed traits in 2 DNA pools （large pool， LP； small pool， SP） with extremely significant differences on related traits. Subsequently， by combining linkage mapping results and transcriptome data of RIL parents in previous study， the consistent genetic loci and candidate genes related to pod and seed traits across multiple environments via different methods and controlling diverse traits were discovered. The results showed that there were significant differences in the pod and seed related traits between the 2 DNA pools， LP and SP， under different environments. The average blasting percentage and coverage percentage of DNA sequences obtained from BSA-seq were above 98% and 94% with Williams 82 reference genome. Based on this， 27， 41 and 51 associated genome regions at 0.95 confident level were identified through the SNP-index， Euclidean distance （ED） and G-statistic methods， and 5， 15， 16 associated genome regions on 11 chromosomes （chromosomes 1， 3， 4， 6， 7， 12， 13， 15， 17， 19 and 20） at 0.99 confident level were obtained. Further analysis showed that the consistent genetic loci under multiple environments through the BSA-seq and linkage mapping methods were detected on chromosomes 7， 19 and 20. Moreover， some SNP markers， such as ss715637629 on chromosome 20 and ss715635705， ss715635755， ss715635827 and ss715635844 on chromosome 19， were detected by BSA-seq and linkage mapping， simultaneously. Furthermore， 40 candidate genes in the consistent association regions were found to present non-synonymous mutations in exons， and 15 of them showed high expression levels in the pod and seed of RIL parents. Among these 15 genes， 3 of them showed increasing expressions with the developmental stages of soybean pod and seed， which might have important functions in the formation of soybean pod and seed traits. Thus， the results in the present study provided novel insight for the genetic basis dissection of soybean pod and seed traits， and also provided the useful markers and genes for the genetic improvement of pod and seed traits in soybean breeding programme.

Key words: soybean, pod and seed traits, BSA-seq, linkage mapping, QTLs, candidate genes

中图分类号:

S565.1

孙亚倩, 陈士亮, 褚佳豪, 李喜焕, 张彩英. 基于BSA-seq结合连锁分析发掘大豆荚粒性状QTLs及候选基因[J]. 中国农业科技导报, 2023, 25(7): 29-42.

Yaqian SUN, Shiliang CHEN, Jiahao CHU, Xihuan LI, Caiying ZHANG. Mining of QTLs and Candidate Genes for Pod and Seed Traits via Combining BSA-seq and Linkage Mapping in Soybean[J]. Journal of Agricultural Science and Technology, 2023, 25(7): 29-42.

图/表 10

参考文献 29

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项目 Item	环境 Environment	荚长 PL/cm	荚宽 PW/cm	荚重 PWT/g	粒长 SL/cm	粒宽 SW/cm	粒重 SWT/g
LP	E1	4.68	1.21	1.81	1.41	0.86	0.51
	E2	5.22	1.20	2.25	1.42	0.86	0.64
	E3	4.82	1.21	1.88	1.33	0.83	0.55
	E4	5.13	1.21	2.07	1.38	0.85	0.57
	均值Mean	4.96	1.21	2.00	1.39	0.85	0.57
SP	E1	4.06	1.10	1.49	1.30	0.79	0.45
	E2	4.52	1.09	1.74	1.31	0.81	0.54
	E3	4.22	1.07	1.53	1.22	0.76	0.48
	E4	4.35	1.07	1.58	1.24	0.78	0.47
	均值Mean	4.29	1.08	1.59	1.27	0.79	0.49
显著性 Significance （P-value）	E1	4.3e-07	5.5e-07	4.2e-06	3.5e-05	6.0e-07	8.7e-04
	E2	1.1e-12	1.3e-08	3.7e-09	6.4e-07	2.7e-07	2.1e-06
	E3	3.8e-10	7.1e-10	1.5e-05	2.6e-07	4.2e-07	1.3e-03
	E4	7.7e-11	9.5e-09	2.8e-08	2.2e-07	4.2e-06	5.1e-06
	多环境All environments	5.6e-03	3.0e-06	1.0e-02	5.8e-03	2.6e-03	4.2e-02

项目 Item	环境 Environment	荚长 PL/cm	荚宽 PW/cm	荚重 PWT/g	粒长 SL/cm	粒宽 SW/cm	粒重 SWT/g
LP	E1	4.68	1.21	1.81	1.41	0.86	0.51
	E2	5.22	1.20	2.25	1.42	0.86	0.64
	E3	4.82	1.21	1.88	1.33	0.83	0.55
	E4	5.13	1.21	2.07	1.38	0.85	0.57
	均值Mean	4.96	1.21	2.00	1.39	0.85	0.57
SP	E1	4.06	1.10	1.49	1.30	0.79	0.45
	E2	4.52	1.09	1.74	1.31	0.81	0.54
	E3	4.22	1.07	1.53	1.22	0.76	0.48
	E4	4.35	1.07	1.58	1.24	0.78	0.47
	均值Mean	4.29	1.08	1.59	1.27	0.79	0.49
显著性 Significance （P-value）	E1	4.3e-07	5.5e-07	4.2e-06	3.5e-05	6.0e-07	8.7e-04
	E2	1.1e-12	1.3e-08	3.7e-09	6.4e-07	2.7e-07	2.1e-06
	E3	3.8e-10	7.1e-10	1.5e-05	2.6e-07	4.2e-07	1.3e-03
	E4	7.7e-11	9.5e-09	2.8e-08	2.2e-07	4.2e-06	5.1e-06
	多环境All environments	5.6e-03	3.0e-06	1.0e-02	5.8e-03	2.6e-03	4.2e-02

样品Sample	过滤后数据 Clean data/bp	Q20/%	Q30/%	GC/%	总读段 Total reads	读段比对率Mapped reads/%	覆盖度 Coverage rate/%
样品Sample	过滤后数据 Clean data/bp	Q20/%	Q30/%	GC/%	总读段 Total reads	读段比对率Mapped reads/%	1×	4×	10×	20×
KN7	30 185 672 700	97.29	92.63	36.42	200 007 218	98.08	98.20	97.28	94.97	79.68
C813	30 445 756 200	97.28	92.59	36.54	201 728 350	98.92	97.67	96.63	94.35	80.95
SP	31 026 257 700	97.42	92.91	36.32	205 658 536	98.98	98.54	97.79	95.76	82.33
LP	29 921 497 800	97.23	92.50	36.58	198 179 602	98.97	98.67	97.63	94.93	79.99

样品Sample	过滤后数据 Clean data/bp	Q20/%	Q30/%	GC/%	总读段 Total reads	读段比对率Mapped reads/%	覆盖度 Coverage rate/%
样品Sample	过滤后数据 Clean data/bp	Q20/%	Q30/%	GC/%	总读段 Total reads	读段比对率Mapped reads/%	1×	4×	10×	20×
KN7	30 185 672 700	97.29	92.63	36.42	200 007 218	98.08	98.20	97.28	94.97	79.68
C813	30 445 756 200	97.28	92.59	36.54	201 728 350	98.92	97.67	96.63	94.35	80.95
SP	31 026 257 700	97.42	92.91	36.32	205 658 536	98.98	98.54	97.79	95.76	82.33
LP	29 921 497 800	97.23	92.50	36.58	198 179 602	98.97	98.67	97.63	94.93	79.99

项目 Item		C813	KN7	LP	SP
SNP数量 SNP number		1 619 731	1 953 331	2 412 499	2 397 371
SNP所在基因组位置 SNP genome position	下游 Downstream	82 313	97 262	123 627	122 700
	外显子区 Exonic	61 800	72 657	91 369	89 863
	基因间Intergenic	1 171 840	1 421 376	1 736 589	1 728 224
	内含子区Intronic	163 852	195 463	249 905	246 732
	可变剪切位点 Splicing	456	543	697	685
	上游Upstream	96 035	114 708	143 985	144 040
	3’-UTR	21 461	25 807	33 101	32 432
	5’-UTR	14 584	16 642	21 941	21 530
	外显子＆可变剪切Exonic ＆ splicing	25	23	36	35
	上游＆下游Upstream ＆ downstream	7 342	8 811	11 195	11 072
	5’-UTR ＆ 3’-UTR	23	39	54	58
SNP变异类型SNP mutation type	非同义突变 Non-synonymous	34 542	40 700	51 060	50 279
	终止获得 Stop-gain	726	894	1 089	1 077
	终止丢失 Stop-loss	144	182	223	216
	同义突变Synonymous	26 386	30 881	39 002	38 302
	未知 Unknown	27	23	31	24