基于BSA-seq方法挖掘大豆再生相关候选基因

doi:10.3724/SP.J.1006.2023.24276

Abstract

Abstract:

Transgenic breeding technology can improve soybean directionally, and provide a new idea for improving soybean yield. In order to search for the genes related to soybean regeneration, explore the rules of soybean regeneration, and improve the efficiency of genetic transformation, we conducted soybean organogenesis experiment with 200 materials including DN50 (a material with strong regeneration ability), Keburi (a material with weak regeneration ability), and RILs of its offspring with strong regeneration ability. Compared the differences in regeneration ability between different genotypes, 20 extreme materials each were screened. Preliminary localization of soybean regeneration candidate genes by BSA-seq (bulked segregant analysis sequencing) technology, 88.04 G clean data were obtained in the 2 Mb interval with an average sequencing depth of 20.03 ×. The differentially expressed genes were mainly enriched in 20 items such as cellulose microfiber tissue, plant type cell wall tissue, or biogenesis, among which there were 6 genes in plant type cell wall tissue or biogenesis item significantly enriched. The tissue expression analysis of 6 genes showed that the relative expression level was high during cluster bud elongation, which indicating that it played a role in the process of soybean regeneration and might be the key gene affecting soybean regeneration. This study provides the basic materials for breeding new regenerated soybean varieties, and confirms the feasibility of BSA-seq technology in mining regenerated genes.

Key words: soybean, regeneration, organogenesis, BSA-seq

ZHAO Yu-Jing, ZHANG Bin-Shuo, SU An-Yu, YU Zhen-Hai, LI Jia-Huan, LIN Yang, ZHANG Yan-Ting, WU Xiao-Xia, ZHAO Ying. Mining candidate genes related to soybean regeneration based on BSA-seq method[J].Acta Agronomica Sinica, 2023, 49(11): 2935-2948.

Figures/Tables 15

Fig. 1

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Table 1

Table 2

Principal component value, membership function value, and D-value of extreme materials"

株系 Strain line	主成分 Comprehensive index		隶属函数 Membership function		D值 D-value	综合评价 Comprehensive valuation
株系 Strain line	F1	F2	F1*	F2*	D值 D-value	综合评价 Comprehensive valuation
Z14	9.05	-0.34	1.00	0.36	6.55	强再生Strong regenerative ability
Z6	7.22	2.47	0.82	0.95	5.95	强再生Strong regenerative ability
Z1	6.22	-0.6	0.72	0.30	4.41	强再生Strong regenerative ability
Z142	5.51	-0.58	0.65	0.31	3.88	强再生Strong regenerative ability
Z20	5.02	-0.14	0.60	0.40	3.65	强再生Strong regenerative ability
Z77	4.10	2.32	0.51	0.92	3.63	强再生Strong regenerative ability
Z168	4.33	0.76	0.53	0.59	3.38	强再生Strong regenerative ability
Z2	5.22	-1.75	0.62	0.06	3.36	强再生Strong regenerative ability
Z35	5.07	-1.53	0.61	0.11	3.32	强再生Strong regenerative ability
Z98	4.38	0.08	0.54	0.45	3.24	强再生Strong regenerative ability
Z16	4.04	-0.94	0.50	0.23	2.72	强再生Strong regenerative ability
Z3	3.69	-0.01	0.47	0.43	2.70	强再生Strong regenerative ability
Z155	3.56	0.11	0.46	0.45	2.64	强再生Strong regenerative ability
Z53	3.25	0.49	0.42	0.53	2.52	强再生Strong regenerative ability
Z30	3.12	0.71	0.41	0.58	2.48	强再生Strong regenerative ability
Z184	2.36	1.84	0.34	0.82	2.22	强再生Strong regenerative ability
Z176	2.93	0.10	0.39	0.45	2.18	强再生Strong regenerative ability
Z4	3.12	-0.80	0.41	0.26	2.08	强再生Strong regenerative ability
Z52	2.49	0.26	0.35	0.49	1.90	强再生Strong regenerative ability
Z149	2.37	0.53	0.34	0.54	1.88	强再生Strong regenerative ability
Z39	-0.71	-1.88	0.03	0.03	-1.03	弱再生Weak regenerative ability
Z153	-0.71	-1.88	0.03	0.03	-1.03	弱再生Weak regenerative ability
Z180	-0.71	-1.88	0.03	0.03	-1.03	弱再生Weak regenerative ability
Z152	-0.73	-1.63	0.03	0.08	-0.97	弱再生Weak regenerative ability
Z95	-0.74	-1.47	0.03	0.12	-0.94	弱再生Weak regenerative ability
Z161	-0.74	-1.47	0.03	0.12	-0.94	弱再生Weak regenerative ability
Z165	-0.74	-1.47	0.03	0.12	-0.94	弱再生Weak regenerative ability
Z193	-0.74	-1.47	0.03	0.12	-0.94	弱再生Weak regenerative ability
Z44	-0.76	-1.22	0.03	0.17	-0.88	弱再生Weak regenerative ability
Z84	-0.76	-1.22	0.03	0.17	-0.88	弱再生Weak regenerative ability
Z147	-0.76	-1.22	0.03	0.17	-0.88	弱再生Weak regenerative ability
Z197	-0.77	-1.14	0.03	0.19	-0.87	弱再生Weak regenerative ability
Z25	-0.77	-1.05	0.03	0.21	-0.85	弱再生Weak regenerative ability
Z62	-0.77	-1.05	0.03	0.21	-0.85	弱再生Weak regenerative ability
Z96	-0.77	-1.05	0.03	0.21	-0.85	弱再生Weak regenerative ability
Z108	-0.77	-1.05	0.03	0.21	-0.85	弱再生Weak regenerative ability
Z111	-0.77	-1.05	0.03	0.21	-0.85	弱再生Weak regenerative ability
Z120	-0.77	-1.05	0.03	0.21	-0.85	弱再生Weak regenerative ability
Z127	-0.77	-1.05	0.03	0.21	-0.85	弱再生Weak regenerative ability
Z129	-0.77	-1.05	0.03	0.21	-0.85	弱再生Weak regenerative ability

Table 2

Table S1

Fig. 8

Table 3

Fig. 9

Table S2

Fig. 10

References 47

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性状 Trait	主成分Comprehensive index
性状 Trait	F₁	F₂
再生潜力Regeneration potential	-0.07	0.99
再生能力Regeneration ability	0.57	-0.10
再生效率Regeneration efficiency	0.58	0.08
再生率Regeneration rate	0.58	0.03
特征值Eigen values	2.81	1.02
贡献率Contributive ratio (%)	70.15	25.42
累计贡献率Cumulative contributive ratio (%)	70.15	95.57

样本 Sample	原始序列数 Raw reads	原始碱基数 Raw bases	过滤后序列数 Clean reads	过滤后碱基数 Cleans bases	GC含量 Clean_GC_Rate%	Clean Q30%	平均测序深度 Average depth	基因组覆盖率 genome coverage (1X)	基因组覆盖率 genome coverage (5X)
DN50	150971394	22796680494	148291154	22248395146	35.0791	89.6463	20.25	95.57	93.65
Keburi	145486518	21968464218	143073908	21437801507	35.8134	88.8524	19.87	95.76	93.87
高池Highly pool	151202346	22831554246	147403650	22105336093	35.173	89.5264	19.79	96.75	95.01
低池Lowly pool	151325210	22850106710	148367314	22245451907	35.4202	90.2708	20.22	96.73	94.96

染色体号 Chromosome ID	起始位置 Start position	终止位置 End position	区间距离 Size (Mb)	基因数目 Number of genes
Chr. 18	54560000	55560000	1	185
Chr. 18	55910000	56910000	1	194
总计Total	—	—	2	379

基因号 Gene ID	NCBI登录号 RefSeq ID	物理位置Physical position (bp)	拟南芥同源基因Arabidopsis homologous gene	CDS 长度 CDS length (bp)	氨基酸长度 ORF (aa)	分子量大小 Molecular weight (kD)	pI	亚细胞定位预测Subcellular localization prediction	基因注释 Gene function prediction
Glyma.18G260900	XM_003552487.5	Gm18：54674591-54677015	AT3G29030	798	266	29.1	9.51	细胞壁 Cell wall	Pollen allergen (Pollen_allerg_1)
Glyma.18G265400	XM_041012184.1	Gm18： 55017312-55018922	AT5G39280	687	229	25.26	8.66	细胞壁 Cell wall	EXPANSIN-A21-RELATED
Glyma.18G271900	XM_014770962.3	Gm18： 55493601-55497764	AT5G60920	1263	421	47.06	9.64	细胞膜 Cell membrane	COBRA-LIKE EXTRACELLULAR GLYCOSYL-PHOSPHATIDYL INOSITOL-ANCHORED FAMILY PROTEIN-RELATED
Glyma.18G272000	XM_003552552.4	Gm18： 55503155-55508039	AT5G60920	1347	449	49.87	8.9	细胞膜 Cell membrane	COBRA-like protein (COBRA)
Glyma.18G272100	NM_001255848.2	Gm18： 55506268-55509726	AT5G15630	1296	432	48.44	9	细胞膜 Cell membrane	COBRA-LIKE PROTEIN 4
Glyma.18G272200	XM_003552554.5	Gm18： 55515800-55518328	AT3G02230	1062	354	40.12	5.61	高尔基体 Golgi apparatus	UDP-ARABINOPYRANOSE MUTASE 1-RELATED

Mining candidate genes related to soybean regeneration based on BSA-seq method

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