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

作物学报 ›› 2023, Vol. 49 ›› Issue (11): 2935-2948.doi: 10.3724/SP.J.1006.2023.24276

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

基于BSA-seq方法挖掘大豆再生相关候选基因

赵宇晶¹(), 张滨烁¹, 苏安玉², 于振海¹, 李佳欢¹, 林洋¹, 张艳婷¹, 武小霞¹^,^*(), 赵莹¹^,^*()

¹东北农业大学农学院, 黑龙江哈尔滨 150030
²东北农业大学资环学院, 黑龙江哈尔滨 150030

收稿日期:2022-12-12 接受日期:2023-04-17 出版日期:2023-11-12 网络出版日期:2023-05-12
通讯作者: 武小霞, E-mail: xxwu2012@126.com; 赵莹, E-mail: tianshi198937@126.com
作者简介:赵宇晶, E-mail: yujingzhao99@163.com第一联系人：
**同等贡献
基金资助:
国家自然科学基金项目(31971899);国家自然科学基金项目(32272093);国家重点研发计划项目(2021YFD120160204);国家重点研发计划项目(2021YFD12011040202);黑龙江省自然科学基金项目(TD2022C003);黑龙江省自然科学基金项目(JJ2022YX0475)

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

ZHAO Yu-Jing¹(), ZHANG Bin-Shuo¹, SU An-Yu², YU Zhen-Hai¹, LI Jia-Huan¹, LIN Yang¹, ZHANG Yan-Ting¹, WU Xiao-Xia¹^,^*(), ZHAO Ying¹^,^*()

¹College of Agronomy, Northeast Agricultural University, Harbin 150030, Heilongjiang, China
²College of Resources and Environment, Northeast Agricultural University, Harbin 150030, Heilongjiang, China

Received:2022-12-12 Accepted:2023-04-17 Published:2023-11-12 Published online:2023-05-12
About author:First author contact:
**Contributed equally to this study
Supported by:
National Natural Science Foundation of China(31971899);National Natural Science Foundation of China(32272093);National Key Research and Development Program of China(2021YFD120160204);National Key Research and Development Program of China(2021YFD12011040202);Heilongjiang Natural Science Foundation(TD2022C003);Heilongjiang Natural Science Foundation(JJ2022YX0475)

摘要/Abstract

摘要：

转基因生物育种技术可以定向改良大豆品种, 为大豆定向育种提供一种新思路。为寻找与大豆再生相关的基因, 探索大豆再生规律、提高遗传转化效率, 本研究利用再生能力强材料东农50、再生能力弱材料Keburi及其子代RIL群体的200份材料进行大豆器官发生试验, 比较不同基因型之间再生能力的差异, 筛选出极端材料各20份, 后通过BSA-seq (bulked segregant analysis sequencing)技术对大豆再生候选基因进行初步定位, 共获得88.04 G的clean data, 平均测序深度为20.03×, 定位到2 Mb区间, 利用GO等数据库对区间内基因进行富集分析, 差异表达基因主要富集在纤维素微纤维组织、植物型细胞壁组织或生物发生等20个条目中, 其中被显著富集的植物型细胞壁组织或生物发生条目中共有6个基因, 对6个基因进行组织表达量分析, 在丛生芽伸长期间表达水平较高, 说明其在大豆再生过程中发挥作用, 可能为影响大豆再生的关键基因。本研究为大豆再生机制研究提供了重要的候选基因信息与必要的材料基础。

关键词: 大豆, 再生, 器官发生, BSA-seq

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

赵宇晶, 张滨烁, 苏安玉, 于振海, 李佳欢, 林洋, 张艳婷, 武小霞, 赵莹. 基于BSA-seq方法挖掘大豆再生相关候选基因[J]. 作物学报, 2023, 49(11): 2935-2948.

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.

图/表 15

图1

图2

图3

图4

图5

图6

图7

表1

表2

极端材料的主成分值、隶属函数值、D值"

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

表2

附表1

图8

表3

图9

附表2

图10

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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

基于BSA-seq方法挖掘大豆再生相关候选基因

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

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