BSA-Seq结合RNA-Seq技术挖掘大豆叶片提前黄化衰老基因

doi:10.3724/SP.J.1006.2023.34062

作物学报 ›› 2024, Vol. 50 ›› Issue (2): 294-309.doi: 10.3724/SP.J.1006.2023.34062

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

BSA-Seq结合RNA-Seq技术挖掘大豆叶片提前黄化衰老基因

李世宽¹^,^2,**(), 洪慧龙^2,**, 付佳祺¹^,², 谷勇哲¹^,², 孙如建³, 邱丽娟¹^,²^,^*()

¹哈尔滨师范大学生命科学与技术学院, 黑龙江哈尔滨 150025
²中国农业科学院作物科学研究所, 北京 100081
³呼伦贝尔市农业科学研究所, 内蒙古扎兰屯 162650

收稿日期:2023-03-23 接受日期:2023-06-29 出版日期:2024-02-12 网络出版日期:2023-07-14
通讯作者: *邱丽娟, E-mail: qiulijuan@caas.cn
作者简介:李世宽, E-mail: 1327605644@qq.com
^**同等贡献
基金资助:
国家自然科学基金项目(32172005)

Mine the genes of premature yellowing and aging in soybean leaves by BSA-seq combined with RNA-seq technology

LI Shi-Kuan¹^,^2,**(), HONG Hui-Long^2,**, FU Jia-Qi¹^,², GU Yong-Zhe¹^,², SUN Ru-Jian³, QIU Li-Juan¹^,²^,^*()

¹College of Life Sciences and Technology, Harbin Normal University, Harbin 150025, Heilongjiang, China
²Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
³Hulun Buir Institution of Agricultural Sciences, Zhalantun 162650, Inner Mongolia, China

Received:2023-03-23 Accepted:2023-06-29 Published:2024-02-12 Published online:2023-07-14
Contact: *E-mail: qiulijuan@caas.cn
About author:^**Contributed equally to this work
Supported by:
National Natural Science Foundation of China(32172005)

摘要/Abstract

摘要：

大豆产量与其生殖生长的持续时间呈正相关, 延缓其开花后叶片的衰老, 提高其生理功能, 有利于增加植物的粒重。叶片黄化是植物衰老的显著特征之一。关于在大豆鼓粒后期叶片黄化的研究鲜见报道。本研究对鼓粒后期叶片提前黄化突变体ly和野生型ofc杂交组合进行遗传分析, 结果表明, 大豆鼓粒后期叶片提前黄化性状受单隐性核基因控制。通过BSA-Seq在19号染色体得到一个2.23 Mb的初定位区间, 经开发标记图位克隆后将区间缩短至1.75 Mb, 区间内有219个基因, 再结合RNA-Seq分析, 得到了区间内12个候选基因, 其中有4个SNP变异基因和8个差异表达基因。本研究结果为大豆鼓粒后期叶片黄化衰老基因的克隆奠定了基础。

关键词: 大豆, 突变体, BSA-Seq, RNA-Seq, 图位克隆

Abstract:

The yield of soybean is positively correlated with the duration of reproductive growth, which delays the aging of leaves after flowering, enhances their physiological performance, and supports growing plants with heavier grains. Leaf yellowing is one of the distinctive features of plant aging. Studies on leaf yellowing at the late stage of soybean drum grain have rarely been reported. The early yellowing feature of soybean late tympanic leaves was controlled by a single recessive nuclear gene, according to the genetic analysis of the hybrid of the early yellowing mutant ly and wild-type ofc in this study. Using Molecular Marker to Map-Based Cloning, a 2.23 Mb preliminary localization interval on chromosome 19 was obtained. The interval was shortened to 1.75 Mb and contained 219 genes. When this interval was combined with RNA-Seq analysis, 12 candidate genes were detected, including 4 SNP variant genes and 8 differentially expressed genes. The findings of this study provides the framework for the cloning of genes that cause aging and yellowing during the filling later period in soybean.

Key words: soybean, mutant, BSA-Seq, RNA-Seq, map-based cloning

李世宽, 洪慧龙, 付佳祺, 谷勇哲, 孙如建, 邱丽娟. BSA-Seq结合RNA-Seq技术挖掘大豆叶片提前黄化衰老基因[J]. 作物学报, 2024, 50(2): 294-309.

LI Shi-Kuan, HONG Hui-Long, FU Jia-Qi, GU Yong-Zhe, SUN Ru-Jian, QIU Li-Juan. Mine the genes of premature yellowing and aging in soybean leaves by BSA-seq combined with RNA-seq technology[J]. Acta Agronomica Sinica, 2024, 50(2): 294-309.

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引物名称 Primer name	上游引物序列 Forward primer (5°-3°)	下游引物序列 Reverse primers (5°-3°)
Glyma.19G223400	TGATTGGTTTAGCCGTGTTTC	AACATCTCGGTTGTAAGTCCTCT
Glyma.19G228000	ATGTAACAGAAGGACGCG	TGTTGGTAAATAGAGTAATG
Glyma.19G236800	TAACATCACTCAAATAAAAC	AACCAGCTTAACAACCTCAA
Glyma.19G237000	AGAAGACATGGTTCCAGCAC	CCCGAGGAATCTCAAAGGA
Glyma.19G216000	CACTTTGCTTTCTTCTCCCTT	TACCAGCATTGATGCAGTTT
Glyma.19G233000	AATAGCCATTTCGCTTATC	ACAAACTTGTTGGGAGTAG
Glyma.19G219600	AGTAGTATAGAAGTGCGTAA	CTTCTCAACTACTTATTAAGATATG
Glyma.19G241500D	ATGCGTATGGACTTCCCCAA	TGGCATTCGTTGGGCACTAA

引物名称 Primer ID	上游引物序列 Forward primer (5°-3°)	下游引物序列 Reverse primers (5°-3°)
216000	Fam: GAAGGTGACCAAGTTCATGCTTGGAGGGAGGGGGACTGg Hex: GAAGGTCGGAGTCAACGGATTGTGGAGGGAGGGGGACTGa	ACTTTGCTTTCTTCTCCCTTTTATCTTTCT
223400	Fam: GAAGGTGACCAAGTTCATGCTATAGGACCATTAAGAGGCGGCATt Hex: GAAGGTCGGAGTCAACGGATTTAGGACCATTAAGAGGCGGCATc	AAGACAGATGCTGCAGCTGCG
228000	Fam: GAAGGTGACCAAGTTCATGCTCGCCGAAATATAGTCACCGg Hex: GAAGGTCGGAGTCAACGGATTAACGCCGAAATATAGTCACCGa	CTGAGATTCGGAGAAGCTTGC
219600	Fam: GAAGGTGACCAAGTTCATGCTGGTGTCAATTTTCTAAAAATTCATACTCTTc Hex: GAAGGTCGGAGTCAACGGATTGGTGTCAATTTTCTAAAAATTCATACTCTTt	TTGTGACTTTCTCTTCTCAACTACTTATTAAG
233000	Fam: GAAGGTGACCAAGTTCATGCTATAAGTTCTTCAGTGTTCACTTGAGAGg Hex: GAAGGTCGGAGTCAACGGATTAATAAGTTCTTCAGTGTTCACTTGAGAGa	TCATTAGTCCTTAACCCGTCACATAG
237000	Fam: GAAGGTGACCAAGTTCATGCTATGCATCACGGGTCATTCTg Hex: GAAGGTCGGAGTCAACGGATTAAATGCATCACGGGTCATTCTa	CAAAGAAAGAAAGTAACTACTGAAGTGTACG
241500D	ATGCGTATGGACTTCCCCAA	ATCAGTTTTGTAAATAATAGTTGTgA

取样时间 Time	(种子鲜重-种子干重)/荚果重 (Seed fresh weight-Seed dry weight)/Pod weight			种子干重/荚果重 Seed dry weight/Pod weight
取样时间 Time	野生型 Wild-type	突变型 Mutant	t检验的P值 P-value by t-test	野生型 Wild-type	突变型 Mutant	t检验的P值 P-value by t-test
第1天Day 1	0.31	0.31	P=0.91	0.24	0.28	P=0.04<0.05
第2天Day 2	0.31	0.31		0.26	0.29
第3天Day 3	0.29	0.31		0.28	0.30
第4天Day 4	0.34	0.35		0.26	0.30
第5天Day 5	0.34	0.34		0.33	0.31
第6天Day 6	0.35	0.34		0.35	0.34
第7天Day 7	0.36	0.34		0.35	0.39
第8天Day 8	0.22	0.23		0.50	0.52

生长条件 Growth conditions	性状 Characters		株高 Plant height (cm)	分枝 Branch	节数 Number of sections	荚数 Number of pods	粒数 Number of grains	百粒重 100-grain weight (g)
短日照条件 Short-day conditions	矮皱叶 Dwarf-wrink led leaves	野生型 Wild	34.32±3.98^*	0.37±0.51	12.75±1.42	27.36±7.33^*	42.93±14.08^*	34.81±4.18^**
	矮皱叶 Dwarf-wrink led leaves	突变型 Mutant	29.31±3.45^*	0.27±0.53	11.07±1.93	22.34±10.58^*	37.18±18.32^*	29.44±2.21^**
	高展叶 Tall-flat leaves	突变型 Mutant	45.28±6.20^**	1.52±1.72^*	11.91±1.26^*	32.06±16.13	61.96±31.66	30.72±3.14^**
	高展叶 Tall-flat leaves	野生型 Wild	52.32±5.02^**	0.77±1.14^*	13.32±1.65^*	32.75±14.60	65.55±34.33	37.44±3.51^**
长日照条件 Long-day conditions	矮皱叶 Dwarf-wrink led leaves	野生型 Wild	65.66±7.52	0.44±0.42	19.51±2.47	35.97±10.02^*	51.37±13.93	27.74±1.50
	矮皱叶 Dwarf-wrink led leaves	突变型 Mutant	67.30±7.38	0.55±0.40	21.71±1.05	42.89±9.42^*	51.27±6.29	27.32±1.20
	高展叶 Tall-flat leaves	突变型 Mutant	122.15±6.23	4.31±1.11^*	23.26±0.95	114.50±25.84	194.39±51.06^*	33.62±1.82^**
	高展叶 Tall-flat leaves	野生型 Wild	124.79±9.12	2.95±1.06^*	22.67±1.32	85.81±17.37	173.87±29.41^*	37.42±1.72^**

关联分析类型 Association analysis type	染色体 Chr.	起始位置 Start position	终止位置 Termination location	区间大小 Region size (Mb)	区间内基因数量 Gene number in the regions
SNP关联结果SNP correlates results	Chr19	45760000	50720000	4.96	636
InDel关联结果InDel correlates results	Chr11	0	250000	0.25	44
	Chr05	20110000	21450000	1.34	10
	Chr12	16090000	16560000	0.47	16
	Chr16	21180000	23610000	2.43	51
	Chr17	24150000	25190000	1.04	12
	Chr17	35420000	35660000	0.24	2
	Chr18	26340000	26470000	0.13	3
	Chr18	9180000	9390000	0.21	15
	Chr19	46440000	49040000	2.60	326
2个关联结果交集 The intersection of the two association results	Chr19	46810000	49040000	2.23	277

BSA-Seq结合RNA-Seq技术挖掘大豆叶片提前黄化衰老基因

Mine the genes of premature yellowing and aging in soybean leaves by BSA-seq combined with RNA-seq technology

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值 Value	表型 Phenotype	基因型 Genotype	标记Marker
值 Value	表型 Phenotype	基因型 Genotype	216000	219600	223400	228000	233000	237000	241500D
理论值 Theoretical value	绿色Green	A	197	195	259	197	180	197	214
理论值 Theoretical value	黄色Yellow	B	149	196	183	194	183	149	163
实际值 Actual value	绿色Green	A	67	182	181	257	176	171	145
实际值 Actual value	黄色Yellow	B	115	119	193	166	189	176	102
χ²值χ²-value			31.13	6.91	0.97	1.50	2.24	0.63	22.25
P值P-value			0	0.01	0.31	0.21	0.12	0.40	0

基因ID Gene ID	SNP位点 SNP loci	碱基类型 Base type		突变类型 Mutation type	突变的影响 Effects of mutations
基因ID Gene ID	SNP位点 SNP loci	野生型 Wild type	突变型 Mutant	突变类型 Mutation type	突变的影响 Effects of mutations
Glyma.19G223400	47582632	A	G	基因的3'UTR内 Gene within 3' UTR	改变RNA二级结构 Change the RNA secondary structure
Glyma.19G233000	48299560	A	G	基因的3'UTR内 Gene within 3' UTR	改变RNA二级结构 Change the RNA secondary structure
Glyma.19G236800	48581005	A	G	非同义SNV Non-synonymous SNV	脯氨酸变成亮氨酸, 改变蛋白结构 Proline becomes leucine, changing the protein structure
Glyma.19G237000	48622649	T	C	基因的5'UTR内 Gene within 5' UTR	改变RNA二级结构 Change the RNA secondary structure

基因编号 Gene number	野生型子代FPKM值 Wild type offspring FPKM value	突变型子代FPKM值 Mutant offspring FPKM value	野生型亲本FPKM值 FPKM values of wild type parents	突变型亲本FPKM值 FPKM values of mutant parents	野生型vs突变型 Wild type vs mutant
Glyma.19G223400	5.97	8.20	6.65	7.87	no
Glyma.19G233000	15.50	17.41	15.19	14.89	no
Glyma.19G236800	22.14	14.91	19.63	19.63	no
Glyma.19G237000	2.35	4.24	1.97	2.89	no

基因编号 Gene number	野生型子代FPKM值 Wild type offspring FPKM value	突变型子代FPKM值 Mutant offspring FPKM value	野生型亲本FPKM值 FPKM values of wild type parents	突变型亲本FPKM值 FPKM values of mutant parents	野生型vs突变型 Wild type vs mutant
Glyma.19G222400	1.141	0.311	1.959	0.334	up
Glyma.19G224500	2.857	15.923	1.933	10.123	down
Glyma.19G225900	0.446	0.083	0.959	0.039	up
Glyma.19G226800	14.965	3.935	11.658	2.708	up
Glyma.19G228200	1.935	0.051	1.032	0.200	up
Glyma.19G228300	4.934	0.775	4.272	0.751	up
Glyma.19G230600	1.125	0.470	1.485	0.329	down
Glyma.19G241200	59.422	137.849	45.382	103.845	down

类型 Type	基因编号 Gene number	具有同源序列的蛋白 Proteins with homologous sequences
SNP突变 SNP mutations	Glyma.19G223400	G蛋白β亚基(含WD40重复蛋白同源序列)、丝氨酸/苏氨酸激酶受体相关蛋白以及醌血红素蛋白β亚基 G protein β subunits (containing WD40 repeat homologous sequences), serine/threonine kinase receptor-associated proteins, and quinone heme protein β subunits
	Glyma.19G233000	乙酰肝素-α-氨基葡萄糖苷N-乙酰转移酶Heparan-α-glucosaside N-acetyltransferase
	Glyma.19G236800	E3泛素蛋白连接酶、和锌指结构域 E3 ubiquitin protein ligase, and zinc finger domains
	Glyma.19G237000	PLP依赖的D-天冬氨酸氨基转移酶, 核苷三磷酸水解酶的P环 PLP-dependent D-aspartate aminotransferase, the P loop of nucleoside triphosphate hydrolases
差异表达基因 Differentially expressed genes	Glyma.19G222400	果胶乙酰酯酶家族蛋白Pectin acetylesterase family proteins
	Glyma.19G224500	果胶乙酰酯酶家族蛋白Pectin acetylesterase family proteins
	Glyma.19G225900	碳水化合物代谢过程碳水化合物结合X8结构域蛋白 Carbohydrate metabolism process, carbohydrate binding to X8 domain protein
	Glyma.19G226800	核苷三磷酸水解酶的P环P loop of nucleoside triphosphate hydrolases
	Glyma.19G228200	类似ECH相关蛋白1的Kelch Kelch-like ech-associated protein 1
	Glyma.19G228300	转录因子MEIS1和相关HOX结构域蛋白 Transcription factor MEIS1 and associated HOX-domain proteins
	Glyma.19G230600	丝氨酸/苏氨酸特异性蛋白磷酸酶, 蛋白磷酸酶2C Serine/threonine-specific protein phosphatase, protein phosphatase 2C
	Glyma.19G241200	转移酶活性, 转移氨基酰基以外的酰基 Transferase activity, transfer of acyl groups other than aminoacyl

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