利用高世代转录组测序挖掘控制南方大豆皱叶症候选基因

doi:10.3864/j.issn.0578-1752.2024.15.002

摘要/Abstract

摘要：

【目的】南方大豆皱叶症（southern soybean crinkle leaf disease，SSCLD）严重时可导致大豆减产40%左右，利用高世代分离群体转录组测序技术（high-generation segregating populations RNA-seq，HGRNA-seq）挖掘控制SSCLD的候选基因，为揭示SSCLD发生的分子机制提供数据支撑。【方法】以2个南方皱叶症症级差异材料及其衍生的F_2:7株系为研究材料，对双亲进行重测序，对12个F_2:7单株（7个皱叶，5个正常叶）进行单个样本转录组测序，利用转录组及亲本的SNP/InDel数据进行混池法定位分析，利用转录组表达量差异数据进行GO和KEGG功能注释和富集分析，并在定位区间附近开发7个KASP分子标记，利用230个F₂群体构建局部连锁图谱，对转录组数据定位结果进行验证。联合基因定位和转录组分析结果，筛选控制SSCLD的候选基因。【结果】利用混池法把控制皱叶症的基因位点CL12定位在大豆第12染色体末端39 231 651-40 705 115 bp的1 473 464 bp区间内，利用F₂群体将控制皱叶症的基因定位于39 743 275-40 948 295 bp的1 205 020 bp区间内，与混池法定位结果基本一致。GO注释结果显示，代谢过程包括免疫系统过程，以及对刺激的反应等，细胞组分主要与膜等相关，KEGG注释结果显示，生物系统通路中主要包括植物-病原菌互作和环境适应等通路。GO富集的表达差异基因主要同跨膜受体蛋白活性、蛋白磷酸化及信号受体活性等方面相关，KEGG富集到的最多差异表达基因（differently expressed genes，DEGs）主要在植物-病原菌互作和植物MAPK信号通路上。结合南方大豆皱叶症诱因特点，选择定位候选区间内与抗病等相关且在外显子上存在非同义突变或表达量有差异的基因作为候选基因，结合qRT-PCR验证，最终确定GLYMA_12G223100、GLYMA_12G223900、GLYMA_12G224100、GLYMA_12G231800和GLYMA_12G233000等5个基因为控制南方大豆皱叶症的候选基因。【结论】HGRNA-seq实现了RNA-seq和BSA-seq相结合，成功挖掘了控制SSCLD的候选基因。

关键词: 大豆, 转录组, 皱叶症, 定位, 分子标记, 二代测序

Abstract:

【Objective】 Severe SSCLD can lead to about 40% yield reduction of soybean. In this study, candidate genes associated with controlling SSCLD were identified by HGRNA-seq technology to provide data support for revealing the molecular mechanisms of SSCLD.【Method】Two parents with SSCLD resistance difference and their derived F_2:7 lines were used as the experimental materials. Two parents and 12 F_2:7 lines (7 crinkled leaves and 5 normal leaves) were separately subjected to re-sequenced and transcriptome sequencing. Mixed pool method was carried out for gene localization by using SNP/InDel data from the parents and their progenies. GO and KEGG functional annotation and enrichment analysis was carried out by using differentially expressed genes (DEGs) from RNA-seq. 7 KASP molecular markers were developed in the vicinity of localization interval, and 230 F₂ populations were used to construct a localized linkage map to verify the localization results of the transcriptome data. Candidate genes controlling SSCLD were screened by combining gene mapping and transcriptome analysis.【Result】The gene controlling SSCLD named CL12 was located on chromosome 12 within a 1 473 464 bp interval by using mixed pool method, ranging from 39 231 651 bp to 40 705 115 bp. At the same time, the gene was localized in the 1 205 020 bp interval from 39 743 275 to 40 948 295 bp by using the F₂ population, which was basically consistent with the results of the mixed-pool method. The GO annotation results showed that the metabolic processes included immune system processes and responses to stimuli, and cellular components were mainly related to membranes, etc. The KEGG annotation results showed that the biosystem pathways mainly included plant-pathogen interaction and environmental adaptation, etc. GO-enriched DEGs were mainly related to the activity of transmembrane receptor proteins, protein phosphorylation, and signaling receptors, etc., KEGG enriched DEGs were mainly related to plant-pathogen interaction and MAPK signaling pathway. Combined with the characteristics of the causal factors of SSCLD, genes within the candidate interval associated with disease resistance which had non-synonymous mutations in the coding exons, or showed differential expression were selected as candidate genes for SSCLD resistance. GLYMA_12G223100, GLYMA_12G223900, GLYMA_12G224100, GLYMA_12G231800 and GLYMA_12G233000 were finally identified as the candidate genes controlling SSCLD by qRT-PCR.【Conclusion】HGRNA-seq realized the combination of RNA-seq and BSA-seq, and successfully mined the candidate genes controlling SSCLD.

Key words: soybean, transcriptome, crinkle leaf disease, localization, molecular markers, second generation sequencing

陈文杰, 陈渊, 韦清源, 汤复跃, 郭小红, 陈淑芳, 覃夏燕, 韦荣昌, 梁江. 利用高世代转录组测序挖掘控制南方大豆皱叶症候选基因[J]. 中国农业科学, 2024, 57(15): 2914-2930.

CHEN WenJie, CHEN Yuan, WEI QingYuan, TANG FuYue, GUO XiaoHong, CHEN ShuFang, QIN XiaYan, WEI RongChang, LIANG Jiang. Identification of Candidate Genes Controlling SSCLD by Utilizing High-Generation Segregating Populations RNA-seq[J]. Scientia Agricultura Sinica, 2024, 57(15): 2914-2930.

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标记 Markers	Primer_AlleleFAM (5′-3′)	Primer_AlleleHEX (5′-3′)	Primer_Common (5′-3′)	AlleleFAM	AlleleHEX
Chr.12_38720327	gaaggtgaccaagttcatgctaaagcatgctttaatattttggA	gaaggtcggagtcaacggattaagcatgctttaatattttggG	GCAATTGAAAACATAAGTCACGCCTAC	A	G
Chr.12_39553364	gaaggtgaccaagttcatgcttaaatcgtgtttcttttcctcctgC	gaaggtcggagtcaacggatttgtaaatcgtgtttcttttcctcctgT	CCCAACGGCTGCAATGGTC	C	T
Chr.12_39643002	gaaggtgaccaagttcatgctatggttggaccagacaggtaaggG	gaaggtcggagtcaacggattatggttggaccagacaggtaaggA	CTCTTTGCGCTGCTCGTCATTACTC	G	A
Chr.12_39743275	gaaggtgaccaagttcatgctgggagtccttgttatgtattatgatggC	gaaggtcggagtcaacggattgagggagtccttgttatgtattatgatggT	ATCCCCTAAGAAGTTGATACCACAT	C	T
Chr.12_40239112	gaaggtgaccaagttcatgctggaggcgactaaaattgtttccaaT	gaaggtcggagtcaacggattggaggcgactaaaattgtttccaaC	AAATTAGCCAAACACGCATGGTTCA	T	C
Chr.12_40443647	gaaggtgaccaagttcatgctccgaattgtgttttaagtcatatcT	gaaggtcggagtcaacggattccgaattgtgttttaagtcatatcC	GCAATATCAAGAATGAAACTTCT	T	C
Chr.12_40948295	gaaggtgaccaagttcatgctattgaaccatgttagcacagcagtT	gaaggtcggagtcaacggattattgaaccatgttagcacagcagtA	TAACAGAATGCTAACTTGTGATACT	T	A

样本 Sample	过滤得到的reads数 Clean reads	过滤后的总碱基数 Clean bases	Q30 (%)	GC含量 GC content (%)	定位到基因组总Reads占比 Mapped Reads radio (%)
GC8	37329976	11235520244	91.72	35.15	99.54
Y2017-1	36749923	11054768304	92.28	35.69	98.64
C_10	48149604	7164122179	96.23	45.71	94.20
C_11	47618902	7090388779	96.57	45.95	94.19
C_18	45335046	6734925207	96.65	45.6	95.18
C_23	49603440	7381504895	96.56	46.14	95.12
C_24	49929876	7430549886	96.52	45.69	95.35
C_3	41997550	6249241100	96.59	45.94	94.70
C_6	45105078	6703705471	96.55	46.07	94.78
N_1	48155648	7150543340	96.44	45.87	95.00
N_3	44567696	6617278005	96.44	45.86	93.82
N_5	47817526	7091029900	96.66	46.07	94.92
N_6	42285914	6296598949	95.26	45.57	94.52
N_7	40742934	6065420513	96.49	46.24	94.91

染色体 Chromosome	长度 Length (bp)	来自GC8 From GC8	来自Y2017-1 From Y2017-1	相同位点 Same site	杂合位点 Hybridization site	SNP/InDel数 No. of SNP/ InDel site	位点总数 Total numbers of SNP/InDel site	纯合率 Purity ratio (%)	标记密度 Density of SNP/ InDel (SNP/Mb)
Chr.1	57932355	150	285	607	11	435	1053	98.96	7.70
Chr.2	50400358	143	218	616	10	361	987	98.99	7.36
Chr.3	46951866	68	334	1107	159	402	1668	90.47	11.95
Chr.4	51203389	159	156	602	26	315	943	97.24	6.66
Chr.5	42274530	299	1	854	239	300	1393	82.84	12.75
Chr.6	50945864	326	217	1196	54	543	1793	96.99	11.72
Chr.7	44949256	479	125	1091	188	604	1883	90.02	17.62
Chr.8	47227184	119	501	820	476	620	1916	75.16	23.21
Chr.9	50572668	339	201	984	284	540	1808	84.29	16.29
Chr.10	51638687	84	229	616	70	313	999	92.99	7.42
Chr.11	39643745	35	277	584	27	312	923	97.07	8.55
Chr.12	41531199	106	77	690	131	183	1004	86.95	7.56
Chr.13	45225048	159	88	1399	422	247	2068	79.59	14.79
Chr.14	49893278	240	176	679	103	416	1198	91.40	10.40
Chr.15	53754295	84	271	955	185	355	1495	87.63	10.05
Chr.16	38112070	637	191	1193	157	828	2178	92.79	25.84
Chr.17	41740656	24	425	597	23	449	1069	97.85	11.31
Chr.18	58286270	404	357	1975	159	761	2895	94.51	15.78
Chr.19	51272880	94	94	924	25	188	1137	97.80	4.15
Chr.20	47846026	323	14	776	16	337	1129	98.58	7.38
总计/平均 Total/mean	961401624	4272	4237	18265	2765	8509	29539	91.61	11.73

基因编号 Gene ID	标记 Marker	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	退火温度 Tm (℃)
GLYMA_12G223900	qPCR-12G223900	TGTGTAGTGGCTTGTCATAT	CTTAGTCTTATGGAGCAATCAC	60
GLYMA_12G224000	qPCR-12G224000	ATTGATGTTGGTGCTTCTTG	TGAATGTGTTGGTGCTTCTA	60
GLYMA_12G224100	qPCR-12G224100	CATCAACCCAACTCACTCAA	GTGGCAACATAAACTCTTGT	60
GLYMA_12G233000	qPCR-12G233000	GCTTCAACCGATCAACTTAT	CCACCATACATATAGCAAGC	60