干旱胁迫下小粒咖啡SNP位点与可变剪接分析

doi:10.13304/j.nykjdb.2023.0903

摘要/Abstract

摘要：

为丰富小粒咖啡的SNP（single nucleotide polymorphism）位点，并阐明可变剪接（alternative splicing，AS）事件在小粒咖啡响应干旱胁迫中的作用，以小粒咖啡‘Catimor’为供试材料，分别进行7和14 d的干旱胁迫处理，利用RNA-seq技术结合生物信息学方法对小粒咖啡抗旱相关SNP位点和AS事件进行统计分析。结果表明，在所有样品中共鉴定到265 047个SNP位点，转换率和颠换率分别为60.72%和39.28%，分布于11条染色体，其中2号染色体上的SNP最多。位于基因外显子和内含子的SNP位点分别为110 582和100 936个。对SNP位点序列注释发现，SNP位点主要富集于氨基酸合成、运输及碳代谢等方面。AS事件分析表明，在干旱0 d与干旱7 d、干旱7 d与干旱14 d的比较中，分别鉴定到3 809、4 142个差异AS，其中外显子跳跃（skipped exon，SE）是最主要的剪接方式，这些发生AS事件的基因主要与剪接体、mRNA监控等途径显著关联。经差异表达基因与AS基因的比较分析，筛选出LOC113699257、LOC113712703、LOC113741326、LOC113730764共4个在干旱胁迫下持续显著上调的剪接因子，这些因子可能在调控咖啡响应干旱胁迫或根系发育中发挥重要作用。以上研究结果为进一步揭示小粒咖啡响应干旱胁迫的分子机制奠定了基础。

关键词: 干旱胁迫, 小粒咖啡, SNP位点, 可变剪接

Abstract:

To enrich the information on single nucleotide polymorphism （SNP） loci and elucidate the role of alternative splicing （AS） events in response to drought stress in Coffea arabica L.， C. arabica cultivar ‘Catimor’was employed as the experimental material for drought stress treatment for 7 and 14 d， respectively. RNA-seq technology combined with bioinformatics methods was utilized to statistically analyze the drought-resistant SNP loci and AS events in C.arabica. The results showed that a total of 265 047 SNP loci were identified in all the samples， with a transition rate of 60.72% and a transversion rate of 39.28%， distributed on 11 chromosomes， with the most SNPs on Chr. 2. The numbers of SNP loci located in exons and introns of the gene were 110 582 and 100 936， respectively. Sequence annotation revealed that the SNP loci were mainly enriched in amino acid synthesis， transport and carbon metabolism. The results of AS event analysis showed that 3 809 and 4 142 different AS events were identified in the comparison groups of 2 drought stages （0 d of drought vs 7 d of drought； 7 d of drought vs 14 d of drought）， respectively. Among them， SE was found to be the most prevalent splicing mode. The genes responsible for these AS events showed significant associations with spliceosome and mRNA surveillance pathway. Through comparative analysis between differential expression genes and AS genes， 4 splicing factors （LOC113699257，LOC113712703， LOC113741326 and LOC113730764） consistently demonstrated significant up-regulation under drought stress conditions. These drought-induced splicing factors might play crucial roles in regulating coffee response to drought stress or root development. Above results laid a foundation for further revealing the molecular mechanism of C.arabica in response to drought stress.

Key words: Coffea arabica L., drought stress, SNP loci, alternative splicing

中图分类号:

S571.2

喻好好, 董相书, 赵颢, 李忠贤, 胡发广, 李亚男, 娄予强, 何飞飞. 干旱胁迫下小粒咖啡SNP位点与可变剪接分析[J]. 中国农业科技导报, 2025, 27(6): 72-82.

Haohao YU, Xiangshu DONG, Hao ZHAO, Zhongxian LI, Faguang HU, Yanan LI, Yuqiang LOU, Feifei HE. Analysis of SNP Loci and Alternative Splicing Events in Coffea arabica L. Under Drought Stress[J]. Journal of Agricultural Science and Technology, 2025, 27(6): 72-82.

图/表 6

图1 SNP基本特征A：SNP突变类型统计； B：SNP在染色体上的分布情况； C：SNP在参考基因组功能元件上的分布

Fig. 1 Basic characteristics of SNPA： Statistics of SNP mutation types； B： Distribution of all SNP on chromosomes； C： Distribution of SNP on functional elements of the reference genome

表1 编码区SNP变异位点类型分布

Table 1 Distribution of SNP variant loci types in coding region

样品Sample	移码替换Frameshift substitution	非移码替换Nonframeshift substitution	同义单核苷酸突变 Synonymous SNV	非同义单核苷酸突变 Nonsynonymous SNV	终止密码子增加 Stopgain	终止密码子减少 Stoploss	未知类型Unknown
CK1	1 620	374	36 596	34 741	111	42	630
CK2	1 634	379	37 284	35 619	114	39	632
CK3	1 612	347	35 002	32 972	103	39	605
CK4	1 609	382	36 612	34 849	109	35	646
D1	1 461	298	30 708	28 819	98	29	587
D2	1 440	306	30 213	28 156	97	29	541
D3	1 550	295	30 583	28 769	103	33	600
D4	1 404	282	27 954	26 104	85	27	533
HD1	1 251	255	25 352	23 035	70	26	473
HD2	1 281	221	23 966	21 613	64	25	475
HD3	1 208	239	22 441	20 450	61	29	390
HD4	1 294	248	24 817	22 921	70	25	475

图2 SNP变异位点功能注释A：GO生物过程富集； B：GO细胞组分过程富集； C：GO分子功能富集； D：KEGG注释

Fig. 2 Functional annotation of SNP variant lociA： Biological process of GO enrichment； B： Cell component of GO enrichment； C： Molecular function of GO enrichment； D： KEGG annotation

图3 干旱胁迫7 d后差异可变剪接事件统计与功能分析A：显著差异可变剪接事件分类统计； B：显著差异可变剪接事件韦恩分析； C：发生显著差异可变剪接事件的基因功能注释。A3SS—3’端可变剪接；A5SS—5’端可变剪接；RI—内含子保留；MXE—外显子互斥；SE—外显子跳跃

Fig. 3 Statistics and functional analysis of differential AS events after 7 d of drought stress.A： Categorical statistics of significant differential AS events； B： Venn analysis of significant differential AS events； C： Functional annotation of genes with significant differential AS events. A3SS—Alternative 3’ splice site； A5SS—Alternative 5’ splice site； RI—Retained intron； MXE—Mutually exclusive exon； SE—Skipped exon

图4 干旱胁迫14 d后差异可变剪接事件统计与功能分析A：显著差异可变剪接事件分类统计； B：显著差异可变剪接事件韦恩分析； C：发生显著差异可变剪接事件的基因功能注释。A3SS—3’端可变剪接；A5SS—5’端可变剪接；RI—内含子保留；MXE—外显子互斥；SE—外显子跳跃

Fig. 4 Statistics and functional analysis of differential AS events after 14 d of drought stress.A： Categorical statistics of significant differential AS events； B： Venn analysis of significant differential AS events； C： Functional annotation of genes with significant differential AS events. A3SS—Alternative 3’ splice site； A5SS—Alternative 5’ splice site； RI—Retained intron； MXE—Mutually exclusive exon； SE—Skipped exon

图5 剪接体相关基因在不同条件下的差异表达和可变剪接分析A：剪接体相关基因的韦恩图； B：剪接体相关基因差异表达分析； C：剪接体相关差异基因的可变剪接模式； D：4个剪接体基因在不同条件下的表达模式。A3SS—3’端可变剪接；A5SS—5’端可变剪接；RI—内含子保留；MXE—外显子互斥；SE—外显子跳跃

Fig. 5 Differential expression and AS analysis of spliceosome related genes under different conditionsA： Venn analysis of spliceosome related genes； B： Differential expression analysis of spliceosome related genes； C： AS patterns of spliceosome related differential genes； D： Expression patterns of four spliceosome genes under different conditions. A3SS—Alternative 3’ splice site； A5SS—Alternative 5’ splice site； RI—Retained intron； MXE—Mutually exclusive exon； SE—Skipped exon

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