普通菜豆镰孢菌枯萎病抗性种质资源筛选及全基因组关联分析

doi:10.11983/CBB22149

摘要/Abstract

摘要： 普通菜豆镰孢菌枯萎病是严重制约菜豆(Phaseolus vulgaris)产量的主要病害之一。采用下胚轴双孔注射法对601份普通菜豆种质资源进行枯萎病抗性鉴定, 共筛选出4份高抗材料。在此基础上, 基于分布在全基因组上的3 765 456个单核苷酸多态性(SNP)标记, 进行全基因组关联分析, 以P<1×10^-5为阈值。结果检测到57个显著关联的SNP位点, 分布于1、2、6、8和11号染色体上; 共获得8个显著关联区域, 其中位于1号染色体上的区域1包含SNP最多(48个), 最显著SNP P值为2.18E-07。在8个显著关联区域中, 共检测到186个基因, 其中157个基因有注释信息, 编码过氧化物酶、抗病蛋白、转录因子和蛋白激酶等。结合KEGG富集分析和序列同源性比对, 鉴定出9个候选基因可能与抗性相关。

关键词: 普通菜豆, 镰孢菌枯萎病, 全基因组关联分析, 抗性基因/种质资源

Abstract: Fusarium wilt is one of the main diseases that seriously restrict the yield of common bean in China. In this study, the hypocotyl double hole injection method was used to identify the resistance of 601 common bean germplasm resources to fusarium wilt, and 4 highly resistance materials were screened out. Based on 3 765 456 single nucleotide polymorphisms (SNPs) distributed on the whole genome, the genome-wide association study was conducted with P<1×10^-5 as the threshold. A total of 57 significant SNPs were detected, which are distributed on chromosomes 1, 2, 6, 8, and 11 respectively. A total of 8 significant associated regions were obtained, of which region 1 on chromosome 1 contained up to 48 SNPs and the most significant SNP P value was 2.18E-07. Furthermore, 186 genes were detected in 8 significant association regions, of which 157 were annotated genes, including encoding peroxidase, disease resistance protein, transcription factor and protein kinase genes. Combing KEGG enrichment analysis and homology comparison, 9 candidate genes were identified that might be related to resistance.

Key words: common bean, fusarium wilt, genome-wide association study, resistance gene/germplasm resources

李园, 常玉洁, 王兰芬, 王述民, 武晶. 普通菜豆镰孢菌枯萎病抗性种质资源筛选及全基因组关联分析. 植物学报, 2023, 58(1): 51-61.

Yuan Li, Yujie Chang, Lanfen Wang, Shumin Wang, Jing Wu. Screening of Resistance Germplasm Resources and Genome-wide Association Study of Fusarium Wilt in Common Bean. Chinese Bulletin of Botany, 2023, 58(1): 51-61.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22149

https://www.chinbullbotany.com/CN/Y2023/V58/I1/51

图/表 10

图1 普通菜豆不同抗性等级维管束病变程度 0、1、3、5、7和9代表病情分级标准。Bar=4 cm

Figure 1 Common bean vascular bundle lesions of different grades 0, 1, 3, 5, 7 and 9 represent the criteria for disease classification. Bar=4 cm

表1 下胚轴双孔注射法接种普通菜豆枯萎病的病情分级标准

Table 1 Disease grading standard of common bean fusarium wilt inoculated by hypocotyl double-hole injection

Rating scale	System description
0	No symptom
1	<20% lesions or discoloration of hypocotyledonary axis
3	20%-50% lesions or some discoloration of hypocotyledonary axis
5	50%-100% lesions or heavy discoloration of hypocotyledonary axis
7	>100% lesions or severe discoloration of hypo- cotyledonary axis
9	Plants wilted and died

图2 病变程度频率分布直方图及正态分布

Figure 2 Frequency distribution histogram and normal distribution of the lesion degree

表2 普通菜豆自然群体枯萎病抗性水平表型描述

Table 2 Phenotype description of fusarium wilt resistance levels in natural populations of common bean

Mean	Max	Min	Standard deviation	Skewness	Kurtosis
0.65	1.00	0.18	0.19	-0.10	-0.62

图3 普通菜豆种质资源枯萎病抗性等级分布 HR: 高抗; MR: 中抗; I: 中间型; MS: 中感; HS: 高感

Figure 3 Distribution of common bean germplasm resources for resistance to fusarium wilt HR: Highly resistant; MR: Medium resistant; I: Intermediate; MS: Medium susceptible; HS: Highly susceptible

图4 普通菜豆枯萎病全基因组关联分析 (A) 曼哈顿图; (B) QQ图; (C) 区段1的局部曼哈顿图

Figure 4 Genome-wide association study of fusarium wilt in common bean (A) Manhattan plots; (B) QQ plots; (C) Local Manhattan plot of the Region 1

表3 普通菜豆枯萎病全基因组关联分析位点

Table 3 Loci related with common bean fusarium wilt by genome-wide association study

Region	Chr.	Interval location^a	P-value
1	1	2583702-2807552	2.18E-07
2	1	50017907-50231907	4.11E-06
3	2	26253984-26467984	5.72E-06
4	2	29642890-29856890	7.61E-06
5	6	26400983-26614983	8.82E-06
6	8	58863473-59077488	5.40E-06
7	11	46491343-46705343	6.48E-06
8	11	49598097-49814082	7.73E-06

表4 候选基因及注释

Table 4 Candidate genes and annotation

Gene ID	Chr.	Start base-end base^a (bp)	Predicted function
Phvul.001G240400	1	50026833-50031313	RNI-like superfamily protein
Phvul.001G241500	1	50126351-50130393	Transmembrane amino acid transporter family protein
Phvul.001G242200	1	50180058-50185697	Cytochrome P450 superfamily protein
Phvul.002G133400	2	26437427-26440702	NB-ARC domain-containing disease resistance protein
Phvul.002G133600	2	26447784-26451915	NB-ARC domain-containing disease resistance protein
Phvul.002G154800	2	29644994-29651836	PAS domain-containing protein tyrosine kinase family protein
Phvul.006G150900	6	26403392-26405713	Protein kinase superfamily protein
Phvul.008G285400	8	59055451-59060614	Heat shock protein 89.1
Phvul.011G212100	11	49695779-49697294	Leucine-rich repeat (LRR) family protein

图5 9个抗性候选基因在普通菜豆不同组织中的表达模式 Stem_10为播种10天后于茎部取样, Stem_19为播种19天后于茎部取样, Roots_10为播种10天后于根部取样, Roots_19为播种19天后于根部取样。数据来源于 https://phytozome-next.jgi.doe.gov/info/Pvulgaris_v2_1。

Figure 5 Expression patterns of 9 resistance candidate ge- nes in common bean different tissues Stem_10 is to take samples from the stem 10 days after planting, Stem_19 is to take samples from the stem 19 days after planting, Roots_10 is to take samples from the root 10 days after planting, Roots_19 is to take samples from the root 19 days after planting. Data is from https://phytozome-next.jgi.doe.gov/info/Pvulgaris_v2_1.

图6 Phvul.001G212100单倍型分析 (A) Phvul.001G212100基因结构和单倍型(线性基因结构显示5'UTR (青色矩形)、CDS区域(黑色矩形)和3'UTR (红色矩形); 黑色虚线表示SNP在基因中的位置); (B) 4个单倍型在育成品种和地方品种中的发生频率; (C) 单倍型间病变程度的差异显著性分析(箱线图上方数字为P-value值, P<0.05表示差异显著。n为种质资源份数。用双尾T检验进行显著性检验)。CDS: 编码序列; UTR: 非翻译区

Figure 6 Haplotype analysis of Phvul.001G212100 (A) The genome structure and haplotypes of the Phvul.001G212100 (the linear gene structure showing 5'UTR (cyan rectangle), CDS region (black rectangle), 3'UTR (red rectangle); the black dotted line indicate the position of the SNP in the gene); (B) Occurrence frequencies of 4 haplotypes in breeding lines and landraces; (C) Significant differences in the degree of lesions between haplotypes (the numbers above the boxplot are P-values, P<0.05 indicate significant differences. n represent the number of germplasm recourses. The significance was tested with two-tailed T-tests). CDS: Coding sequence; UTR: Untranslated region

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