Genome-wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.)

---

发表期刊：Plant Biotechnology Journal (2017) 15, pp. 1520–1532

发表单位：中国农科院和新疆农垦科学院

背景：黄萎病（Verticillium wilt）是土传病害，感染后，棉花产量和品质会大幅下降。

一、材料和方法

材料：

A total of 299 cotton accessions were collected from major breeding institutes across China and the germplasm gene bank of CRI-CAAS.

方法：

1 . 大田和温室鉴定

A scale of 0–4 was used to rate disease severity based on the percentage of diseased leaves.

将病害分为0-4个等级，并转化为病情指数。 

2. SLAF测序

The GATK (McKenna et al., 2010) and SAMtools (Li et al., 2009) packages were used to perform SNP calling. All SNPs called by GATK and SAMtools were designated high-quality.

3. LD和群体结构分析

LD was calculated as the squared correlation coefficient (r2) of alleles using Haploview 4.2 (Barrett et al., 2005). Parameters in the program included MAF (≥0.05) and the missing rate of each SNP (≤50%). A total of 85 630 SNPs distributed evenly across the entire genome were selected for genetic relatedness analyses. PCA based on the same SNP set was carried out using the EIGENSOFT software (Price et al., 2006). The phylogenetic tree of the 299 accessions was constructed using MEGA 5.1 program with neighbour-joining methods (1000 bootstraps) (Tamura et al., 2011). The relative kinship matrix of 299 cotton lines was computed using SPAGeDi software (Hardy and Vekemans, 2002).

4. 全基因组关联分析

A total of 85 630 SNPs were used for GWAS with a compressed mixed linear model (CMLM) implemented in the GAPIT software (Lipka et al., 2012). The first three PCA values were used as fixed effects in the model to correct for stratification (Price et al., 2006). The GWAS threshold was set to P < 1.17 9 10–5 (1/total SNPs used, –log10P = 4.93).genes that were located within the region 300 kb upstream or downstream of trait-associated SNPs were identified as candidate associated genes.

利用GAPIT进行全基因组关联分析，模型为PCA+K，关联位点两侧300kb内的基因为候选基因。

5. 候选基因的验证

qRT-PCR analysis and VIGS analysis

---

二、结果

结果共分为三部分：表型鉴定，关联分析和候选基因验证

1. 表型鉴定

We evaluated resistance to VW in the 299 accessions in a glasshouse (RDIG2015) and in a field screening nursery in 2015 (RDIF2015) and 2016 (RDIF2016), with three replicates per environment.

An analysis of variance (ANOVA) of the RDI in three environments revealed significant differences among the genotypes (F = 8.26, P < 0.0001)

三环境三重复鉴定

2.全基因组关联分析

关联结果与以往研究结果的比对；共关联到17个显著关联位点，其中3个(A04_2334165, A04_3445154 and A04_3516941)定位于已知QTL区域内

We performed trait–SNP association analysis for the three environments (RDIG2015, RDIF2015 and RDIF2016) with the CMLM, and which accounts for both population structure and familial relatedness.

A total of 3, 3 and 11 significant SNPs were detected at P < 1.17 9 10–5 (P = 1/ 85 630, –log10P = 4.93) in RDIG2015, RDIF2015 and RDIF2016, respectively（图1）.

Three significant SNPs (A04_2334165, A04_3445154 and A04_3516941) on A04 were mapped to regions where VW resistance QTL (qVWR-08-c4-1) had been previously reported (Zhang et al., 2015a)

图1. Manhattan 图

 有利变异（favourable SNP alleles）

To understand the effects of allelic variation on VW resistance, we selected three significant SNPs (A10_99672586, A10_98859056 and A10_99071906) on A10 as identified favourable alleles.

For the first peak SNP (A10_99672586), the average RDI of accessions with favourable alleles (T) was 27.81, which was lower than the average RDI (48.68) of accessions with unfavourable alleles (C) investigated in RDIF2016. ......

The average RDI values for accessions with two or three favourable alleles were lower significantly than those with zero or a single favourable allele (P < 0.01). These results indicate that favourable SNP alleles had significant pyramiding effects on VW resistance（图2）.

图2 有利变异和非有利变异的比对

---

3. 候选基因及验证

The first peak SNP (A10_99672586) on A10 exhibited the minimum P-value (–log10P = 6.21) in RDIF2016, and may be a major genetic locus responsible for VW resistance in cotton. 

Haplotype block structure analysis for A10 indicated that the candidate gene regions were 99.38–99.75 Mb, and a total of 22 genes were found on A10.

A gene ontology analysis showed that three genes were without any definite annotation concerning their biological function and four genes had unpredicted pathways. Four genes were linked to biological pathways involved in plant stress response, including disease resistance, and the other genes were predicted to be involved in transport, translational regulation, transcription regulation and signal transduction, among other processes.

作者选取一个P-value最小的位点(A10_99672586)进行分析注释，单倍型分析（Haplotype block structure analysis）A10_99672586位于99.38–99.75 Mb之间，包含22个基因（图3），并对其进行了注释。

图3 单倍型分析

---

To investigate which genes were responsible for resistance to VW, we used quantitative real-time PCR (qRT-PCR) analysis to determine differential gene expression in candidate genes identified by GWAS analysis. 

The results showed that five genes (CG02, CG03, CG12, CG13 and CG19) were up-regulated in the resistant (R) genotype Zhongzhimian2 (ZZM2) and susceptible (S) genotype Jimian11 (JM11) at different times after V. dahliae inoculation.

Two genes (CG02 and CG13) specific to the R genotype were implicated in resistance against V. dahliae in cotton.

Arabidopsis homologues of CG02 and CG13 encode TIR-NBS-LRR protein and WOX family transcription factors, respectively, which have been proposed to be involved in disease resistance response and signal transduction.

作者对上述22个候选基因进行qRT-PCR分析，发现有5个基因(CG02, CG03, CG12, CG13 and CG19)在抗感基因型中表达不同，其中CG02和CG13在抗性基因型中上调表达，在感病材料中下调表达（图4）。

图4   qRT-PCR分析

---

To further investigate the function of the five R genotype upregulated genes in VW resistance, we performed VIGS, constructing recombinant viruses including pTRV2:CG02, pTRV2:CG03, pTRV2:CG12, pTRV2:CG13 and pTRV2:CG19 to silence endogenous genes, with pTRV1 serving as a mock treatment.

When plants infiltrated with pTRV2:CLA1 showed bleaching in newly emerged leaves, we used qRT-PCR to confirm the silencing of the genes, which exhibited lower expression levels in five genes in infiltrated pTRV2:CG02, pTRV2:CG03, pTRV2:CG12, pTRV2:CG13 and pTRV2:CG19 plants than the control (ZZM2, WT and pTRV2:00, CK).

We inoculated these plants by dip infection with conidial suspension (5 9 106 conidia/mL). After 3 weeks, the control plants seldom exhibited leaf wilting; approximately 15% of plants were diseased.

The vascular wilt symptoms varied; the stems of CG02-silenced plants turned brown, whereas other gene-silenced plants showed no wilt symptoms in stems.

Fungal biomass qRT-PCR analysis demonstrated that CG02-silenced plants developed significantly higher fungal biomass than control plants, 4.3-fold higher than that in WT and 3.9-fold higher than in CK, whereas fungal biomass in CG03-, CG12-, CG13- and CG19- silenced plants did not differ significantly from that in control plants.

为进一步确认上调表达基因的功能，作者利用VIGS实验沉默内源基因（pTRV2:CG02, pTRV2:CG03, pTRV2:CG12, pTRV2:CG13 and pTRV2:CG19）。

qRT-PCR表明转化pTRV2:CG02, pTRV2:CG03, pTRV2:CG12, pTRV2:CG13 and pTRV2:CG19后的植株对应的基因表达水平低于对照植株(ZZM2, WT and pTRV2:00, CK)（图5）。

图5 转化植株和对应基因表达水平

接种后，对照植株病情（WT和CK）较轻，约有15%的植株染病（图6a）。CG02-silenced plants茎杆变褐，其他转化植株茎杆未变色（图6b）。

图6 植株接种后表现

图7为接种后病害叶片比率（c）和真菌生物量比对（d）。

图7

这篇文章总体思路中规中矩的，创新点在于最后利用VIGS进行候选基因的验证，这也可能是本文能发到PBJ的原因吧！