Genome-wide association study discovered candidate
genes of Verticillium wilt resistance in upland cotton
(Gossypium hirsutum L.)
發(fā)表期刊：Plant Biotechnology Journal (2017) 15, pp. 1520–1532
發(fā)表單位：中國農(nóng)科院和新疆農(nóng)墾科學(xué)院
背景：黃萎病（Verticillium wilt）是土傳病害有序，感染后筹我，棉花產(chǎn)量和品質(zhì)會大幅下降弥锄。
一溅固、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 . 大田和溫室鑒定
A scale of 0–4 was used to rate disease severity based on the percentage?of diseased leaves.
將病害分為0-4個等級遇伞，并轉(zhuǎn)化為病情指數(shù)字旭。?
2. SLAF測序
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和群體結(jié)構(gòu)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. 全基因組關(guān)聯(lián)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進(jìn)行全基因組關(guān)聯(lián)分析，模型為PCA+K莺奔，關(guān)聯(lián)位點兩側(cè)300kb內(nèi)的基因為候選基因欣范。
5. 候選基因的驗證
qRT-PCR analysis and VIGS analysis
二、結(jié)果
結(jié)果共分為三部分：表型鑒定令哟，關(guān)聯(lián)分析和候選基因驗證
1. 表型鑒定
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)
三環(huán)境三重復(fù)鑒定
2.全基因組關(guān)聯(lián)分析
關(guān)聯(lián)結(jié)果與以往研究結(jié)果的比對恼琼；共關(guān)聯(lián)到17個顯著關(guān)聯(lián)位點，其中3個(A04_2334165, A04_3445154 and A04_3516941)定位于已知QTL區(qū)域內(nèi)
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（圖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)
?有利變異（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（圖2）.
3. 候選基因及驗證
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.
作者選取一個P-value最小的位點(A10_99672586)進(jìn)行分析注釋屏富，單倍型分析（Haplotype block structure analysis）A10_99672586位于99.38–99.75 Mb之間晴竞，包含22個基因（圖3），并對其進(jìn)行了注釋狠半。
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.
作者對上述22個候選基因進(jìn)行qRT-PCR分析噩死，發(fā)現(xiàn)有5個基因(CG02, CG03, CG12, CG13 and CG19)在抗感基因型中表達(dá)不同，其中CG02和CG13在抗性基因型中上調(diào)表達(dá)神年，在感病材料中下調(diào)表達(dá)（圖4）已维。
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.
為進(jìn)一步確認(rèn)上調(diào)表達(dá)基因的功能，作者利用VIGS實驗沉默內(nèi)源基因（pTRV2:CG02, pTRV2:CG03, pTRV2:CG12, pTRV2:CG13 and pTRV2:CG19）已日。
qRT-PCR表明轉(zhuǎn)化pTRV2:CG02, pTRV2:CG03, pTRV2:CG12, pTRV2:CG13 and pTRV2:CG19后的植株對應(yīng)的基因表達(dá)水平低于對照植株(ZZM2, WT and pTRV2:00, CK)（圖5）垛耳。
接種后，對照植株病情（WT和CK）較輕，約有15%的植株染舶纭（圖6a）。CG02-silenced plants莖桿變褐占婉，其他轉(zhuǎn)化植株莖桿未變色（圖6b）泡嘴。
圖7為接種后病害葉片比率（c）和真菌生物量比對（d）。
這篇文章總體思路中規(guī)中矩的逆济，創(chuàng)新點在于最后利用VIGS進(jìn)行候選基因的驗證酌予，這也可能是本文能發(fā)到PBJ的原因吧！
