柑橘溃疡病抗性SNP验证及其相关钙依赖性蛋白激酶基因诱导表达

Verification of SNPs Associated with Citrus Bacterial Canker Resistance and Induced Expression of SNP-Related

【目的】前期根据转录组数据挖掘柑橘单核苷酸多态性(single nucleotide polymorphism,SNP)位点,通过关联分析获得14个与柑橘溃疡病耐/感性关联的SNP。以此为基础,本研究利用柑橘杂交群体验证这些位点与柑橘溃疡耐/感性的相关性,以期获得显著相关的SNP,并对其相关的基因进行柑橘溃疡病菌(Xanthomonas citri subsp.citri,Xcc)和植物激素诱导表达分析。【方法】以抗病和敏感柑橘品种及其杂交F1代群体共143个材料为试材,采用离体叶片针刺接种法进行溃疡病抗性鉴定;利用高分辨率熔解曲线(high resolution melting,HRM)技术,对F1群体进行SNP分型;使用DPS软件对F1群体的溃疡病耐/感性表型和SNP基因型进行相关分析;实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)分析其中一个SNP相关的柑橘钙依赖性蛋白激酶基因(calcium-dependent protein kinase gene,CDPK)的诱导表达模式。【结果】供试杂交F1代群体的病斑面积在0.75—3.29 mm2;病情指数在30.2—100,而抗病品种‘金弹’病情指数为11.1,敏感品种‘冰糖橙’病情指数为100。病情指数较低的杂交后代,其亲本至少有1个为耐病性品种,母本耐病性强的居多。根据病情指数确定免疫材料1个,高抗17个,中抗31个,中感38个,高感56个。SNP分型结果显示,14个SNP位点在143个供试材料间均有多态性,可分为3种不同的基因型,2种纯合型和1种杂合型。简单相关分析结果表明,多个SNP位点的基因型与病斑面积及病情指数相关性显著。典型相关分析结果显示,其中5个SNP位点与病斑面积相关性较高,相关系数绝对值均>0.2,其编号分别为HP31、HP42、HP85、HP87、HP170,可利用这5个SNP位点的基因型预测柑橘溃疡病耐性的强弱。其中SNP位点HP31位于CsCDPK(CAP ID:Cs4g10370)的编码区,对柑橘离体叶片接种溃疡病菌诱导处理6、12、24、48和72 h后进行基因相对表达量分析,发现‘金弹’(高抗)、‘新生系3号椪柑’(中抗)和‘冰糖橙’(高感)中该基因的表达量均呈先升后降趋势,且均在48 h其相对表达量达到最高;接菌处理后12 h,‘金弹’中该基因的相对表达量为对照的3倍,而‘新生系3号椪柑’和‘冰糖橙’中无明显差异。此外,CsCDPK受水杨酸(SA)、茉莉酸甲酯(MeJA)和脱落酸(ABA)诱导表达,且在不同溃疡病耐/感性品种中该基因诱导表达的模式不同。【结论】获得5个与溃疡病耐/感性显著相关的SNP,可用作柑橘溃疡病耐/感性筛选标记。SNP位点HP31相关的CsCDPK受溃疡病菌和SA、MeJA和ABA诱导表达,该基因可能在柑橘应答溃疡病菌侵染的信号转导过程中具有重要功能。

【Objective】In the previous study,single nucleotide polymorphisms(SNPs)of citrus varieties were screened based on transcriptome,and 14 SNPs were defined to be associated with citrus bacterial canker(CBC)resistance via association analysis.In this study,it is aimed to verify the correlation between these SNP loci and CBC resistance in order to obtain significantly related SNPs and to find the inducible expression profiles of corresponding genes by plant hormones and Xanthomonas citri subsp.citri(Xcc)infection.【Method】The sensitive and resistant varieties and their F1 populations were used for CBC resistance identification via in vitro acupuncture inoculation and SNP-based genotyping was conducted via high resolution melting(HRM)technology.The phenotypes and genotypes were then associated by software DPS,and inducible expression profiles of SNP(HP031)related calcium-dependent protein kinase gene(CDPK)were analyzed by quantitative real-time PCR(qRT-PCR).【Result】The lesion areas of F1 populations ranged from 0.75 to 3.29 mm2,and the disease index ranged from 30.2 to 100,while the resistant variety Jindan had a disease index of 11.1,and disease index of the susceptible variety Bingtangcheng was 100.For the offspring of hybrids with a low disease index,at least one of their parents is a disease-tolerant variety,and most of the female parents have strong disease-tolerance.According to the disease index,the populations could be grouped into immune(1 variety),highly resistant(17),moderately resistant(31),moderately sensitive(38),and highly sensitive(56).The SNP typing results showed that all the 14 SNP loci were polymorphic among 143 test materials,which could be divided into 3 different genotypes,2 homozygous types and 1 heterozygous type.The results of simple correlation analysis showed that the genotypes of multiple SNP loci were significantly correlated with the lesion area and disease index.The results of canonical correlation analysis showed that the correlation between the 5 SNP loci and the lesion area was high,the absolute values of the correlation coefficients were all>0.2,and their numbers were HP31,HP42,HP85,HP87,and HP170.The genotypes of these 5 SNP loci could be used to predict the tolerance to CBC.SNP HP31 located in the coding region of CsCDPK(CAP ID:Cs4g10370).The expression of CsCDPK was analyzed at 6,12,24,48 and 72 hpi(hours post inoculation),it was found that the expression of CsCDPK all increased first and then decreased,and reached the highest relative expression at 48 hpi in Jindan(highly resistant),Xinshengxi No.3(moderately resistant),and Bingtangcheng(highly sensitive)varieties.At 12 hpi,the relative expression level of CsCDPK in Jindan was 3 times of that in the control,but there was no significant difference in Xinshengxi No.3 and Bingtangcheng.Besides,CsCDPK was also differently induced by salicylic acid(SA),methyl jasmonate(MeJA),and abscisic acid(ABA)in CBC resistant and sensitive varieties.【Conclusion】Five SNPs associated with CBC resistance were verified,which can be used for marker-assistant selection.SNP HP31 related gene CsCDPK can be induced by Xcc and phytohormones,which may play an important role in the signal transduction process of citrus response to Xcc.