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

doi:10.3864/j.issn.0578-1752.2020.09.010

Abstract

Abstract:

【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.

Key words: citrus bacterial canker (CBC), Xanthomonas citri subsp. citri (Xcc), single nucleotide polymorphism (SNP), calcium-dependent protein kinase gene (CDPK), induced expression

Yun PENG,TianGang LEI,XiuPing ZOU,JingYun ZHANG,QingWen ZHANG,JiaHuan YAO,YongRui HE,Qiang LI,ShanChun CHEN. Verification of SNPs Associated with Citrus Bacterial Canker Resistance and Induced Expression of SNP-Related Calcium-Dependent Protein Kinase Gene[J].Scientia Agricultura Sinica, 2020, 53(9): 1820-1829.

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SNP编号 SNP number	SNP位点 SNP loci	正向引物序列 Forward primer sequence (5′-3′)	反向引物序列 Reverse primer sequence (5′-3′)
HP02	GGTTTTT GGTGTTT	AGAGCTGCCTTAGTGGTGTC	CAGTCTCTACTGCCCCGTTC
HP17	ATATCCC ATAGCCC	ACCAGTTACTCTCCGTCCGA	TTAGGGTTTGGCGAGGACG
HP18	GATCGAA GATTGAA	CAGAACATTATCCGGCATGGG	CCTGAATGAGAGGTGCTGATTT
HP31	GCCATAT GCCGTAT	TGGCAGAATGTTGGGTAGGG	CCACTCAAGAGCCGCCTTAT
HP40	ACGCTCA ACGTTCA	ATCCTCCTTCTCCCGTCACC	TGGGTTTGGTCATGTCCTTCC
HP42	ATTCTCC ATTTTCC	ATCAAGTAACACACTCCCTTTACG	TGAGACTTTATATTTGTGATTGCGA
HP54	TGCCCTT TGCTCTT	GGGTTCCCCGTTTCAGATGT	CCACCTCCGATGGAAAGAAGT
HP75	TTACTCC TTATTCC	TCCTTCTGCCCGGATGATGA	TCCTTCTGCCCGGATGATGA
HP85	TTGCCC TTGTCC	CACAAGTCACAAGCCGCAAT	AAGTACGATTGCCCGAGGTC
HP87	ACAGTAG ACATTAG	ATCAATGGGGTCACCAACCC	ATTGGTGGTGGCGTTAGTCC
HP108	CATACCAA CATATCAA	TGAAAGTGAACTTCAACGATGC	TCGTTGAGGAGAGGTCCAAGA
HP127	CTCTTTCC CTCTCTCC	TCGGCTTTTGTCGCGTTTAC	TAGCTGACACGACACCGTTT
HP128	ATCCCTTT ATCACTT	TGGATCCCACCATGGAATTGT	AGCGATGAGTTTAAGGAGAAGGG
HP170	AACTCGA AACCCGA	ATGGATCCTTTTGAGAGAAGAAAACTGT	TGTCATTCTTGCCTTTTCCTTTCT

基因Gene	上游引物序列Upstream primer sequence (5′-3′)	下游引物序列Downstream primer sequence (5′-3′)
CsCDPK	CTACCCGGTTTGCCTTGCTA	CAGCTCTGTTGCCTGACTGA
actin	CATCCCTCAGCACCTTCC	CCAACCTTAGCACTTCTCC

	相关系数 Correlation coefficient	Wilks统计量 Wilks Lambda	卡方值 Chi-Square	自由度 Degree of freedom (df)	P值 P value
病斑面积Lesion area	0.4849	0.6547	55.2766	28	0.0016
病情指数Disease index	0.3796	0.8559	20.2999	13	0.088

	HP 02	HP 17	HP 18	HP 31	HP 40	HP 42	HP 54	HP 75	HP 85	HP 87	HP 108	HP 127	HP 128	HP 170
病斑面积 Lesion area	0.13	0.12	-0.01	-0.30	-0.16	-0.30	0.01	0.07	0.24	-0.28	-0.14	-0.04	0.11	0.20
病情指数 Disease index	0.09	0.05	0.04	-0.23	-0.12	-0.23	0.06	0.01	0.27	-0.24	-0.15	-0.02	0.01	0.24

Verification of SNPs Associated with Citrus Bacterial Canker Resistance and Induced Expression of SNP-Related Calcium-Dependent Protein Kinase Gene

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