Verification Of Citrus Canker Resistance Related SNPs And Expression Analysis Of The Associated Genes

Citrus bacterial canker disease(CBCD)caused by Xanthomonas citri subsp.citri(Xcc)is a systemic and quarantine bacterial disease that affects citrus plantations globally.Citrus bacterial canker disease can lead to deciduous,fruit drop,yield and quality decline of citrus trees.Cultivating disease-resistant varieties is one of the most fundamental and effective ways to prevent and control citrus canker.Due to the low breeding efficiency of traditional hybrid breeding methods and the food safety problems of genetically modified citrus,a more efficient and safe breeding method is urgently needed.Marker-assisted selection(MAS)is a breeding method based on genotype selection.It has high accuracy and is not affected by environmental conditions.It can greatly improve the selection efficiency and shorten the breeding period.Therefore,the development of molecular markers associated with citrus canker disease is of great significance for accelerating citrus canker resistance breeding.Previously,more than 170 differentially expressed and single nucleotide polymorphism(SNP)kinase genes were mined from the transcriptome analysisof Kumquat,Sijiju,Taiwan Ponkan and Newhall.These SNPs were used to screen association of citrus canker disease resistance and SNP genotyping with More than 100 citrus varieties(natural groups),14 associated SNPs were identified.CCD resistance phenotyping via in vitro acupuncture inoculation and Using SNP typing via high resolution melting curve(HRM)technology were used to verify the accuracy of these SNPs to obtain molecular markers for citrus breeding of citrus canker disease.One SNP located in a calcium-dependent protein kinase(CDPK)gene is significantly correlated with citrus canker resistance/susceptibility.It is speculated that this gene may play a role in the signal transduction of citrus in response to Xcc infection.Therefore,and inducible expression profiles of SNP(HP031)related calcium-dependent protein kinase(CDPK)gene was analyzed by quantitative real-time PCR(q RT-PCR).The main results are as follows:1.The lesion sizes of the 143 materials ranged from 0.75 to 3.29 mm~2,and the disease indexes ranged from 30.2 to 100.The disease index disease-resistant variety'Jindan'was 11.1,while the susceptible variety'Bingtang Orange'was100.For the offspring of hybrids with a low disease index,at least one of their parents is a disease-tolerant variety,and most of them have strong disease-tolerance.The F1 generation obtained from the cross between a susceptible female parent with a strong disease-resistant male parent showed strong disease resistance,such as'Shatianyou'×'Liangpingyou','Or'×'Nanfeng Mitang'combination.In this study,the populations can be grouped into immune(0 variety),highly resistant(17),moderately resistant(30),moderately sensitive(38),and highly sensitive(55)based on the disease index valuas.2.The SNP typing results showed that 14 SNP sites were polymorphic among 143 test materials,which can be divided into three different genotypes,two homozygous types and one heterozygous type.Each SNP site of the test material has a specific genotype.The DPS software was used to analyze the correlation of the genotype value,lesion area and morbidity index of the tested materials.The results of simple correlation analysis show that the genotypes of multiple SNP loci are significantly correlated with the lesion sizes and the disease index.Typical correlation analysis showed that the 5 SNPs(HP31,HP42,HP85,HP87,and HP170)have a high correlation to the lesion sizes with absolute values of the correlation coefficients 0.2.A regression equation was established based on the genotype and lesion sizes of the test materials.The genotypes of these 5 SNP loci can be used to predict the tolerance of citrus canker disease of citrus samples.3.SNP HP31 located in the coding region of citrus calcium-dependent protein kinase(CAP ID:Cs4g10370).The expression of Cs CDPK at 6,12,24,48and 72 hpi(hours post inoculation)displayed a similar pattern with increase first to the top level at 48 hpi and followed by decrease in Jindan(highly resistant),Xinshengxi No.3(moderately sensitive),and Bingtangcheng(highly sensitive).At 12 hpi,the relative expression level of Cs CDPK in Jindan was 3 times of pre-inoculated sample,whilst there was no obvious induction found in Xinshengxi No.3 and Bingtangcheng.Besides,Cs CDPK was also differently induced by salicylic acid(SA),methyl jasmonate(Me JA),and abscisic acid(ABA)in CCD resistant and sensitive varieties.This indicates that the expression of this gene plays an important role in the signal transduction of citrus in response to biological stress such as canker disease.In this study,5 SNPs associated with CCD were verified which could be useful to marker-assistant selection.SNP HP31 related calcium-dependent protein kinase Cs CDPK could be induced by Xcc and phytohormones and could play important roles in the responses and signaling process against Xcc infection.This study laid a certain foundation for the molecular marker assisted breeding and resistance gene mining of citrus canker resistance.