Identification Of AFLP Markers Linked To Bacterial Wilt Resistance In Tomato And Isolation Of Related Genes

Solanaceae plant bacterial wilt(BW)incited by Ralstonia solanacearum is a constraint on crop production in tropical,sub-tropical and temperate humid regions of the world.Ralstonia solanacearum is a kind of soilborne bacterium which could cause a serious disease mainly harmful to Solanaceae plants including 44 families of more than 300 kinds of plants,such as tomato, potato,eggplant,sesame,peanut,soybean,carrot,pepper and other vegetables, as well as tobacco,mulberry,banana and other economic crops.In China, Fujian,Guangdong,Taiwan,Guangxi,Sichuan,Yunnan,Hunan,Jiangxi, Zhejiang,Shanghai,Jiangsu,Anhui and other provinces(autonomous regions) have witnessed this serious disease.Tomato is easily infected by the bacterium in its general growth period,especially in the case that it begins to form young tomatoes when encountering with the rainy season featuring high temperature and humidity.At present,yet no effective way has been found to control this disease.The researchers have paid increasing attention to pathogenesis of this bacterium and plants' resistance mechanisms for this bacterium.(1)To evaluate the pathogenicity of the six strains and select a strong virulence strain for the following studies,the OD value changes in the process of growth,EPSI,pectinase and cellulose activity were studied.The results indicated that FJ is a strong virulence strain.A highly susceptible tomato T9230 plants were inoculated with the six strains,and the bacterial wilt incidence and disease index were investigated.The bacterial wilt incidence of FJ strain-inoculated plants reached 100%and disease index 100.The long-term preservation in laboratory might cause the decrease of pathogenicity of other strains by slowly shifting strain from virulence ones to avirulence.(2)Eight tomato cultivars were screened with the virulence stain FJ and they can be grouped in three types:two susceptible tomatoes(T9230 and T903),three middle-resistant type tomatoes(T9178,T9176 and T0504C)and three 3 resistant tomatoes(T01254,T01028 and T51A).The two susceptible tomatoes have the bacterial wilt incidence of 100%and the average disease index 98.54 and 91.77 resp(?)tively.There were no significant differences in 0.05 and 0.01 levels on the bacterial wilt incidence,and have significant differences in 0.05 and 0.01 levels on the disease index.The average disease index of T9178,T9176 and T0504C were 20-60,and the average disease index of the three screened resisiant varieties were lower than 20.In the all tomato cultivars T51A has the strongest resistance with an average disease index of 3.96.(3)Genetic analysis for BW resistance showed that resistance to the bacterial strain emoloyed here was incomplete dominance to susceptibility and the resistance to the strain used in the present work was conferred by two incomplete dominant genes,and the two genes have complementary effect.We have designated the two genes as TRSR-1.In the following studies,we present the results of a research aimed at the identification of PCR-based markers amplified fragment length polymorphism (AFLP)linked to the genes that confer resistance to tomato BW.To this purpose,bulked segregant analysis was applied to an F₂ population segregating for the BW resistant gene and derived from the pair-cross between a BW resistant cultivar T51A and the susceptible cultivar T9230.Genetic ##原图像不清晰 analysis indicated that tomato BW was conferred by two incomplete dominant genes.A CTAB method for total DNA extraction,developed by Murray and Thompson with some modifications was used to isolate the infected tomato leaves.There were 19200 bands were screened between T51A and T9230 with 256 primer combinations,and only 238 differentially expressed fragments have been screened with 122 combinations.Thirteen differential fragments were detected between the B_A,B_B,B_R and B_S pools using 122 primer combinations, and two AFLP markers(T-E2M4 and T-E10M13)were linked to the BW resistance.The sequence of the two AFLP markers have been submitted to NCBI and the GenBank number of the two markers are EU260055 and EU260056,respectively.Blast research showed that EU260055 has no similarity sequence in NCBI and EU260056 has a high similarity with the sequence of rrn4.5 gene.Subsequently,the AFLP markers were converted to co-dominant SCAR markers,named TSCARAAT/CGA and TSCARAAG/CAT.Linkage analysis showed that the two markers are on the contralateral side of TRSR-1.Genetic distance between TSCARAAT/CGA and TRS-1 was estimated to 4.6 cM, while 8.4 cM between TSCARAAG/CAT and TRSR-1.(4)According to the manufacturer's manual,total RNA was isolated from leaves of frozen sample using Trizol reagent before treated.The 1st- and the 2nd-strand cDNA were synthesized using the SMARTTM PCR cDNA synthesis kit(Gibco BRL)according to its instructions.The cDNA from 10 extremely resistant lines and 10 extremely susceptible lines of the F₂ population was pooled to form bulked-resistant(CB_R)samples and bulked-susceptible(cB_S)samples,respectively.Bulked-resistant(cB_A) samples and bulked-susceptible(cB_B)samples for the parents T51A and T9230 were also made by 10 plants.Four pool cDNA used for further analysis of cDNA-AFLP.cDNA-AFLP analysis was used 256 pairs of EcoRI and MseI primer combinations and about 30,000 transcription fragments with a length 50-500 bp were obtained.525 differentially expressed transcript derived fragments (DE-TDFs)that were present in only CB_R and cB_A pools and absent in cB_B,cB_S pools were detected,and 215 of them were then re-amplified from the excised gel for cloning and sequencing.102 DE-TDFs were successfully sequenced,and 11 DE-TDFs have false positive after with the RT-PCR confirming.The remaining 91 DE-TDFs can be divided into 9 categories according to their functional.Most of the DE-TDFs are related to stress reaction(29.67%)and metabolic(20.88%) related,and the rest are unknown or presumed protein(11 DE-TDFs),signal transduction(8 DE-TDFs),the transmembrane channel(7 DE-TDFs),Phage (3 DE-TDFs),Protein-protein interaction related proteins(4 DE-TDFs)and the transcription factor(2-DE TDFs),no similarity 97 DE-TDFs).(5)A DE-TDF058 with a length of approximately 300 bp was found in resistant pools and absent in susceptible pools when using the primer combination E12M7.This gene showed similarity to a gene called CCoAOMT, which was previously isolated from S.tuberosum,N.tabacum and other plants. Hence,the gene is designated tomato CCoAOMT.According to the sequence of potato and tobacco CCoAOMT gene CDS,primers P3/P4 were designed to amplify the CCoAOMT gene in Tomato,including the initiation codon ATG and termination codon TAA.The complete cds of tomato CCoAOMT gene was 729 bp in length encoding 242 amino acids.This full-length sequence has been submitted to the NCBI database(GenBank accession no.EU161983). The full-length sequence of DNA was 816 bp with four introns(23 bp,15 bp, 22 bp and 27 bp).Comparin of the tomato's CCoAOMT gene nucleotide sequence with other solanaceae plants' CCoAOMT gene nueleotide sequences shows that tomato CCoAOMT gene had high identities to the CCoAOMT gene of potato and tobacco.The deduced protein contains one conserved domain Methyltransf₃. Members of this family are O-methyltransferases.The family includes catechol o-methyltransferase,caffeoyl-CoA O-methyltransferase and a family of bacterial O-methyltransferases that may be involved in antibiotic production(pfam01596).Methylates caffeoyl-CoA to feruloyl-CoA and 5-hydroxyferuloyl-CoA to sinapoyl-CoA plays a role in the synthesis of feruloylated polysaccharides.Involved in the reinforcement of the plant cell wall,responding to wounding or pathogen is challenged by the increased formation of cell wall-bound ferulic acid polymers.Expression analysis was performed using q1/q2 primer pairs and equal amounts of cDNA templates prepared from total RNA of different genotypes before and after bacterium inoculation by means of RT-PCR using actin as external control:T51A,T01028,T01254,1364,T9230,Hezuo,T9178,T9176, T0504C and BR,BS pools.A fragment size about 730 bp was amplified in all genotypes before bacterium inoculation and resistant genotypes after inoculation.There were no expression in susceptible genotypes after inoculation and weak expression in moderately resistant genotype.The expression results indicated that CCoAOMT gene expressed inhibitted only after bacterium inoculation in susceptible tomatoes.(6)To research the basic physiological mechanism in resistant tomato bacterial wilt,different type tomato cultivars were inoculated with the virulence pathogen FJ.The PAL activity,SOD activity,the lignin and total phenolics contents in the roots and leaves were investigated.Results showed that the PAL activity of resistant tomato T51A were always higher than that of middle-resistant cultivar T9178 and the latter was always higher than that of susceptible type T9230.The enzyme activity of PAL in leaves and roots of all the cultivars ascended after inoculation and T51A has a fastest ascending speed and the highest peak values.The SOD activity in leaves and roots,of all cultivars were under homeostasis without inoculation in seedling.The SOD activity in roots and leaves of all cultivars increased after inoculation.The resistant tomato T51A has a higher SOD activity than T9178 and T9230.After the peak value,the SOD activity started descending,T51A and T9178 have a higher activity than those of CK and T9230 has a lower activity than those of CK at the 10^thday.The lignin contents in leaves and roots of all cultivars were under homeostasis without inoculation in seedling.The lignin contents in roots and leaves of all cultivars increased after inoculation.The lignin contents of resistant tomato T51A were always higher than that of middle-resistant cultivar T9178 and the latter was always higher than that of susceptible type T9230.T51A has a fastest ascending speed and the highest peak values,while T9230 was reverse to others.The total phenolics contents in roots and leaves of all cultivars increased after inoculation.T51A has a fastest ascending speed and has two peak values after inoculation.The total phenolics of T51A were always higher than that of middle-resistant cultivar T9178 and the latter was always higher than that of susceptible type T9230.The total phenolics in roots of T9230 ascended at the first four days,and then decreased with a higher level than CK.3 days after inoculation,the total phenolics in roots of T9230 has a higher level than the CK of T51A and this results indicated that T9230 has an unresponsive than that of resistant cultivar.