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Establishment Of Molecular Markers For Resistance Genes And Screening Of Disease-resistant Germplasm For Tomato Gray Leaf Spot And Fusarium Crown And Root Rot

Posted on:2023-10-26Degree:MasterType:Thesis
Country:ChinaCandidate:Y G HaoFull Text:PDF
GTID:2543306830465594Subject:Biology
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In this study,the molecular markers of disease resistance genes were studied for the increasingly serious gray leaf spot and Fusarium crown and root rot in tomato production.The tomato germplasm resources with known disease-resistant genotypes were used as experimental materials,and specific primers were designed with reference to the RFLP markers sequence closely linked to the Sm gene,and the PCR amplification products were sequenced to find the restriction site.Finally,a new CAPS marker"T280"closely linked with the disease resistance gene Sm was established.Referring to relevant literature and designing specific primers for PCR amplification,a SCAR marker"SCAR200"closely linked to the disease resistance gene Frl was obtained by screening.On this basis,two multiplex PCR technology systems for simultaneous detection of two disease resistance genes were established,and the above molecular markers and multiplex PCR technologies were applied to identify and screen disease-resistant germplasm resources.(1)A co-dominant CAPS marker closely linked with the resistance gene Sm of gray leaf spot was created and named as T280.The primer sequences were T280 F(5’-TAG ATG ATG GTC AAG CTG TAG ATA TAA GG-3’)and T280 R(5’-TTC TGC AGA AGG CAT TGC AGG GCC AA-3’).PCR amplification of all materials produced a specific band of655 bp.After Mme I digestion,the disease-resistant homozygous material still retained a specific band of 655 bp.The susceptible homozygous material produced two specific bands of 206 bp and 449 bp.The disease-resistant heterozygous material produced three specific bands of 206 bp,449 bp and 655 bp.The verification results show that the marker is stable and reliable,and can be used for molecular marker-assisted breeding.(2)A molecular marker closely linked to the Fusarium crown and root rot resistance gene Frl was obtained by screening,namely SCAR200.The primer sequences were SCAR200F(5’-TCG GTC CAA ATT CAC TTC AA-3’)and SCAR200 R(5’-ACT CCT CCA CTT GCA TAC CC-3’).The PCR amplification results showed that the disease-resistant homozygous material produced a specific band of 290 bp,the susceptible homozygous material produced a specific band of 340 bp,and the disease-resistant heterozygous material produced two specific bands of 290 bp and 340 bp.SCAR200is a codominant SCAR marker.(3)On the basis of single gene molecular markers,by optimizing the reaction system and conditions such as primer ratio,annealing temperature and DNA template weight,two multiplex PCR technology systems for simultaneous detection of disease resistance genes Sm and Frl and simultaneous detection of disease resistance genes Mi and Frl was established.The results of multiplex PCR amplification showed the superposition of the amplification results of a single pair of primers,and the identification results of enzyme digestion were the same as those of single PCR.These established multiplex PCR technology systems can simultaneously detect two disease resistance genes of gray leaf spot and Fusarium crown and root rot,or simultaneously detect two disease resistance genes of root-knot nematode disease and Fusarium crown and root rot.These technologies greatly reduce the detection cost and significantly improve the detection efficiency.(4)The disease-resistant genotypes of 98 tomato germplasm materials were preliminarily identified using the established disease-resistant gene molecular markers.25homozygous genotype materials for resistance to gray leaf spot and 54 homozygous genotype materials for resistance to Fusarium crown and root rot were screened out.Using the multiplex PCR technology systems to detect two disease resistance genes at the same time,14 homozygous genotype materials with resistance to gray leaf spot and Fusarium crown and root rot,24 homozygous genotype materials with resistance to root-knot nematode disease and Fusarium crown and root rot,7 homozygous genotype materials with resistance to gray leaf spot,root-knot nematode disease and Fusarium crown and root rot were further screened.The established molecular markers for resistance genes of gray leaf spot and Fusarium crown and root rot,and the multiplex PCR technologies for simultaneous detection of the two disease resistance genes provide technical support for future tomato disease resistance breeding work.The disease-resistant germplasm resources obtained by screening have laid a solid material foundation for further breeding of new tomato varieties with multiple resistance and high quality.
Keywords/Search Tags:Tomato, Gray leaf spot, Fusarium crown and root rot, Molecular marker, Multiplex PCR, Screening of disease-resistant germplasm
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