| Bitter melon(Momordica charantia L.)is one of the Cucurbitaceae vegetables,it is suitable for cultivating in tropical and temperate regions,powdery mildew is widespread disease in the world,when it appears will lead severely affected to bitter's yield and quality,therefore the research for disease-resistant breeding of bitter gourd is one of the main goals of breed breeding.In this study,we use the whole genome sequencing of bitter gourd to develop SSR primers,and the SSR-PCR reaction system of bitter gourd was optimized by orthogonal design of L16(44).Using four kinds of bitter melon as templates,the SSR primers developed in this laboratory were screened by the optimized system.,screened the polymorphic primers,and using the polymorphic primers PCR cucumbers,melons,watermelons,loofah,wax gourd and pumpkin.To study the versatility of bitter gourd SSR primers in the six melon crops.High-resistant powdery mildew wild-type bitter gourd 04-17 and high-level powdery mildew cultivar 25 as parents,by using SSR molecular marker technology to build genetic map of SSR,and located powdery mildew of F2:3 family.The experimental results are as follows:1.Clean base 3.133G was obtained by this sequencing,a total of 12,533,97 Clean reads,99.02%efficiency,a base error rate of 0.04%,and a base GC content of 35.61%.According to the sequencing quality analysis results,Q20 ? 94.69%and Q30 ? 90.28%.It can be seen that the quality of sequencing is relatively high.The average site depth was 22.59 and the coverage reached 99.21%,reflecting the homogeneity of the sequencing data and the consistency of the assembled genome.The sample assembly contig was filtered to obtain a 100bp SSR fragment at both ends.The total number of primers designed was 7292,the effective primers were 6234,the design rate of the primers was 85.49%.2.The optimized SSR reaction system was:dNTPs(2.5 mmol/L)0.5 1.L,Taq DNA polymerase(5 U/?L)0.1 1.L,lOxPCR-Buffer 1.5 ?L,SSR primer(10 ?mol/L)0.6 ?L The template DNA(100 ng/?L)was 70 ng and the total reaction system was 10 ?L.In 300 pairs of SSR primers of bitter gourd,49 pairs of bands were screened for polymorphic primers,and the polymorphism rate was 16.3%.3.Using JoinMap 4.0 software to analyze the 175 SSR data and construct the SSR genetic map of bitter gourd.Contains 4 and more than 4 markers as a linkage group,there are 10,each linkage group numerals 4-12,linkage group length 23.0-106.7cM,the map coverage genomic length was 545.9cM,average pitch 7.58 cM.The 10th linkage group with the largest average distance between markers includes 8 SSR markers with an average distance of 13.34 cM.The smallest average pitch is third linkage group,including 12 SSR markers,it is a genetic map that constructed the most marker,the average pitch 3.02cM,the minimum spacing of 0.8cM,the maximum spacing of 6.5cm.4.In the two-year and two-season cultivation mode,the disease index was QTL 5 and one QTL locus was obtained,located between the SSR3124 and SSR3734 markers of the third linkage group,can explain 11.5%phenotypic variation..This is the first time to use the simplified genome sequencing technology(RAD-seq)developed SSR markers for bitter gourd to build the genetic map,and QTL the bitter melon powdery mildew disease index,it lays the foundation for the genetics of bitter gourd and the disease-resistance of bitter gourd... |
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