| Peach[Prunus persica?L.?Batsch]has excessive vigorous sprout and vigor,dense crown,which causes waste of photosynthate,pruning workload and increase of production.Developing limited dwarfing peach varieties to reduce work time and workload is a main solution.Prior study conformed the PpTssd is controlled by temperature.Based on previous studies?targeted interval 270 Kb?,determination of hormones,fine mapping of Pp Tssd gene,molecular identification of hybrids,SNP/InDel analysis and structure variation of candidate region were concluded.The main results were as follows:We evaluated the effect of different temperatures on internode length and brassinosteroids.We identified the phenotype of individuals by the double-temperature?22?and 30??treatment.275hybrids were obtained from a cross between female parent?‘09-8-25'?and male parent?‘zhongtaobaiyu'?.As a result of phenotype identification of 275 progenies,the PpTssd showed significant difference under different temperatures.At 22?,PpTssd showed severe dwarfing with very short internode.At 30?,it was nearly the same length as the standard type with long internode.For standard type,there was no significant difference under different temperature.The results of the effects of different temperatures?22?,26?and 30??on the Castaster-One level of the stem tip of PpTssd showed there was no Brassinolide detected in the tip of the new stem.The difference in the content of Castaster-One under three temperature treatment conditions was not obvious,only was 1.329 times the content of fresh weight at 30?to 22?;The content of Typ-hasterol was significantly different under different temperature,the content of Typ-hasterol at 30?was 2.917 times that of 22?,and the difference of Typ-hasterol content may be related to the formation of the phenotypes.At 22?,the content of Typ-hasterol is 0.096 ng/g FW,0.133 ng/g FW at 26?and 0.280 ng/g FW at 30?.We established a high throughput method of DNA extraction.The population were obtained from a cross between female parent‘09-1-112'?PpTssd Type?and male parent‘CN8'?Standard Type?.DNA extraction were carried out on the young leaves of two parents and 500 progenies by procedure using1.2 mL eight-row drainage tube instead of a single centrifuge tube.The detection results showed the concentration of DNA was about 25200 ng.L-1,and OD260nm/OD280nm80nm was between 1.811.98.The HRM technique distinguished the 96 individuals using SNP maker SNPPp033758620 into two groups,respectively.InDelPp033829009 was developed and the results of electrophoresis of polyacrylamide gel showed that PCR amplification was bright and clear,with obvious polymorphism in PpTssd type and ST type.Peach leaf DNA in a short period of time,in a simple way and with a low cost,was obtained with this extraction method.Based on re-sequencing of‘09-8-25'and‘zhongtaobaiyu',Pp Tssd were genotyped using double population.16 of 38 pairs of primers which were designed based on genome re-sequencing data were effective and linked to target trait.As a result of genotyping on 275 Seedlings,we obtained two SNP makers tightly linked to desired trait,Pp03-SNP3480000 and Pp03-SNP3553771.The target locus was between these two markers,an approximate physical distance of 74 Kb which contains 8transcripts?7 candidate genes?.Based on the fluorescence SSR primers developed in our laboratory we identified 6 recombinant strains.10 of 16 pairs of SSR primers were polymorphic,and alleles were all from the parents with100%accuracy.The genetic variation in the candidate interval was analyzed on the basis of 5 SNP markers.Sanger sequencing was amplified on F1 progeny of 9 plants by 6 SNP markers linked target traits.And we obtained two plants with AA genotype.Based on the second-generation sequencing data,the genomic DNA sequence of the AA and aa genotypes were compared to analyze the SNP,InDel and structural variation.It was found that there was an approximately 377 bp deletion in the 5'UTR of Purpe.3G049700,which was only present in PpTssd,possible candidate genes. |
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