Fine Mapping And Candidate Gene Analysis For Tssd Gene Regulating The Peach Internode Length

Peach [Prunus persica(L.) Batsch] is a deciduous perennial fruit crop with excessive sprout and tree vigor. Perfecting the tree architecture is the best way to solve the problem. Tree height, one key component of tree architecture affects many agronomic characters. Reducing tree height can increase planting density, obtain higher yields, reduce labor cost in fruit production, and also improve grower‘s benefit. Yet, little is known about the genetic and molecular regulation basis of the height in peach tree. In this study, a temperature-sensitive semi-dwarf peach were selected as research material and we used map-based cloning method for fine mapping and identifying candidate gene regulating internode length. In addition, molecular markers were developed to identify the trait. The main results were as follows:1. The results demonstrated that internode length of ST was insensitive to temperature and there was no significant difference under different temperatures(22℃, 26℃, 28℃, 30℃ and 33℃). By contrast, the Tssd showed significant difference under different temperatures. The internode length was greatly reduced at 23℃, showing severe dwarfing. At 26 and 28℃, average internode length was 2.3 mm with 1/4 reduction of ST. However, it was nearly the same length as the standard type at 30 and 33℃ with about 8.4 mm internode length. Genetic analysis indicates that this trait is controlled by one dominate gene. Based on these results, we named the gene controlling this trait as temperature-sensitive semi-dwarf type in Prunus Persica(Pp Tssd).2. We evaluated the effect of different temperatures(23℃, 26℃, 30℃) on internode length, node diameter, shoot elongation rate, cell size and IAA level in ST and Tssd. No significant difference was observed under different temperatures in ST. By contrast, the Tssd showed significant differences between different treatments. At 23℃, the seedling showed dwarfing type. The internode length and shoot elongation rate was almost 0 mm with the biggest diameter(3.43 mm), smallest cell length(12.89μm) and lowest IAA level(23ng/g). At 26℃, internode length and node diameter were 2.64 mm and 2.49 mm and shoot elongation rate was 8.03 mm/3 day. The IAA level in shoot tip was 50ng/g. Otherwise at 30℃ the Tssd almost showed the same phenotype with ST, with internode length of 8.27 mm, node diameter of 1.716 mm, shoot elongation rate of 25.97 mm/3 day, cell length of 30.86 μm and IAA level of 88 ng/g.3. Based on phenotype and genetic characteristics, SLAF-seq technique was used to locate the related genes. Finally, 3037 SLAF tags including SNP, Indel and SV were generated we try to locate the trait locus using SLAF-seq. totally, we generated 3037 SLAF tags including SNP, Indel and SV and mapped the locus in scaffold 3 of peach genome with interval of 750 Kb validated using HRM and Sanger sequencing. Further SNPs were developed for relative fine mapping the locus, which narrowed an interval of 500 Kb and contains 69 predicted protein-coding gene models. Also tightly linked SNP markers were developed, and employed HRM analysis to conduct SNP genotyping. As the key factors for genotyping, we established the proper concentration of temperate DNA and Mg2+ in PCR reaction system. HRM analysis could discriminate four types of SNPs(A/T、A/G、A/C and C/G) occurring via single nucleotide mutation and divided semi-dwarf and standard type individuals into two groups.4. Within 500 Kb, using Sanger sequence we developed SNP markers tightly linked to the trait, and narrowed it into 2.70Mbp~2.97 Mbp, containing 36 candidate genes. Using next-generation sequencing with 67.99 X coverage we selected the Aa genotype and found 13 genes with heterozygous sequence. One gene IAAd related to IAA distribution and level was with 15 bp deletion in upstream of Tssd. Finally, we validated the deletion in other individuals.5. Within fine mapping region, SNP and Indel markers were developed and used to identify the phenotype of offsprings in several segregation populations with 100% accuracy. The results will play an important role in marker-assisted selection(MAS) for Tssd.