Marker Development And Physical Map Construction Of Tuber Shape Gene Locus In Potato (Solanum Tuberosum L.)

Potato tuber shape is controlled by a major locus Ro on chromosome X that could explain over 70%variance.In previous study in this project,a tuber shape segregation population of diploid potato was built and evaluated on tuber appearance in last four years.In this study,using whole genome sequencing combining with BSA,polymorphic markers linked to Ro-locus were developed.A BAC library was constructed with mixed genomic DNA of 8 typical round-shaped progenies,and the library was screened with makers linked to Ro-locus.Several positive BAC mono-clones were selected.Through BAC end sequencing and location verification of these mono-clones,a 180 kb physical distance contigs of Ro-locus were constructed.The main results achieved are as follows:1.Tuber shape of 487 progenies from a segregation population has been evaluated for two years.Combining with phenotype data last four years,a number of 227 individuals with round tubers(I<1.2),105 individuals with oval tubers(1.2≤I<1.5)and 155 individuals with long tubers(I≥1.5)were obtained.Among them,there are 151 extreme round tuber(0.9≤I≤1.1)individuals and 59 extreme long tuber(I≥1.8)individuals.2.Ten markers linked to tuber shape were developed.A total of 106 individuals with extreme round or long tubers were selected to construct round tuber shape DNA bulk and long tuber shape DNA bulk for high-throughput simplified genome sequencing.According to the density distribution of unique tags between the two bulks,the density distribution map of specific unique tags in was obtained,and the result showed that a big difference block of specific unique tags located on chromosome X.According to the sequence of specific unique tags in this block,1 polymorphic markers linked to tuber shape was developed.And then,a total of 66 individuals with extreme round or long tubers were selected to construct round tuber shape DNA bulk and long tuber shape DNA bulk for whole genome sequencing.Through identification and screening of particular SNPs,a total of 17 different blocks in the range of 47.31-49.40 Mb on chromosome X were selected,and the primers were designed according to the SNP locus and flanking sequence in 17 intervals.9 polymorphic markers linked to tuber shape were finally developed.3.A physical map of Ro-locus was established through construction and screening of round tuber shape BAC library.Based on phenotypic data last four years,8 individuals with typically round tubers were picked out and their genomic DNA was extracted and mixed for the construction of round tuber shape BAC library.By screening the BAC library with markers linked to Ro-locus,eight positive BAC mono-clones were distinguished.Two BAC end markers were developed by BAC end sequencing.A 180 kb physical map of Ro-locus was established by chromosome walking.By integrating the polymorphic markers linked to tuber shape and the BAC end markers,the linkage map of the Ro locus was constructed.Combining with the position of the physical map,the Ro gene was repositioned between two molecular markers,which laid a foundation for the final cloning.