A rapid method for assembly of single chromosome and identification of sex determination region based on single-chromosome sequencing.

Abstract

The sex-determining-region (SDR) may offer the best prospects for studying sex-determining gene, recombination suppression, and chromosome heteromorphism. However, current progress of SDR identification and cloning showed following shortcomings: large near-isogenic lines need to be constructed, and a relatively large population is needed; the cost of whole-genome sequencing and assembly is high. Herein, the X/Y chromosomes of Spinacia oleracea L. subsp. turkestanica were successfully microdissected and assembled using single-chromosome sequencing. The assembly length of X and Y chromosome is c. 192.1 and 195.2 Mb, respectively. Three large inversions existed between X and Y chromosome. The SDR size of X and Y chromosome is c. 13.2 and 24.1 Mb, respectively. MSY region and six male-biased genes were identified. A Y-chromosome-specific marker in SDR was constructed and used to verify the chromosome assembly quality at cytological level via fluorescence insitu hybridization. Meanwhile, it was observed that the SDR located on long arm of Y chromosome and near the centromere. Overall, a technical system was successfully established for rapid cloning the SDR and it is also applicable to rapid assembly of specific chromosome in other plants. Furthermore, this study laid a foundation for studying the molecular mechanism of sex chromosome evolution in spinach.