Isolation and sequencing of a single copy of an introgressed chromosome from a complex genome for gene and SNP identification

Abstract

The large complex genomes of many crops constrain the use of new technologies for genome-assisted selection and genetic improvement. One method to simplify a genome is to break it into individual chromosomes by ow cytometry, however, in many crop species most chromosomes cannot be isolated individually. Flow sorting of a single copy of a chromosome has been developed in wheat and here we demonstrate its use to identify markers of interest in an Erianthus/Sacchurum hybrid. Erianthus/Saccharum hybrids are of interest because Erianthus is known to be highly resistant to soil borne diseases which cause extensive sugarcane yield losses in Australia. Sugarcane (Saccharum) cultivars are autopolyploids with a highly complex genome and over 100 chromosomes. Flow cytometry for sugarcane, as in most crops, does not resolve individual chromosomes to a karyotype peak for sorting. To isolate a single chromosome, we used genomic in situ hybridisation (GISH) to identify the ow karyotype region containing the Erianthus chromosomes, ow sorted single chromosomes from this region, PCR screened for the Erianthus chromosomes and sequenced them. One Erianthus chromosome ampli ed and sequenced well, and from this data we could identify 57 resistant type genes and SNPs in nearly half of these genes. We developed KASP SNP assays and demonstrated that the identi ed SNP markers segregated as expected in a small introgression population. The pipeline we developed here to ow sort and sequence single chromosomes could be used in any crop with a large complex genome to rapidly discover and develop markers to important loci. Key MessageThis manuscript describes the identi cation, isolation and sequencing of a single chromosome containing high value resistance genes from a complex polyploid where sequencing the whole genome is too costly.

The complex polyploid genome architecture of sugarcane

“…The analysis revealed that chromosomes could be separated into a few size fractions and while the sorted populations were 100% pure chromosomes, it was not possible to sort individual sugarcane chromosomes. To overcome this problem and prepare samples of chromosome-specific DNA for sequencing, single copies of chromosomes were sorted and their DNA amplified 52 . This strategy for preparing sugarcane chromosomes for flow cytometry was first described in ref.…”

“…Third, additional clustering was performed using genetic markers derived from single flow-sorted chromosome libraries sequenced from R570 (refs. 52 , 53 ). After making initial joins, both simplex and single-chromosome genetic markers were re-aligned putative chromosomes to investigate misjoins, which were broken and corrected.…”