Development of KASP markers linked to apomixis in Eragrostis curvula

Abstract

Recently, our working group has built a new linkage map of the diplosporous grass Eragrostis curvula. For this purpose, a mapping population composed of 109 individuals was generated from the cross between the tetraploids OTA (sexual) and Don Walter (facultative apomictic pollen donor). This new population was genotyped with SNPs markers obtained through the DArT-seq technology. This technique has multiple advantages over other similar sequencing techniques (eg. GBS), since DArT-seq generates thousands of markers with high efficiency, due to the specific pipeline, the high fidelity restriction enzymes used and the intrinsic system of duplicate samples. Like other techniques, multiple pooled samples can be analyzed, reducing costs and time required, and it is possible to work without a reference genome.In this new E. curvula linkage map it was possible to identify a region associated with apomeiosis (APO-locus) and three closely linked (100%) SNPs markers. Primers designed from the sequence of these SNPs allowed the development of KASP (Kompetitive allele specific PCR) markers able to differentiate between sexual and apomictic E. curvula plants (phenotyping).KASP are codominant markers and the results obtained can be observed by fluorescence at the end of a PCR in a PHERAstar Plus equipment available at the GENeTyC laboratory (CERZOS-CONICET). First, the sequence of each SNP markers linked to the APO-locus was identified (69 bp) and the most appropriate regions for primer design were chosen. The Primer3Plus program was used and 5 sets of KASP primers were designed with specific "tails'' for each one of the FAM and HEX alleles for the paternal and maternal alleles, respectively. These 5 sets of KASP markers were tested on the population parental lines and on a set of sexual and apomictic cultivars (20). Based on its efficiency one of these KASPs markers (K277) was selected and used to validate the phenotype of the offspring. The KASP marker designed, despite being codominant, demonstrated its reliability to perform the phenotypic characterization, distinguishing between sexual and apomictic individuals. Sequences corresponding to SNP markers 100% linked to apomeiosis showed homology with genes, pointing at such genes as potential candidates to be involved in the regulation of the reproductive mode of this grass.