Utilizing next-generation sequencing to study homeologous polymorphisms and herbicide-resistance-endowing mutations in Poa annua acetolactate synthase genes.

Abstract

Detection of single nucleotide polymorphisms (SNPs) related to herbicide resistance in non-model polyploid weed species is fraught with difficulty owing to the gene duplication and lack of reference sequences. Our research seeks to overcome these obstacles by Illumina HiSeq read mapping, SNP calling and allele frequency determinations. Our focus is on the acetolactate synthase (ALS) gene, the target site of ALS-inhibiting herbicides, in Poa annua, an allotetraploid weed species originating from two diploid parents, P. supina and P. infirma. ALS contigs with complete coding regions of P. supina, P. infirma and P. annua were assembled and compared with ALS genes from other plant species. The ALS infirma-homeolog of P. annua showed higher levels of nucleotide sequence variability than the supina-homeolog. Comparisons of read mappings of P. annua and a simulated P. supina × P. infirma hybrid showed high resemblance. Two homeolog-specific primer pairs were designed and used to amplify a 1860 bp region covering all resistance-conferring codons in the ALS gene. Four P. annua populations, GN, RB, GW and LG, showed high resistance to two ALS inhibitors, bispyribac-sodium and foramsulfuron, and two populations, HD and RS, showed lower resistance in the rate-response trial. Mutations conferring Trp-574-Leu substitution were observed in the infirma-homeolog of GN and RB and in the supina-homeolog of GW and LG, but no resistance-conferring mutation was observed in the two populations of lower resistance, HD and RS. In this study we have demonstrated the use of NGS data to study homeologous polymorphisms, parentage and herbicide resistance in an allotetraploid weed species, P. annua. Complete coding sequences of the ALS gene were assembled for P. infirma, P. supina, infirma-homeolog and supina-homeolog in P. annua. A pipeline consisting of read mapping, SNP calling and allele frequency calculation was developed to study the parentage of P. annua, which provided a new perspective on this topic besides the views of morphology, karyotype and phylogeny. Our two homeolog-specific primer pairs can be utilized in future research to separate the homeologs of the ALS gene in P. annua and cover all the codons that have been reported to confer herbicide resistance.