Perennial ryegrass (Lolium perenne L.) is one of the most important pasture grasses in New Zealand. However, seed production is negatively impacted by seed shattering (shedding). Recently, genes involved in the shattering process have been isolated and functionally characterised in several crop species, including qSH1 and SH4 in rice and SH1 in sorghum. The aim of this project was to identify the critical genes involved in the seed shattering process in perennial ryegrass, and then to screen for mutations in the target gene in a perennial ryegrass EcoTILLING population. Additionally, seed size is an aspect of seed production of interest to perennial ryegrass breeders. CKX1 has been identified as playing a key role in seed size in rice and wheat, so the LpCKX1 was added as a target gene for mutation screening.

DNA sequences of genes involved in seed shattering and seed size in the Poaceae were used to identify and isolate target genes in perennial ryegrass using a comparative genomics strategy. The candidate ryegrass shattering genes were identified using an ‘in-house’ perennial ryegrass transcriptome database. The relative expression levels of candidate ryegrass shattering genes were determined using RT-qPCR during the floret and seed developmental stages. A genetic model for seed shattering in perennial ryegrass is outlined. LpSH1 and LpCKX1 were selected as the target genes for mutation screening in the ryegrass EcoTILLING population, and the full-length DNA sequences of both target genes were amplified.

Several methods designed to optimise the EcoTILLING approach were developed. For mutation screening, plant genomic DNA was isolated using regenerated silica columns. The reliability of using regenerated silica columns was tested in terms of both yield and purity. A new method combining high resolution melting (HRM) analysis to detect CEL I digestion products was developed to reduce the time taken for mutation screening. The CEL I+HRM method was tested to screen mutations in exon 1 of LpCKX1, but due to the high degree of genetic diversity in perennial ryegrass and the high GC-content in LpCKX1, this method was not suitable for perennial ryegrass, but could be used for self-pollinated species, such as wheat and pea. A homemade HRM master mix was optimised, and HRM analysis was used to screen for mutations in LpSH1 and LpCKX1 in the EcoTILLING population. Multiple mutations in LpSH1 and LpCKX1 were identified from the EcoTILLING population. These were then sequenced to detect if the mutations were likely to cause a change in protein structure and function.

The strategy adopted in this project allowed for the application of genetic knowledge from well-studied domesticated plants to be applied to a lesser-studied crop plant, and shows the potential for detecting useful mutations for future plant breeding.