Sylvia Malory

High-throughput sequencing and mutagenesis to accelerate the domestication of Microlaena stipoides as a new food crop.

Global food demand, climatic variability and reduced land availability are driving the need for domestication of new crop species. The accelerated domestication of a rice-like Australian dryland polyploid grass, Microlaena stipoides (Poaceae), was targeted using chemical mutagenesis in conjunction with high throughput sequencing of genes for key domestication traits. While M. stipoides has previously been identified as having potential as a new grain crop for human consumption, only a limited understanding of its genetic diversity and breeding system was available to aid the domestication process. Next generation sequencing of deeply-pooled target amplicons estimated allelic diversity of a selected base population at 14.3 SNP/Mb and identified novel, putatively mutation-induced polymorphisms at about 2.4 mutations/Mb. A 97% lethal dose (LD97) of ethyl methanesulfonate treatment was applied without inducing sterility in this polyploid species. Forward and reverse genetic screens identified beneficial alleles for the domestication trait, seed-shattering. Unique phenotypes observed in the M2 population suggest the potential for rapid accumulation of beneficial traits without recourse to a traditional cross-breeding strategy. This approach may be applicable to other wild species, unlocking their potential as new food, fibre and fuel crops.

Characterizing homologues of crop domestication genes in poorly described wild relatives by high‐throughput sequencing of whole genomes

Wild crop relatives represent a source of novel alleles for crop genetic improvement. Screening biodiversity for useful or diverse gene homologues has often been based upon the amplification of targeted genes using available sequence information to design primers that amplify the target gene region across species. The crucial requirement of this approach is the presence of sequences with sufficient conservation across species to allow for the design of universal primers. This approach is often not successful with diverse organisms or highly variable genes. Massively parallel sequencing (MPS) can quickly produce large amounts of sequence data and provides a viable option for characterizing homologues of known genes in poorly described genomes. MPS of genomic DNA was used to obtain species-specific sequence information for 18 rice genes related to domestication characteristics in a wild relative of rice, Microlaena stipoides. Species-specific primers were available for 16 genes compared with 12 genes using the universal primer method. The use of species-specific primers had the potential to cover 92% of the sequence of these genes, while traditional universal primers could only be designed to cover 80%. A total of 24 species-specific primer pairs were used to amplify gene homologues, and 11 primer pairs were successful in capturing six gene homologues. The 23 million, 36-base pair (bp) paired end reads, equated to an average of 2X genome coverage, facilitated the successful amplification and sequencing of six target gene homologues, illustrating an important approach to the discovery of useful genes in wild crop relatives.