Frances M Shapter

Domestication to Crop Improvement: Genetic Resources for Sorghum and Saccharum (Andropogoneae)

Background Both sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum) are members of the Andropogoneae tribe in the Poaceae and are each others closest relatives amongst cultivated plants. Both are relatively recent domesticates and comparatively little of the genetic potential of these taxa and their wild relatives has been captured by breeding programmes to date. This review assesses the genetic gains made by plant breeders since domestication and the progress in the characterization of genetic resources and their utilization in crop improvement for these two related species. Genetic Resources The genome of sorghum has recently been sequenced providing a great boost to our knowledge of the evolution of grass genomes and the wealth of diversity within S. bicolor taxa. Molecular analysis of the Sorghum genus has identified close relatives of S. bicolor with novel traits, endosperm structure and composition that may be used to expand the cultivated gene pool. Mutant populations (including TILLING populations) provide a useful addition to genetic resources for this species. Sugarcane is a complex polyploid with a large and variable number of copies of each gene. The wild relatives of sugarcane represent a reservoir of genetic diversity for use in sugarcane improvement. Techniques for quantitative molecular analysis of gene or allele copy number in this genetically complex crop have been developed. SNP discovery and mapping in sugarcane has been advanced by the development of high-throughput techniques for ecoTILLING in sugarcane. Genetic linkage maps of the sugarcane genome are being improved for use in breeding selection. The improvement of both sorghum and sugarcane will be accelerated by the incorporation of more diverse germplasm into the domesticated gene pools using molecular tools and the improved knowledge of these genomes.

Robust allele-specific polymerase chain reaction markers developed for single nucleotide polymorphisms in expressed barley sequences

Many methods have been developed to assay for single nucleotide polymorphisms (SNPs), but generally these depend on access to specialised equipment. Allele-specific polymerase chain reaction (AS-PCR) is a method that does not require specialised equipment (other than a thermocycler), but there is a common perception that AS-PCR markers can be unreliable. We have utilised a three primer AS-PCR method comprising of two flanking-primers combined with an internal allele-specific primer. We show here that this method produces a high proportion of robust markers (from candidate allele specific primers). Forty-nine inter-varietal SNP sites in 31 barley (Hordeum vulgare L.) genes were targeted for the development of AS-PCR assays. The SNP sites were found by aligning barley expressed sequence tags from public databases. The targeted genes correspond to cDNAs that have been used as restriction fragment length polymorphic probes for linkage mapping in barley. Two approaches were adopted in developing the markers. In the first approach, designed to maximise the successful development of markers to a SNP site, markers were developed for 18 sites from 19 targeted (95% success rate). With the second approach, designed to maximise the number of markers developed per primer synthesised, markers were developed for 18 SNP sites from 30 that were targeted (a 60% success rate). The robustness of markers was assessed from the range of annealing temperatures over which the PCR assay was allele-specific. The results indicate that this form of AS-PCR is highly successful for the development of robust SNP markers.

Genome diversity in wild grasses under environmental stress

Patterns of diversity distribution in the Isa defense locus in wild-barley populations suggest adaptive selection at this locus. The extent to which environmental selection may act at additional nuclear-encoded defense loci and within the whole chloroplast genome has now been examined by analyses in two grass species. Analysis of genetic diversity in wild barley (Hordeum spontaneum) defense genes revealed much greater variation in biotic stress-related genes than abiotic stress-related genes. Genetic diversity at the Isa defense locus in wild populations of weeping ricegrass [Microlaena stipoides (Labill.) R. Br.], a very distant wild-rice relative, was more diverse in samples from relatively hotter and drier environments, a phenomenon that reflects observations in wild barley populations. Whole-chloroplast genome sequences of bulked weeping ricegrass individuals sourced from contrasting environments showed higher levels of diversity in the drier environment in both coding and noncoding portions of the genome. Increased genetic diversity may be important in allowing plant populations to adapt to greater environmental variation in warmer and drier climatic conditions.

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.

Whole grain morphology of Australian rice species

The grain morphology of 17 wild rice relatives were studied by light and scanning electron microscopy and compared to two cultivated rice varieties ( Oryza sativa cv. Nipponbare and O. sativa cv. Teqing). Observations were made of the grain colour, size and shape. Grains from wild rice species exhibited a variety of colours that have potential aesthetic and nutritional value. The grains of these species exhibited a wide array of sizes and shapes, but still fell within the standard classification scale that rice breeders use for routine breeding evaluation. These results highlight the potential of these species as whole grain foods or as sources of novel alleles in conventional rice breeding programmes.

Analysis of adaptive ribosomal gene diversity in wild plant populations from contrasting climatic environments

Plant populations may contain variation that reflects adaptation to local environmental conditions. Clues to adaptive evolution of plants may be found in the genomes of species growing in diverse environments or across steep environmental gradients, and under stress. We have examined populations of wild relatives of barley and rice across diverse environmental gradients. Greater diversity, in a nuclear biotic stress defense gene and in chloroplast genes, was found in the more stressed, hotter and dryer environments. This may reflect the greater heterogeneity of these environments. Adaptation of plants to different abiotic stresses (temperatures and levels of water availability) may also require significant adaptation to the different biotic (pest and disease) pressures in these environments.1 Plants growing across environmental gradients revealed greater diversity in a defense gene (Isa) in more stressed, hotter and dryer environments.2 Chloroplast genome diversity also exhibited a similar variation with environment.3 We now report analysis of nuclear ribosomal genes from the same wild population. Two contrasting environments did not show significant differences in the level of diversity. However the pattern of SNP distribution within the rDNA did vary with greater SNP density in the RNA coding sequences compared with the internal transcribed spacers.

Aleurone and subaleurone morphology in native Australian wild cereal relatives

The pericarp and aleurone layer of cereal grains are associated with the accumulation of anti-nutritional factors, vitamins, high-value proteins and trace elements. Variations in these tissues may be associated with important differences in the nutritional and functional value of cereals as human or animal feeds. Wild crop relatives (WCR) have been successfully utilised in breeding programs to improve agronomic traits such as dwarfism and pest and disease resistance. Australia’s undomesticated grass species (Poaceae) provide a unique and genetically diverse array of WCRs and therefore the grains of 17 Australian WCRs were examined by scanning electron microscopy (SEM). Aleurone of each WCR was compared with that of its nearest domesticated cereal relative, with little significant morphological variation observed to this structure. A novel subaleurone morphology was observed in the Sorghum WCRs which had the appearance of being a very dense protein matrix only sparsely embedded with small starch granules or completely lacking starch granules. Histochemical analysis of a subsample of the specimens confirmed that the described morphology was lacking starch granules and had a proteinaceous matrix. Such morphological variations within Australian wild crop relatives of commercial cereals may provide novel sources of genetic diversity for future grain improvement programs.

A new look at the origins of gibbon ape leukemia virus

Is the origin of gibbon ape leukemia virus (GALV) human after all? When GALV was discovered and found to cause neoplastic disease in gibbons, it stimulated a great deal of research including investigations into the origins of this virus. A number of publications have suggested that the GALV progenitor was a retrovirus present in one of several species of South East Asian rodents that had close contact with captive gibbons. However, there are no published retroviral sequences from any South East Asian species to support this view. Here we present an alternative hypothesis that the origin of GALV is a virus closely related to Melomys burtoni retrovirus, and that this virus infected human patients in Papua New Guinea from whom biological material was obtained or in some way contaminated these samples. This material we propose contained infectious MbRV-related virus that was then unwittingly introduced into gibbons which subsequently developed GALV infections.

The endosperm morphology of rice and its wild relatives as observed by scanning electron microscopy

While cultivated rice, Oryza sativa, is arguably the world’s most important cereal crop, there is little comparative morphological information available for the grain of rice wild relatives. In this study, the endosperm of 16 rice wild relatives were compared to O. sativa subspecies indica and O. sativa subspecies japonica using scanning electron microscopy. Although the aleurone, starch granules, protein bodies and endosperm cell shapes of the cultivated and non-cultivated species were similar, several differences were observed. The starch granules of some wild species had internal channels that have not been reported in cultivated rice. Oryza longiglumis, Microlaena stipoides and Potamophila parviflora, had an aleurone that was only one-cell thick in contrast to the multiple cell layers observed in the aleurone of the remaining Oryza species. The similarity of the endosperm morphology of undomesticated species with cultivated rice suggests that some wild species may have similar functional properties. Obtaining a better understanding of the wild rice species grain ultrastructure will assist in identifying potential opportunities for development of these wild species as new cultivated crops or for their inclusion in plant improvement programmes.