Molecular Breeding

Climate change and the increasing demand for sustainable energy resources require urgent strategies to increase the accuracy of selection in tree breeding (associated with higher gain). We investigated the combined pedigree and genomic-based relationship approach and its impact on the accuracy of predicted breeding values using data from 5-year-old Eucalyptus grandis progeny trial. The number of trees that can be genotyped in a tree breeding population is limited; therefore, the combined approach can be a feasible and efficient strategy to increase the genetic gain and provide more accurate predicted breeding values. We calculated the accuracy of predicted breeding values for two growth traits, diameter at breast height and total height, using two evaluation approaches: the combined approach and the classical pedigree-based approach. We also investigated the influence of two different trait heritabilities as well as the inclusion of competition genetic effects or environmental heterogeneity in an individual-tree mixed model on the estimated variance components and accuracy of breeding values. The genomic information of genotyped trees is automatically propagated to all trees with the combined approach, including the non-genotyped mothers. This increased the accuracy of overall breeding values, except for the non-genotyped trees from the competition model. The increase in the accuracy was higher for the total height, the trait with low heritability. The combined approach is a simple, fast, and accurate genomic selection method for genetic evaluation of growth traits in E. grandis and tree species in general. It is simple to implement in a traditional individual-tree mixed model and provides an easy extension to individual-tree mixed models with competition effects and/or environmental heterogeneity.

The comparative phenotypic analysis of mutants is often hampered by their diverse and poorly characterised genetic backgrounds. To overcome this problem, a suite of recombinant spring barley lines was developed for four starch biosynthesis genes in a common elite background. Rapid breeding progress was made by combining foreground and background selection with the screening of bulked families. A toolkit of perfect co-dominant PCR assays was developed for the four target genes, based on the causative single nucleotide polymorphisms underlying their starch phenotypes. These were used for foreground selection during backcrossing and selfing, and may be applied to bulks of up to ten plants. Screening bulks meant that large numbers of individuals with known family structure were rapidly assessed and that breeding effort was accurately targeted. These markers were also used for quality control during field multiplication and should be readily transferable to any crosses involving these four mutations. Background selection amongst BC1 progeny known to be heterozygous for the target starch alleles identified individuals which were relatively enriched for the recurrent parent across the rest of the genome. These were further advanced and true-breeding recombinants were selected which carry the target starch mutations in a largely recurrent parent background. The resulting set of BC2F5 pre-breeding lines should enable meaningful analysis of the starch phenotypes and facilitate their transfer into commercial breeding programmes.

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. niveum (Fon), is one of the predominant diseases of watermelon. Resistance to Fon race 1 is conferred by a single major quantitative trait locus (QTL), Fo-1.1, but resolution of this region has been poor due to low marker density. In this study, a combination of whole genome resequencing of bulked segregants (QTL-seq analysis) followed by QTL mapping with kompetitive allele specific PCR (KASP) markers developed across Fo-1.1 successfully increased the resolution from 2.03 to 1.56 Mb and 315 kb, respectively. The linkage of the KASP markers to Fon race 1 resistance across a wide range of watermelon germplasm was validated in a set of elite watermelon cultivars. The linked markers described here provide a breeder-friendly toolkit immediately available for high-throughput genotyping in large-scale breeding programs for fine mapping and incorporation of Fon race 1 resistance in watermelon.

The process of crop domestication occurs through the selection and subsequent propagation of novel alleles that improve traits of interest. Cultivated tomato (Solanum lycopersicum), particularly heirloom varieties, exhibit a wide range of variation in fruit size, shape and color. The green-flesh mutant of tomato possesses a stay-green phenotype resulting in fruits that ripen to a red-brown color, due to the retention of chlorophyll and the simultaneous accumulation of lycopene. The recent identification of the GREEN-FLESH gene provides a molecular tool with which to investigate the origin of a subset of cultivated tomato varieties that resemble the green-flesh mutant. Sequence analysis of the GF locus from 26 varieties revealed the existence of four previously unidentified null alleles. This study illustrates the potential of cultivated tomato varieties, including heritage cultivars, heirlooms, and land races, for uncovering new alleles in genes of interest.

Plant biomass, the most abundant renewable resource on earth, is a potential source of fermentable sugars for production of alternative transportation fuels and other chemicals. Bioconversion of plant biomass to fermentable glucose involves enzymatic hydrolysis of cellulose, a major polysaccharide constituent. Because commercially available microbial cellulases are prohibitively expensive for bioethanol processes, we have investigated the feasibility of producing these enzymes in plants as a low-cost, potentially high-volume alternative to traditional production methods. We have successfully expressed the catalytic domain of a thermostable (T opt=81 °C) endo-1,4-β-D-glucanase from the eubacterium, Acidothermus cellulolyticus, in the apoplast of tobacco BY-2 suspension cells and leaves of Arabidopsis thaliana plants. The apoplast-targeting cassette designed for this work consists of the cauliflower mosaic virus 35S promoter, the tobacco mosaic virus Ω translational enhancer, the sequence encoding the tobacco Pr1a signal peptide, and the polyadenylation signal of nopaline synthase. Recombinant E1 catalytic domain was targeted to the ER by the signal peptide and secreted into the apoplast via the default pathway. Secretion of the enzyme did not detectably affect the growth rate of transgenic BY-2 cells, although the protein was enzymatically active at elevated temperatures. Similarly, transgenic plants exhibited no abnormal phenotypes correlating with expression of the enzyme. Close agreement between independent immunochemical and activity-based assays indicates that the enzyme accumulated to concentrations up to 26% of the total soluble protein in leaves of primary A. thaliana transformants. The amount of functional endoglucanase produced illustrates that plants can accumulate very large quantities of enzyme for commercial biomass conversion.

Microsatellites (SSRs) are widely used in cereal research, and their use in marker assisted breeding has increased the speed and efficiency of germplasm improvement. Central to the application of SSRs for many purposes are methodologies enabling the low-cost acquisition of large quantities of genetic information for gene and genotype identification. In this study, multiplex-ready PCR was evaluated in barley and bread wheat as an approach for rapid and more automated SSR genotyping on a fluorescence-based DNA fragment analyzer. Multiplex-ready PCR is a method that allows SSR genotyping to be performed using a standardized protocol. The method enables flexible fluorescence labeling of SSRs, generates a relatively constant amount of PCR product for each marker, and has a high amenability to multiplex PCR (the simultaneous amplification of several SSRs in the same reaction). A high (92%) compatibility of published SSRs with multiplex-ready PCR is demonstrated, and the usefulness of the method for large scale genotyping is shown by its application for whole genome marker assisted breeding in barley. A database of more than 2,800 barley and wheat SSRs, and a suite of bio-informatic tools were developed to support the deployment of multiplex-ready PCR for various genetic applications, and are accessible at http://www.genica.net.au . Multiplex-ready PCR is broadly applicable to cereal genomics research and marker assisted breeding, and should be transferable to similar analyses of any animal or plant species.

The first microsatellite-based linkage map for Vitis aestivalis was constructed using 183 progeny from the crosses of V. aestivalis-derived “Norton” and V. vinifera “Cabernet Sauvignon”. A total of 1157 simple sequence repeat (SSR) markers were tested, 859 were amplified via PCR and 413 were polymorphic for at least one parent. The map for Norton consisted of 376 markers and covered 1496.6 centimorgan (cM) on 19 chromosomes. The consensus map consisted of 411 markers on 19 linkage groups with a total distance of 1678.6 cM. Although isozyme and SSR marker analyses in 1993 and 2009 provided preliminary evidence that Norton and Cynthiana grapes are genetically identical, only five banding patterns and four microsatellite loci were reported. This study characterized the relationship between these two cultivars using 185 microsatellites spanning 19 linkage groups for a genome-wide analysis. Four accessions of Norton and three accessions of Cynthiana were used; capillary electrophoresis results revealed Norton and Cynthiana to be identical at all selected loci.

Verticillium wilt (VW) is one of the most destructive diseases of cotton that decreases yield and quality. Identification of resistance gene analogs (RGAs) can provide potential candidate gene markers for marker-assisted selection (MAS) for VW resistance and cloning of VW resistance genes. The objective of this study was to convert RGA-targeted RGA–AFLP (amplified fragment length polymorphism) markers into sequence-tagged site (STS) markers. A total of 54 RGA–AFLP markers, including 28 from a backcross inbred line (BIL) population and 26 from a recombinant inbred line (RIL) population, were cloned and sequenced. Of the resulted 86 unique sequences, the majority were found to be homologous to genes, some of which showed similarity to genes encoding resistance-related products. A total of 163 STS primer pairs were designed and screened in the BIL population, 72 of which were also screened in the RIL population, resulting in 21 polymorphic STS markers in the BIL population and seven in the RIL population. Twelve STS markers were mapped onto eight chromosomes or linkage groups in the BIL population, six of which were mapped on the same chromosomes with their original RGA–AFLP markers including two STS markers on c4 flanking two VW resistance quantitative trait loci. These STS markers will be useful in MAS in breeding cotton for VW resistance. This study represents the first attempt to successfully convert RGA–AFLP markers into STS for mapping resistance genes in cotton.

Selection index (SI) theory has been applied to predict the net genetic merit, select parents for the next cycle, and maximize the selection response in plant breeding. However, up to now, SI has not been applied to predict unobserved hybrid performance. In hybrid breeding, it is impossible to test every cross, and accurate prediction can help breeders greatly reduce the experimental cost. Traditional genomic selection (GS) targets single-trait prediction, and useful information about other related traits is ignored. With the data set of 575 rice hybrids subjected to the North Carolina mating design II, this information was utilized to develop a linear SI-based GS method to predict rice hybrid for a more accurate and comprehensive selection. Cross-validation results showed that genetic information of a low-heritability target trait such as grain yield could be greatly aggregated from auxiliary traits using SI. When SI was used for directly predicting the target trait, the SI-direct prediction underperformed the traditional genomic prediction in most cases. However, when the SI-direct prediction was combined with the traditional genomic prediction using a suitable weight ω inferred from the training data set, significantly higher accuracy could be obtained. This method was called as SI-assisted prediction. It provided a promising prediction means for breeding application, which used the phenotypes of auxiliary traits only in the training data set. Additionally, it was found that SI-assisted accuracy increased as the genetic correlation between auxiliary traits and the target trait increased, and high heritability of auxiliary traits could also improve the prediction performance.

Stem rust of wheat, caused by Puccinia graminis f. sp. tritici (Pgt), is a threat to global food security due to its ability to cause total crop failures. The Pgt race TTKSK (Ug99) and its derivatives detected in East Africa carry virulence for many resistance genes present in modern cultivars. However, stem rust resistance gene Sr26 remains effective to all races of Pgt worldwide. Sr26 is carried on the Agropyron elongatum (syn. Thinopyrum ponticum) segment 6Ae#1L translocated to chromosome 6AL of wheat. In this study, a recombinant inbred line (RIL) population derived from a cross between the landrace Aus27969 and Avocet S, which carries Sr26, was used to develop co-dominant kompetitive allele-specific polymerase chain reaction (KASP) markers that co-segregate with Sr26. Four KASP markers (sunKASP_216, sunKASP_218, sunKASP_224 and sunKASP_225) were also shown to co-segregate with Sr26 in four additional RIL populations. When tested on Australian cultivars and breeding lines, these markers amplified alleles alternate to that linked with Sr26 in all cultivars known to lack this gene and Sr26-linked alleles in cultivars and genotypes known to carry Sr26. Genotypes WA-1 and WA-1/3*Yitpi carrying the shortest Sr26 translocation segment were positive only for markers sunKASP_224 and sunKASP_225. Our results suggest the four KASP markers are located on the original translocation and sunKASP_224 and sunKASP_225 are located on the shortened version. Therefore, sunKASP_224 and sunKASP_225 can be used for marker-assisted pyramiding of Sr26 with other stem rust resistance genes to achieve durable resistance in wheat.