Genetic differentiation analysis for the identification of complementary parental pools for sorghum hybrid breeding in Ethiopia

Key message The potential for exploiting heterosis for sorghum hybrid production in Ethiopia with improved local adaptation and farmers preferences has been investigated and populations suitable for initial hybrid development have been identified. Abstract Hybrids in sorghum have demonstrated increased productivity and stability of performance in the developed world. In Ethiopia, the uptake of hybrid sorghum has been limited to date, primarily due to poor adaptation and absence of farmer’s preferred traits in existing hybrids. This study aimed to identify complementary parental pools to develop locally adapted hybrids, through an analysis of whole genome variability of 184 locally adapted genotypes and introduced hybrid parents (R and B). Genetic variability was assessed using genetic distance, model-based STRUCTURE analysis and pair-wise comparison of groups. We observed a high degree of genetic similarity between the Ethiopian improved inbred genotypes and a subset of landraces adapted to lowland agro-ecology with the introduced R lines. This coupled with the genetic differentiation from existing B lines, indicated that these locally adapted genotype groups are expected to have similar patterns of heterotic expression as observed between introduced R and B line pools. Additionally, the hybrids derived from these locally adapted genotypes will have the benefit of containing farmers preferred traits. The groups most divergent from introduced B lines were the Ethiopian landraces adapted to highland and intermediate agro-ecologies and a subset of lowland-adapted genotypes, indicating the potential for increased heterotic response of their hybrids. However, these groups were also differentiated from the R lines, and hence are different from the existing complementary heterotic pools. This suggests that although these groups could provide highly divergent parental pools, further research is required to investigate the extent of heterosis and their hybrid performance.

Inheritance and Characterization of Strong Resistance to Phosphine in Sitophilus oryzae(L.)

Sitophilus oryzae (Linnaeus) is a major pest of stored grain across Southeast Asia and is of increasing concern in other regions due to the advent of strong resistance to phosphine, the fumigant used to protect stored grain from pest insects. We investigated the inheritance of genes controlling resistance to phosphine in a strongly resistant S. oryzae strain (NNSO7525) collected in Australia and find that the trait is autosomally inherited and incompletely recessive with a degree of dominance of -0.66. The strongly resistant strain has an LC50 52 times greater than a susceptible reference strain (LS2) and 9 times greater than a weakly resistant strain (QSO335). Analysis of F2 and backcross progeny indicates that two or more genes are responsible for strong resistance, and that one of these genes, designated Sorph1, not only contributes to strong resistance, but is also responsible for the weak resistance phenotype of strain QSO335. These results demonstrate that the genetic mechanism of phosphine resistance in Soryzae is similar to that of other stored product insect pests. A unique observation is that a subset of the progeny of an F1 backcross generation are more strongly resistant to phosphine than the parental strongly resistant strain, which may be caused by multiple alleles of one of the resistance genes.

Production attributes of Merino sheep genetically divergent for wool growth are reflected in differing rumen microbiotas

Divergent genetic selection for wool growth as a single trait has led to major changes in sheep physiology and metabolism, including variations in rumen microbial protein production and uptake of α-amino nitrogen in portal blood. This study was conducted to determine if sheep with different genetic merit for wool growth exhibit distinct rumen bacterial diversity. Eighteen Merino wethers were separated into groups of contrasting genetic merit for clean fleece weight (CFW; low: WG− and high: WG+) and fed a blend of oaten and lucerne chaff diet at two levels of intake (LOI; 1 or 1.5 times maintenance energy requirements) for two seven-week periods in a crossover design. Bacterial diversity in rumen fluid collected by esophageal intubation was characterized using 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Bacterial diversity estimated by Phylogenetic distance, Chao1 and observed species did not differ significantly with CFW or LOI; however, the Shannon diversity index differed (P=0.04) between WG+ (7.67) and WG− sheep (8.02). WG+ animals had a higher (P=0.03) proportion of Bacteroidetes (71.9% vs 66.5%) and a lower (P=0.04) proportion of Firmicutes (26.6% vs 31.6%) than WG− animals. Twenty-four specific operational taxonomic units (OTUs), belonging to the Firmicutes and Bacteroidetes phyla, were shared among all the samples, whereas specific OTUs varied significantly in presence/abundance (P<0.05) between wool genotypes and 50 varied (P<0.05) with LOI. It appears that genetic selection for fleece weight is associated with differences in rumen bacterial diversity that persist across different feeding levels. Moderate correlations between seven continuous traits, such as methane production or microbial protein production, and the presence and abundance of 17 OTUs were found, indicating scope for targeted modification of the microbiome to improve the energetic efficiency of rumen microbial synthesis and reduce the greenhouse gas footprint of ruminants.

Genetic characterization of field-evolved resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Laemophloeidae: Coleoptera)

Inheritance of resistance to phosphine fumigant was investigated in three field-collected strains of rusty grain beetle, Cryptolestes ferrugineus, Susceptible (S-strain), Weakly Resistant (Weak-R) and Strongly Resistant (Strong-R). The strains were purified for susceptibility, weak resistance and strong resistance to phosphine, respectively, to ensure homozygosity of resistance genotype. Crosses were established between S-strain × Weak-R, S-strain × Strong-R and Weak-R × Strong-R, and the dose mortality responses to phosphine of these strains and their F1, F2 and F1-backcross progeny were obtained. The fumigations were undertaken at 25 °C and 55% RH for 72 h. Weak-R and Strong-R showed resistance factors of 6.3 × and 505 × compared with S-strain at the LC50. Both weak and strong resistances were expressed as incompletely recessive with degrees of dominance of − 0.48 and − 0.43 at the LC50, respectively. Responses of F2 and F1-backcross progeny indicated the existence of one major gene in Weak-R, and at least two major genes in Strong-R, one of which was allelic with the major factor in Weak-R. Phenotypic variance analyses also estimated that the number of independently segregating genes conferring weak resistance was 1 (nE = 0.89) whereas there were two genes controlling strong resistance (nE = 1.2). The second gene, unique to Strong-R, interacted synergistically with the first gene to confer a very high level of resistance (~ 80 ×). Neither of the two major resistance genes was sex linked. Despite the similarity of the genetics of resistance to that previously observed in other pest species, a significant proportion (~ 15 to 30%) of F1 individuals survived at phosphine concentrations higher than predicted. Thus it is likely that additional dominant heritable factors, present in some individuals in the population, also influenced the resistance phenotype. Our results will help in understanding the process of selection for phosphine resistance in the field which will inform resistance management strategies. In addition, this information will provide a basis for the identification of the resistance genes.

Genetic characterization of field-evolved resistance to phosphine in the rusty grain beetle, Cryptolestes ferrugineus (Laemophloeidae: Coleoptera)

Inheritance of resistance to phosphine fumigant was investigated in three field-collected strains of rusty grain beetle, Cryptolestes ferrugineus, Susceptible (S-strain), Weakly Resistant (Weak-R) and Strongly Resistant (Strong-R). The strains were purified for susceptibility, weak resistance and strong resistance to phosphine, respectively, to ensure homozygosity of resistance genotype. Crosses were established between S-strain × Weak-R, S-strain × Strong-R and Weak-R × Strong-R, and the dose mortality responses to phosphine of these strains and their F1, F2 and F1-backcross progeny were obtained. The fumigations were undertaken at 25 °C and 55% RH for 72 h. Weak-R and Strong-R showed resistance factors of 6.3 × and 505 × compared with S-strain at the LC50. Both weak and strong resistances were expressed as incompletely recessive with degrees of dominance of − 0.48 and − 0.43 at the LC50, respectively. Responses of F2 and F1-backcross progeny indicated the existence of one major gene in Weak-R, and at least two major genes in Strong-R, one of which was allelic with the major factor in Weak-R. Phenotypic variance analyses also estimated that the number of independently segregating genes conferring weak resistance was 1 (nE = 0.89) whereas there were two genes controlling strong resistance (nE = 1.2). The second gene, unique to Strong-R, interacted synergistically with the first gene to confer a very high level of resistance (~ 80 ×). Neither of the two major resistance genes was sex linked. Despite the similarity of the genetics of resistance to that previously observed in other pest species, a significant proportion (~ 15 to 30%) of F1 individuals survived at phosphine concentrations higher than predicted. Thus it is likely that additional dominant heritable factors, present in some individuals in the population, also influenced the resistance phenotype. Our results will help in understanding the process of selection for phosphine resistance in the field which will inform resistance management strategies. In addition, this information will provide a basis for the identification of the resistance genes.

Epidemiology and genetic diversity of Tobacco streak virus and related subgroup 1 ilarviruses

A quarter of Australia’s sunflower production is from the central highlands region of Queensland and is currently worth six million dollars ($AUD) annually. From the early 2000s a severe necrosis disorder of unknown aetiology was affecting large areas of sunflower crops in central Queensland, leading to annual losses of up to 20%. Other crops such as mung bean and cotton were also affected. This PhD study was undertaken to determine if the causal agent of the necrosis disorder was of viral origin and, if so, to characterise its genetic diversity, biology and disease cycle, and to develop effective control strategies. The research described in this thesis identified Tobacco streak virus (TSV; genus Ilarvirus, family Bromoviridae) as the causal agent of the previously unidentified necrosis disorder of sunflower in central Queensland. TSV was also the cause of commonly found diseases in a range of other crops in the same region including cotton, chickpea and mung bean. This was the first report from Australia of natural field infections of TSV from these four crops. TSV strains have previously been reported from other regions of Australia in several hosts based on serological and host range studies. In order to determine the relatedness of previously reported TSV strains with TSV from central Queensland, we characterised the genetic diversity of the known TSV strains from Australia. We identified two genetically distinct TSV strains from central Queensland and named them based on their major alternative hosts, TSV-parthenium from Parthenium hysterophorus and TSV-crownbeard from Verbesina encelioides. They share only 81 % total-genome nucleotide sequence identity. In addition to TSV-parthenium and TSV-crownbeard from central Queensland, we also described the complete genomes of two other ilarvirus species. This proved that previously reported TSV strains, TSV-S isolated from strawberry and TSV-Ag from Ageratum houstonianum, were actually the first record of Strawberry necrotic shock virus from Australia, and a new subgroup 1 ilarvirus, Ageratum latent virus. Our results confirmed that the TSV strains found in central Queensland were not related to previously described strains from Australia and may represent new incursions. This is the first report of the genetic diversity within subgroup 1 ilarviruses from Australia. Based on field observations we hypothesised that parthenium and crownbeard were acting as symptomless hosts of TSV-parthenium and TSV-crownbeard, respectively. We developed strain-specific multiplex PCRs for the three RNA segments to accurately characterise the range of naturally infected hosts across central Queensland. Results described in this thesis show compelling evidence that parthenium and crownbeard are the major (symptomless) alternative hosts of TSV-parthenium and TSV-crownbeard. While both TSV strains had wide natural host ranges, the geographical distribution of each strain was closely associated with the respective distribution of their major alternative hosts. Both TSV strains were commonly found across large areas of central Queensland, but we only found strong evidence for the TSV-parthenium strain being associated with major disease outbreaks in nearby crops. The findings from this study demonstrate that both TSV-parthenium and TSV-crownbeard have similar life cycles but some critical differences. We found both TSV strains to be highly seed transmitted from their respective major alternative hosts from naturally infected mother plants and survived in seed for more than 2 years. We conclusively demonstrated that both TSV strains were readily transmitted via virus-infected pollen taken from the major alternative hosts. This transmission was facilitated by the most commonly collected thrips species, Frankliniella schultzei and Microcephalothrips abdominalis. These results illustrate the importance of seed transmission and efficient thrips vector species for the effective survival of these TSV strains in an often harsh environment and enables the rapid development of TSV disease epidemics in surrounding crops. Results from field surveys and inoculation tests indicate that parthenium is a poor host of TSV-crownbeard. By contrast, crownbeard was naturally infected by, and an experimental host of TSV-parthenium. However, this infection combination resulted in non-viable crownbeard seed. These differences appear to be an effective biological barrier that largely restricts these two TSV strains to their respective major alternative hosts. Based on our field observations we hypothesised that there were differences in relative tolerance to TSV infection between different sunflower hybrids and that seasonal variation in disease levels was related to rainfall in the critical early crop stage. Results from our field trials conducted over multiple years conclusively demonstrated significant differences in tolerance to natural infections of TSV-parthenium in a wide range of sunflower hybrids. Glasshouse tests indicate the resistance to TSV-parthenium identified in the sunflower hybrids is also likely to be effective against TSV-crownbeard. We found a significant negative association between TSV disease incidence in sunflowers and accumulated rainfall in the months of March and April with increasing rainfall resulting in reduced levels of disease. Our results indicate that the use of tolerant sunflower germplasm will be a critical strategy to minimise the risk of TSV epidemics in sunflower.

Genetic diversity and epidemiology of Tobacco streak virus and related subgroup 1 Ilarviruses

A quarter of Australia’s sunflower production is from the central highlands region of Queensland and is currently worth six million dollars ($AUD) annually. From the early 2000s a severe necrosis disorder of unknown aetiology was affecting large areas of sunflower crops in central Queensland, leading to annual losses of up to 20%. Other crops such as mung bean and cotton were also affected. This PhD study was undertaken to determine if the causal agent of the necrosis disorder was of viral origin and, if so, to characterise its genetic diversity, biology and disease cycle, and to develop effective control strategies. The research described in this thesis identified Tobacco streak virus (TSV; genus Ilarvirus, family Bromoviridae) as the causal agent of the previously unidentified necrosis disorder of sunflower in central Queensland. TSV was also the cause of commonly found diseases in a range of other crops in the same region including cotton, chickpea and mung bean. This was the first report from Australia of natural field infections of TSV from these four crops. TSV strains have previously been reported from other regions of Australia in several hosts based on serological and host range studies. In order to determine the relatedness of previously reported TSV strains with TSV from central Queensland, we characterised the genetic diversity of the known TSV strains from Australia. We identified two genetically distinct TSV strains from central Queensland and named them based on their major alternative hosts, TSV-parthenium from Parthenium hysterophorus and TSV-crownbeard from Verbesina encelioides. They share only 81 % total-genome nucleotide sequence identity. In addition to TSV-parthenium and TSV-crownbeard from central Queensland, we also described the complete genomes of two other ilarvirus species. This proved that previously reported TSV strains, TSV-S isolated from strawberry and TSV-Ag from Ageratum houstonianum, were actually the first record of Strawberry necrotic shock virus from Australia, and a new subgroup 1 ilarvirus, Ageratum latent virus. Our results confirmed that the TSV strains found in central Queensland were not related to previously described strains from Australia and may represent new incursions. This is the first report of the genetic diversity within subgroup 1 ilarviruses from Australia. Based on field observations we hypothesised that parthenium and crownbeard were acting as symptomless hosts of TSV-parthenium and TSV-crownbeard, respectively. We developed strain-specific multiplex PCRs for the three RNA segments to accurately characterise the range of naturally infected hosts across central Queensland. Results described in this thesis show compelling evidence that parthenium and crownbeard are the major (symptomless) alternative hosts of TSV-parthenium and TSV-crownbeard. While both TSV strains had wide natural host ranges, the geographical distribution of each strain was closely associated with the respective distribution of their major alternative hosts. Both TSV strains were commonly found across large areas of central Queensland, but we only found strong evidence for the TSV-parthenium strain being associated with major disease outbreaks in nearby crops. The findings from this study demonstrate that both TSV-parthenium and TSV-crownbeard have similar life cycles but some critical differences. We found both TSV strains to be highly seed transmitted from their respective major alternative hosts from naturally infected mother plants and survived in seed for more than 2 years. We conclusively demonstrated that both TSV strains were readily transmitted via virus-infected pollen taken from the major alternative hosts. This transmission was facilitated by the most commonly collected thrips species, Frankliniella schultzei and Microcephalothrips abdominalis. These results illustrate the importance of seed transmission and efficient thrips vector species for the effective survival of these TSV strains in an often harsh environment and enables the rapid development of TSV disease epidemics in surrounding crops. Results from field surveys and inoculation tests indicate that parthenium is a poor host of TSV-crownbeard. By contrast, crownbeard was naturally infected by, and an experimental host of TSV-parthenium. However, this infection combination resulted in non-viable crownbeard seed. These differences appear to be an effective biological barrier that largely restricts these two TSV strains to their respective major alternative hosts. Based on our field observations we hypothesised that there were differences in relative tolerance to TSV infection between different sunflower hybrids and that seasonal variation in disease levels was related to rainfall in the critical early crop stage. Results from our field trials conducted over multiple years conclusively demonstrated significant differences in tolerance to natural infections of TSV-parthenium in a wide range of sunflower hybrids. Glasshouse tests indicate the resistance to TSV-parthenium identified in the sunflower hybrids is also likely to be effective against TSV-crownbeard. We found a significant negative association between TSV disease incidence in sunflowers and accumulated rainfall in the months of March and April with increasing rainfall resulting in reduced levels of disease. Our results indicate that the use of tolerant sunflower germplasm will be a critical strategy to minimise the risk of TSV epidemics in sunflower.

Integrating Rapid Phenotyping and Speed Breeding to Improve Stay-Green and Root Adaptation of Wheat in Changing, Water-Limited, Australian Environments

Temperatures have increased and in-crop rainfall decreased over recent decades in many parts of the Australian wheat cropping region. With these trends set to continue or intensify, improving crop adaptation in the face of climate change is particularly urgent in this, already drought-prone, cropping region. Importantly, improved performance under water-limitation must be achieved while retaining yield potential during more favourable seasons. A multi-trait-based approach to improve wheat yield and yield stability in the face of water-limitation and heat has been instigated in northern Australia using novel phenotyping techniques and a nested association mapping (NAM) approach. An innovative laboratory technique allows rapid root trait screening of hundreds of lines. Using soil grown seedlings, the method offers significant advantages over many other lab-based techniques. Another recently developed method allows novel stay-green traits to be quantified objectively for hundreds of genotypes in standard field trial plots. Field trials in multiple locations and seasons allow evaluation of targeted trait values and identification of superior germplasm. Traits, including yield and yield components are measured for hundreds of NAM lines in rain fed environments under various levels of water-limitation. To rapidly generate lines of interest, the University of Queensland “speed breeding” method is being employed, allowing up to 7 plant generations per annum. A NAM population of over 1000 wheat recombinant inbred lines has been progressed to the F5 generation within 18 months. Genotyping the NAM lines with the genome-wide DArTseq molecular marker system provides up to 40,000 markers. They are now being used for association mapping to validate QTL previously identified in bi-parental populations and to identify novel QTL for stay-green and root traits. We believe that combining the latest techniques in physiology, phenotyping, genetics and breeding will increase genetic progress toward improved adaptation to water-limited environments.

Genetic conservation of Phosphine Resistance in the Rice Weevil Sitophilus oryzae (L.)

High levels of resistance to phosphine in the rice weevil Sitophilus oryzae have been detected in Asian countries including China and Vietnam, however there is limited knowledge of the genetic mechanism of resistance in these strains. We find that the genetic basis of strong phosphine resistance is conserved between strains of S. oryzae from China, Vietnam and Australia. Each of four strongly resistant strains has an identical amino acid variant in the encoded dihydrolipoamide dehydrogenase (DLD) enzyme that was previously identified as a resistance factor in Rhyzopertha dominica and Tribolium castaneum. The unique amino acid substitution, Asparagine > Threonine (N505T) of all strongly resistant S. oryzae corresponds to the position of an Asparagine > Histidine variant (N506H) that was previously reported in strongly resistant R. dominica. Progeny (F16 and F18) from two independent crosses showed absolute linkage of N505T to the strong resistance phenotype, indicating that if N505T was not itself the resistance variant that it resided within 1 or 2 genes of the resistance factor. Non-complementation between the strains confirmed the shared genetic basis of strong resistance, which was supported by the very similar level of resistance between the strains, with LC50 values ranging from 0.20 to 0.36 mgL-1 for a 48 hour exposure at 25°C. Thus, the mechanism of high level resistance to phosphine is strongly conserved between R. dominica, T. castaneum and S. oryzae. A fitness cost associated with strongly resistant allele was observed in segregating populations in the absence of selection.

Inventories and significance of the genetic resources of an African mahogany species (Khaya senegalensis (Desr.) A. Juss.) assembled and further developed in Australia.

The forest tree species Khaya senegalensis (Desr.) A. Juss. occurs in a belt across 20 African countries from Senegal-Guinea to Sudan-Uganda where it is a highly important resource. However, it is listed as Vulnerable (IUCN 2015-3). Since introduction in northern Australia around 1959, the species has been planted widely, yielding high-value products. The total area of plantations of the species in Australia exceeds 15,000 ha, mostly planted in the Northern Territory since 2006, and includes substantial areas across 60-70 woodlots and industrial plantations established in north-eastern Queensland since the early-1990s and during 2005-2007 respectively. Collaborative conservation and tree improvement by governments began in the Northern Territory and Queensland in 2001 based on provenance and other trials of the 1960s-1970s. This work has developed a broad base of germplasm in clonal seed orchards, hedge gardens and trials (clone and progeny). Several of the trials were established collaboratively on private land. Since the mid-2000s, commercial growers have introduced large numbers of provenance-bulk and individual-tree seedlots to establish industrial plantations and trials, several of the latter in collaboration with the Queensland Government. Provenance bulks (>140) and families (>400) from 17 African countries are established in Australia, considered the largest genetic base of the species in a single country outside Africa. Recently the annual rate of industrial planting of the species in Australia has declined, and R&D has been suspended by governments and reduced by the private sector. However, new commercial plantings in the Northern Territory and Queensland are proposed. In domesticating a species, the strategic importance of a broad genetic base is well known. The wide range of first- and advanced-generation germplasm of the species established in northern Australia and documented in this paper provides a sound basis for further domestication and industrial plantation and woodlot expansion, when investment conditions are favourable

Molecular Breeding for Complex Adaptive Traits: How Integrating Crop Ecophysiology and Modelling Can Enhance Efficiency

Progress in crop improvement is limited by the ability to identify favourable combinations of genotypes (G) and management practices (M) in relevant target environments (E) given the resources available to search among the myriad of possible combinations. To underpin yield advance we require prediction of phenotype based on genotype. In plant breeding, traditional phenotypic selection methods have involved measuring phenotypic performance of large segregating populations in multi-environment trials and applying rigorous statistical procedures based on quantitative genetic theory to identify superior individuals. Recent developments in the ability to inexpensively and densely map/sequence genomes have facilitated a shift from the level of the individual (genotype) to the level of the genomic region. Molecular breeding strategies using genome wide prediction and genomic selection approaches have developed rapidly. However, their applicability to complex traits remains constrained by gene-gene and gene-environment interactions, which restrict the predictive power of associations of genomic regions with phenotypic responses. Here it is argued that crop ecophysiology and functional whole plant modelling can provide an effective link between molecular and organism scales and enhance molecular breeding by adding value to genetic prediction approaches. A physiological framework that facilitates dissection and modelling of complex traits can inform phenotyping methods for marker/gene detection and underpin prediction of likely phenotypic consequences of trait and genetic variation in target environments. This approach holds considerable promise for more effectively linking genotype to phenotype for complex adaptive traits. Specific examples focused on drought adaptation are presented to highlight the concepts.