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QTL controlling vigour and related traits were identified in a chickpea RIL population and validated in diverse sets of germplasm. Robust KASP markers were developed for marker-assisted selection. To understand the genetic constitution of vigour in chickpea (Cicer arietinum L.), genomic data from a bi-parental population and multiple diversity panels were used to identify QTL, sequence-level haplotypes and genetic markers associated with vigour-related traits in Australian environments. Using 182 Recombinant Inbred Lines (RILs) derived from a cross between two desi varieties, Rupali and Genesis836, vigour QTL independent of flowering time were identified on chromosomes (Ca) 1, 3 and 4 with genotypic variance explained (GVE) ranging from 7.1 to 28.8%. Haplotype analysis, association analysis and graphical genotyping of whole-genome re-sequencing data of two diversity panels consisting of Australian and Indian genotypes and an ICRISAT Chickpea Reference Set revealed a deletion in the FTa1–FTa2–FTc gene cluster of Ca3 significantly associated with vigour and flowering time. Across the RIL population and diversity panels, the impact of the deletion was consistent for vigour but not flowering time. Vigour-related QTL on Ca4 co-located with a QTL for seed size in Rupali/Genesis836 (GVE = 61.3%). Using SNPs from this region, we developed and validated gene-based KASP markers across different panels. Two markers were developed for a gene on Ca1, myo -inositol monophosphatase (CaIMP), previously proposed to control seed size, seed germination and seedling growth in chickpea. While associated with vigour in the diversity panels, neither the markers nor broader haplotype linked to CaIMP was polymorphic in Rupali/Genesis836. Importantly, vigour appears to be controlled by different sets of QTL across time and with components which are independent from phenology.

Phytophthora root rot (PRR) caused by the soilborne oomycete Phytophthora medicaginis is a significant constraint to chickpea (Cicer arietinum) production across the northern grains region of Australia. In flooded soil, which is conducive to PRR disease development, up to 70% yield loss can occur in the most resistant Australian cultivars. Incorporating waterlogging tolerance in soybean (Glycine max) has been shown to improve quantitative resistance to Phytophthora sojae. Root growth of three chickpea genotypes was assessed at the seedling stage under waterlogging, PRR, and the combination of these abiotic and biotic constraints. Levels of waterlogging tolerance in chickpea are inherently low, yet selected genotypes displayed variability in root traits linked to improved waterlogging tolerance. The PRR moderately susceptible chickpea cultivar Yorker and PRR very susceptible Rupali demonstrated an eightfold increase in early adventitious root growth from the epicotyl region under waterlogging stress, compared with the PRR resistant interspecific backcross genotype 04067-81-2-1-1 (C. echinospermum × C. arietinum*2). Selection for primary root depth, which was significantly greater in 04067-81-2-1-1 under waterlogging, appears to improve PRR resistance compared with root replacement traits. Soilborne Phytophthora spp. are reportedly attracted to branch sites and leached exudates. We propose that compromised root barriers at emergence sites of adventitious roots under waterlogging conditions hasten hyphal entry, potentially increasing susceptibility to PRR. Hence, screening for root depth and absence of adventitious root development under waterlogged conditions may offer a novel proxy phenotyping method for PRR resistance traits at early stages of chickpea breeding.

Ascochyta blight disease, caused by the necrotrophic fungus Ascochyta rabiei, is a major biotic constraint to chickpea production in Australia and worldwide. Detailed knowledge of the structure of the pathogen population and its potential to adapt to our farming practices is key to informing optimal management of the disease. This includes understanding the molecular diversity among isolates and the frequency and distribution of the isolates that have adapted to overcome host resistance across agroecologically distinct regions. Thanks to continuous monitoring efforts over the past 6 years, a comprehensive collection of A. rabiei isolates was collated from the major Australian chickpea production regions. To determine the molecular structure of the entire population, representative isolates from each collection year and growing region have been genetically characterized using a DArTseq genotyping-by-sequencing approach. The genotyped isolates were further phenotyped to determine their pathogenicity levels against a differential set of chickpea cultivars and genotype-phenotype associations were inferred. Overall, the Australian A. rabiei population displayed a far lower genetic diversity (average Nei’s gene diversity of 0.047) than detected in other populations worldwide. This may be explained by the presence of a single mating-type in Australia, MAT1-2, limiting its reproduction to a clonal mode. Despite the low detected molecular diversity, clonal selection appears to have given rise to a subset of adapted isolates that are highly pathogenic on commonly employed resistance sources, and that are occurring at an increasing frequency. Among these, a cluster of genetically similar isolates was identified, with a higher proportion of highly aggressive isolates than in the general population. The discovery of distinct genetic clusters associated with high and low isolate pathogenicity forms the foundation for the development of a molecular pathotyping tool for the Australian A. rabiei population. Application of such a tool, along with continuous monitoring of the genetic structure of the population will provide crucial information for the screening of breeding material and integrated disease management packages.

QTL controlling vigour and related traits were identified in a chickpea RIL population and validated in diverse sets of germplasm. Robust KASP markers were developed for marker-assisted selection. To understand the genetic constitution of vigour in chickpea (Cicer arietinum L.), genomic data from a bi-parental population and multiple diversity panels were used to identify QTL, sequence-level haplotypes and genetic markers associated with vigour-related traits in Australian environments. Using 182 Recombinant Inbred Lines (RILs) derived from a cross between two desi varieties, Rupali and Genesis836, vigour QTL independent of flowering time were identified on chromosomes (Ca) 1, 3 and 4 with genotypic variance explained (GVE) ranging from 7.1 to 28.8%. Haplotype analysis, association analysis and graphical genotyping of whole-genome re-sequencing data of two diversity panels consisting of Australian and Indian genotypes and an ICRISAT Chickpea Reference Set revealed a deletion in the FTa1-FTa2-FTc gene cluster of Ca3 significantly associated with vigour and flowering time. Across the RIL population and diversity panels, the impact of the deletion was consistent for vigour but not flowering time. Vigour-related QTL on Ca4 co-located with a QTL for seed size in Rupali/Genesis836 (GVE = 61.3%). Using SNPs from this region, we developed and validated gene-based KASP markers across different panels. Two markers were developed for a gene on Ca1, myo -inositol monophosphatase (CaIMP), previously proposed to control seed size, seed germination and seedling growth in chickpea. While associated with vigour in the diversity panels, neither the markers nor broader haplotype linked to CaIMP was polymorphic in Rupali/Genesis836. Importantly, vigour appears to be controlled by different sets of QTL across time and with components which are independent from phenology.

During the reproductive stage, chilling temperatures and frost reduce the yield of chickpea and limit its adaptation. The adverse effects of chilling temperature and frost in terms of the threshold temperatures, impact of cold duration, and genotype-by-environment-by-management interactions are not well quantified. Crop growth models that predict flowering time and yield under diverse climates can identify combinations of cultivars and sowing time to reduce frost risk in target environments. The Agricultural Production Systems Simulator (APSIM-chickpea) model uses daily temperatures to model basic crop growth but does not include penalties for either frost damage or cold temperatures during flowering and podding stages. Regression analysis overcame this limitation of the model for chickpea crops grown at 95 locations in Australia using 70 years of historic data incorporating three cultivars and three sowing times (early, mid, and late). We modified model parameters to include the effect of soil water on thermal time calculations, which significantly improved the prediction of flowering time. Simulated data, and data from field experiments grown in Australia (2013 to 2019), showed robust predictions for flowering time (n = 29; R2 = 0.97), and grain yield (n = 22; R2 = 0.63–0.70). In addition, we identified threshold cold temperatures that significantly affected predicted yield, and combinations of locations, variety, and sowing time where the overlap between peak cold temperatures and peak flowering was minimal. Our results showed that frost and/or cold temperature–induced yield losses are a major limitation in some unexpected Australian locations, e.g., inland, subtropical latitudes in Queensland. Intermediate sowing maximise yield, as it avoids cold temperature, late heat, and drought stresses potentially limiting yield in early and late sowing respectively.

Ascochyta blight disease, caused by the necrotrophic fungusAscochyta rabiei, is a major biotic constraint to chickpea production in Australia and worldwide. Detailed knowledge of the structure of the pathogen population and its potential to adapt to our farming practices is key to informing optimal management of the disease. This includes understanding the molecular diversity among isolates and the frequency and distribution of the isolates that have adapted to overcome host resistance across agro-geographically distinct regions.Thanks to continuous monitoring efforts over the past six years, a comprehensive collection ofA. rabieiisolates was collated from the major Australian production regions. To determine the molecular structure of the entire population, representative isolates from each collection year and growing region have been genetically characterised using a DArTseq™ genotyping-by-sequencing approach. The genotyped isolates were further phenotyped to determine their pathogenicity levels against a differential set of chickpea cultivars and genotype-phenotype associations were inferred.Overall, the AustralianA. rabieipopulation displayed a far lower genetic diversity (average Nei’s gene diversity of 0.047) than detected in other populations worldwide. This may be explained by the presence of a single mating-type in Australia, MAT1-2, limiting its reproduction to a clonal mode. Despite the low detected molecular diversity, clonal selection appears to have given rise to a subset of adapted isolates that are highly pathogenic on commonly employed resistance sources, and that are occurring at an increasing frequency.To better understand the mechanisms and patterns of the pathogen adaptation, multi-locus genotype analysis was performed and two hypotheses were proposed on how new genotypes emerge. These were: 1) In a local, within-region evolutionary pathway; or 2) Through inter-region dispersal, most likely due to human activities. Furthermore, a cluster of genetically similar isolates was identified, with a higher proportion of highly aggressive isolates than in the general population, indicating the adaptive evolution of a sub-set of isolates that pose a greater risk to the chickpea industry.The discovery of distinct genetic clusters associated with high and low isolate pathogenicity forms the foundation for the development of a molecular pathotyping tool for the AustralianA. rabieipopulation. Application of such a tool, along with continuous monitoring of the genetic structure of the population will provide crucial information for the screening of breeding material and integrated disease management packages.Data SummaryAn online dataset containing all supporting genotyping and phenotyping data and the code required to reproduce the results, summary tables and plots found in this publication, is publicly available at Zenodo via the following links:https://zenodo.org/record/4311477; DOI:10.5281/zenodo.4311477(1).

Ascochyta Blight (AB), caused by Ascochyta rabiei (syn Phoma rabiei), is the major endemic foliar fungal disease affecting the Australian chickpea industry, resulting with potential crop loss and management costs. This study was conducted to better understand the risk posed by the Australian A. rabiei population to current resistance sources and to provide informed decision support for chemical control strategies. Recent changes in the pathogenicity of the population were proposed based on disease severity and histopathological observations on a host set. Controlled environment disease screening of 201 isolates on the host set revealed distinct pathogenicity groups, with 41% of all isolates assessed as highly aggressive and a significant increase in the proportion of isolates able to cause severe damage on resistant and moderately resistant cultivars since 2013. In particular, the frequency of highly aggressive isolates on the widely adopted PBA HatTrick cultivar rose from 18% in 2013 to 68% in 2017. In addition, isolates collected since 2016 caused severe disease on Genesis 090, another widely adopted moderately resistant cultivar and on ICC3996, a commonly used resistance source. Of immediate concern was the 10% of highly aggressive isolates able to severely damage the recently released resistant cultivar PBA Seamer (2016). Histopathology studies revealed that the most aggressive isolates were able to germinate, develop appressoria and invade directly through the epidermis faster than lower aggressive isolates on all hosts assessed, including ICC3996. The fungal invasion triggered a common reactive oxygen species (ROS) and hypersensitive response (HR) on all assessed resistant genotypes with initial biochemical and subsequent structural defence responses initiated within 24 hours of inoculation by the most highly aggressive isolates. These responses were much faster on the less resistant and fastest on the susceptible check host, indicating that speed of recognition was correlated with resistance rating. This will inform fungicide application timing so that infected crops are sprayed with prophylactic chemistries prior to invasion and with systemic chemistries after the pathogen has invaded.

Desi chickpea is a significant export crop for Australia; Australia being the largest exporter globally. Visual appearance of the seed is an economically significant measure of seed quality by the Indian subcontinent, the major importer of desi chickpea worldwide. Any visual blemish on the seed is considered undesirable, regardless of the cause (biotic or otherwise). Literature on biotic causes of seed blemishes, such as ascochyta blight, are available; however, little could be found on abiotic blemishes. Abiotic seed blemishes caused by physiological plant responses are more commonly known as seed markings. Despite the presence of seed markings being confirmed by several chickpea‐producing countries during personal discussions (India, Canada), no scientific literature has been published. The aim of this study was to proactively seek out and characterise different types of seed marking patterns using a wide genetic pool of desi chickpea across a range of environments in Australia. Thirteen different seed marking patterns were identified in desi chickpea and three in kabuli chickpea, including several rare seed markings that were discovered, photographed, and described. Seed markings (blemishes thought to be caused by physiological plant stress) can be characterised as dark patterns on the testa (seed coat) that do not visually affect the underlying cotyledon. In contrast, other seed blemishes (caused by pests and disease, physical damage, or poor storage) were more likely to affect the cotyledons underlying the testa, but not always. This paper classifies and describes various types of seed markings and blemishes for future reference by the global chickpea industry.

Major QTL for Phytophthora root rot resistance have been identified in three mapping populations with independent sources of resistance contributed by C. echinospermum and C. arietinum. Phytophthora root rot (PRR) caused by the oomycete Phytophthora medicaginis is a major soil-borne disease of chickpea in Australia. With no economic in-crop control of PRR, a genetic approach to improve resistance is the most practical management option. Moderate field resistance has been incorporated in the cultivated C. arietinum variety, Yorker, and a higher level of resistance has been identified in a derivative of wild chickpea (C. echinospermum, interspecific breeding line 04067-81-2-1-1). These genotypes and two other released varieties were used to develop one intra-specific and two interspecific F6-derived recombinant inbred line mapping populations for genetic analysis of resistance. The Yorker × Genesis114 (YG), Rupali × 04067-81-2-1-1 (RB) and Yorker × 04067-81-2-1-1 (YB) populations were genotyped using genotyping-by-sequencing and phenotyped for PRR under three field environments with a mixture of 10 P. medicaginis isolates. Whole-genome QTL analysis identified major QTL QRBprrsi01, QYBprrsi01, QRBprrsi03 and QYBprrsi02 for PRR resistance on chromosomes 3 and 6, in RB and YB populations, respectively, with the resistance source derived from the wild Cicer species. QTL QYGprrsi02 and QYGprrsi03 were also identified on chromosomes 5 and 6 in YG population from C. arietinum. Aligning QTL regions to the corresponding chickpea reference genome suggested that the resistance source from C. arietinum and C. echinospermum may be different. The findings from this study provide tools for marker-assisted selection in chickpea breeding and information to assist the development of populations suitable for fine-mapping of resistance loci to determine the molecular basis for PRR resistance in chickpea.

Chickpea (Cicer arietinum L.) is the third most economically important food legume in the world. Its yield potential is often limited by various biotic stresses, including fungal and viral diseases, insects, nematodes and parasitic weeds. Incorporating genetic resistance into cultivars is the most effective and economical way of controlling biotic stresses and this is a major objective in many breeding programs. Extensive searches for resistances have been conducted by screening commercial varieties, landraces and closely related species. Resistances to disease such as Ascochyta blight and Fusarium wilt have been identified and molecular tools are being used to increase the efficiency of gene transfer from wild species into chickpea elite genotypes. Quantitative trait loci for resistance genes have been located on linkage maps and molecular markers associated with these loci can potentially be used for efficient pyramiding of the traits. Significant chickpea genomic resources have been developed in order to investigate resistance genes. Such resources include an integrated genetic map, expressed sequence tag libraries, bacterial artificial chromosome libraries, microarrays and draft genome sequences. Although these resources have yet to be used to improve chickpea cultivars in the field, this is likely to change in the near future. These genomic resources, as well as high-resolution phenotyping tools and cutting-edge technologies such as next-generation sequencing, promise to increase efficiency as work to identify valuable candidate genes continues.

The Australian Ascochyta rabiei (Pass.) Labr. (syn. Phoma rabiei) population has low genotypic diversity with only one mating type detected to date, potentially precluding substantial evolution through recombination. However, a large diversity in aggressiveness exists. In an effort to better understand the risk from selective adaptation to currently used resistance sources and chemical control strategies, the population was examined in detail. For this, a total of 598 isolates were quasi-hierarchically sampled between 2013 and 2015 across all major Australian chickpea growing regions and commonly grown host genotypes. Although a large number of haplotypes were identified (66) through short sequence repeat (SSR) genotyping, overall low gene diversity (Hexp = 0.066) and genotypic diversity (D = 0.57) was detected. Almost 70% of the isolates assessed were of a single dominant haplotype (ARH01). Disease screening on a differential host set, including three commonly deployed resistance sources, revealed distinct aggressiveness among the isolates, with 17% of all isolates identified as highly aggressive. Almost 75% of these were of the ARH01 haplotype. A similar pattern was observed at the host level, with 46% of all isolates collected from the commonly grown host genotype Genesis090 (classified as “resistant” during the term of collection) identified as highly aggressive. Of these, 63% belonged to the ARH01 haplotype. In conclusion, the ARH01 haplotype represents a significant risk to the Australian chickpea industry, being not only widely adapted to the diverse agro-geographical environments of the Australian chickpea growing regions, but also containing a disproportionately large number of aggressive isolates, indicating fitness to survive and replicate on the best resistance sources in the Australian germplasm.

Ascochyta blight (AB) is a fungal disease that can significantly reduce chickpea production in Australia and other regions of the world. In this study, 69 chickpea genotypes were sequenced using whole genome re-sequencing (WGRS) methods. They included 48 Australian varieties differing in their resistance ranking to AB, 16 advanced breeding lines from the Australian chickpea breeding program, four landraces, and one accession representing the wild chickpea species Cicer reticulatum. More than 800,000 single nucleotide polymorphisms (SNPs) were identified. Population structure analysis revealed relatively narrow genetic diversity amongst recently released Australian varieties and two groups of varieties separated by the level of AB resistance. Several regions of the chickpea genome were under positive selection based on Tajima’s D test. Both Fst genome- scan and genome-wide association studies (GWAS) identified a 100 kb region (AB4.1) on chromosome 4 that was significantly associated with AB resistance. The AB4.1 region co-located to a large QTL interval of 7 Mb∼30 Mb identified previously in three different mapping populations which were genotyped at relatively low density with SSR or SNP markers. The AB4.1 region was validated by GWAS in an additional collection of 132 advanced breeding lines from the Australian chickpea breeding program, genotyped with approximately 144,000 SNPs. The reduced level of nucleotide diversity and long extent of linkage disequilibrium also suggested the AB4.1 region may have gone through selective sweeps probably caused by selection of the AB resistance trait in breeding. In total, 12 predicted genes were located in the AB4.1 QTL region, including those annotated as: NBS-LRR receptor-like kinase, wall-associated kinase, zinc finger protein, and serine/threonine protein kinases. One significant SNP located in the conserved catalytic domain of a NBS-LRR receptor-like kinase led to amino acid substitution. Transcriptional analysis using qPCR showed that some predicted genes were significantly induced in resistant lines after inoculation compared to non-inoculated plants. This study demonstrates the power of combining WGRS data with relatively simple traits to rapidly develop “functional makers” for marker-assisted selection and genomic selection.

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Labr., is a highly destructive disease of chickpea (Cicer arietinum L.) on a global basis, and exhibits considerable natural variation for pathogenicity. Different sources of ascochyta blight resistance are available within the cultivated species, suitable for pyramiding to improve field performance. Robust and closely linked genetic markers are desirable to facilitate this approach. A total of 4,654 simple sequence repeat (SSR) and 1,430 single nucleotide polymorphism (SNP) markers were identified from a chickpea expressed sequence tag (EST) database. Subsets of 143 EST–SSRs and 768 SNPs were further used for validation and subsequent high-density genetic mapping of two intraspecific mapping populations (Lasseter × ICC3996 and S95362 × Howzat). Comparison of the linkage maps to the genome of Medicago truncatula revealed a high degree of conserved macrosynteny. Based on field evaluation of ascochyta blight incidence performed over 2 years, two genomic regions containing resistance determinants were identified in the Lasseter × ICC3996 family. In the S95362 × Howzat population, only one quantitative trait locus (QTL) region was identified for both phenotypic evaluation trials, which on the basis of bridging markers was deduced to coincide with one of the Lasseter × ICC3996 QTLs. Of the two QTL-containing regions identified in this study, one (ab_QTL1) was predicted to be in common with QTLs identified in prior studies, while the other (ab_QTL2) may be novel. Markers in close linkage to ascochyta blight resistance genes that have been identified in this study can be further validated and effectively implemented in chickpea breeding programs.

For lentil production to expand further in Australia, adaptation to the less favourable soils of the low to medium rainfall zones is required. To improve adaptation to these regions, varieties are required with increased tolerance to soil constraints such as high concentrations of boron (B), salinity and sodicity. To evaluate the range of B tolerance in lentil germplasm, 310 lines were screened in soil with a high concentration of B and tolerance was assessed at the seedling stage. A wide range in response to high concentrations of soil B was observed in the germplasm tested. Current Australian varieties were generally very intolerant to high concentrations of soil B. High levels of B tolerance was identified in germplasm originating from Afghanistan and Ethiopia. A subsequent experiment comparing lentils with different levels of B tolerance found that tolerant accessions (ILL213A and ILL2024) produced greater above and below ground biomass than intolerant accessions. The tolerant accessions had no significant yield loss under a high B treatment (extractable B = 18.20 mg/kg) compared to the control treatment (extractable B = 1.55 mg/kg). The large improvement in B tolerance, at soil concentrations typical of those found in the target regions, suggests there is potential to improve the tolerance level of adapted varieties and expand lentil production areas to regions with higher concentrations of soil B.