Extensive allele mining discovers novel genetic diversity in the loci controlling frost tolerance in barley

Key message Exome sequencing-based allele mining for frost tolerance suggests HvCBF14 rather than CNV at Fr-H2 locus is the main responsible of frost tolerance in barley. Abstract Wild relatives, landraces and old cultivars of barley represent a reservoir of untapped and potentially important genes for crop improvement, and the recent sequencing technologies provide the opportunity to mine the existing genetic diversity and to identify new genes/alleles for the traits of interest. In the present study, we use frost tolerance and vernalization requirement as case studies to demonstrate the power of allele mining carried out on exome sequencing data generated from > 400 barley accessions. New deletions in the first intron of VRN-H1 were identified and linked to a reduced vernalization requirement, while the allelic diversity of HvCBF2a, HvCBF4b and HvCBF14 was investigated by combining the analysis of SNPs and read counts. This approach has proven very effective to identify gene paralogs and copy number variants of HvCBF2 and the HvCBF4b-HvCBF2a segment. A multiple linear regression model which considers allelic variation at these genes suggests a major involvement of HvCBF14, rather than copy number variation of HvCBF4b-HvCBF2a, in controlling frost tolerance in barley. Overall, the present study provides powerful resource and tools to discover novel alleles at relevant genes in barley.

The Rice Blast Resistance Gene Pid Family has been Strictly Diverged into Indica and Japonica Subspecies

Background: Rice blast (causative agent the fungus Pyricularia oryzae) represents a major constraint over the productivity of one of the world’s most important staple foods. Genes encoding resistance have been identified in both the indica and japonica subspecies genepools, and combining these within new cultivars represents a rational means of combating the pathogen.Results: In this research, a deeper allele mining was carried out on Pid-2, Pid-3, and Pid-4 by their specific FNP markers in the three panels consisting of 70 indica and 58 japonica cultivars. Within Pid-2, three functional and one non-functional alleles were identified; the former were only identified in indica type entries. At Pid-3, four functional and one non-functional alleles were identified, and once again, all of the former were present in indica type entries. However, the pattern of variation at Pid-4 was rather different: here, the five functional alleles uncovered were dispersed across the japonica type germplasm. Among all the 12 candidate functional alleles, both Pid2-ZS and Pid3-ZS were predominant.Conclusions: Variation in both Pid-2 and Pid-3 appears to have evolved in response to pathogen pressure exerted on indica type cultivars, while that in Pid-4 reflects the interaction between the fungus and the host in japonica type crops. Owning to the founder lineage, ‘Zhenzhuai 11-ZS97’, rather limited genotypes of the Pid family have been effective in both indica and japonica rice groups, of which Pid2-ZS+Pid3-ZS is present in a large proportion of Chinese indica cultivars released since the 1960s.

Explore the genetics of weedy traits using rice 3K database

Background Weedy rice, a conspecific weedy counterpart of the cultivated rice (Oryza sativa L.), has been problematic in rice-production area worldwide. Although we started to know about the origin of some weedy traits for some rice-growing regions, an overall assessment of weedy trait-related loci was not yet available. On the other hand, the advances in sequencing technologies, together with community efforts, have made publicly available a large amount of genomic data. Given the availability of public data and the need of “weedy” allele mining for a better management of weedy rice, the objective of the present study was to explore the genetic architecture of weedy traits based on publicly available data, mainly from the 3000 Rice Genome Project (3K-RGP). Results Based on the results of population structure analysis, we have selected 1378 individuals from four sub-populations (aus, indica, temperate japonica, tropical japonica) without admixed genomic composition for genome-wide association analysis (GWAS). Five traits were investigated: awn color, seed shattering, seed threshability, seed coat color, and seedling height. GWAS was conducted for each sub-population × trait combination and we have identified 66 population-specific trait-associated SNPs. Eleven significant SNPs fell into an annotated gene and four other SNPs were close to a putative candidate gene (± 25 kb). SNPs located in or close to Rc were particularly predictive of the occurrence of seed coat color and our results showed that different sub-populations required different SNPs for a better seed coat color prediction. We compared the data of 3K-RGP to a publicly available weedy rice dataset. The profile of allele frequency, phenotype-genotype segregation of target SNP, as well as GWAS results for the presence and absence of awns diverged between the two sets of data. Conclusions The genotype of trait-associated SNPs identified in this study, especially those located in or close to Rc, can be developed to diagnostic SNPs to trace the origin of weedy trait occurred in the field. The difference of results from the two publicly available datasets used in this study emphasized the importance of laboratory experiments to confirm the allele mining results based on publicly available data.

Allele Mining in Diverse Accessions of Urochloa and Megathyrsus spp. Tropical Grasses to Improve Forage Quality and Reduce Environmental Impact

The C4 Urochloa spp (syn. Brachiaria) and Megathyrsus maximus (syn. Panicum maximum) are used as pasture for cattle across vast areas in tropical agriculture systems in Africa and South America. A key target for variety improvement is forage quality: enhanced digestibility could decrease amount of land required per unit production and enhanced lipid content could decrease methane emissions from cattle. For these traits, loss-of-function (LOF) alleles in known gene targets are predicted to improve them, making a reverse genetics approach of allele mining feasible. We studied allelic diversity of 20 target genes (11 for digestibility, 9 for lipid content) in 104 accessions selected to represent genetic diversity and ploidy levels of U. brizantha, U. decumbens, U. humidicola, U. ruziziensis and M. maximum. We used RNAseq and then bait-capture DNA-seq to improve gene models in a U. ruziziensis reference genome to assign polymorphisms with high confidence. We found 953 non-synonymous polymorphisms across all genes and accessions; within these, we identified 7 putative LOF alleles with high confidence, including ones in the non-redundant SDP1 and BAHD01 genes present in diploid and tetraploid accessions. These LOF alleles could respectively confer increased lipid content and digestibility if incorporated into a breeding programme.HighlightWe found gene variants in a collection of tropical grasses that could help reduce environmental impact of cattle production.

Identification of Novel Alleles of the Rice Blast-Resistance Gene Pi9 through Sequence-Based Allele Mining

Background As rice (Oryza sativa) is the staple food of more than half the world’s population, rice production contributes greatly to global food security. Rice blast caused by the fungus Magnaporthe oryzae (M. oryzae) is a devastating disease that affects rice yields and grain quality, resulting in substantial economic losses annually. Because the fungus evolves rapidly, the resistance conferred by most the single blast-resistance genes is broken after a few years of intensive agricultural use. Therefore, effective resistance breeding in rice requires continual enrichment of the reservoir of resistance genes, alleles, or QTLs. Seed banks represent a rich source of genetic diversity; however, they have not been extensively used to identify novel genes and alleles. Results We carried out a large-scale screen for novel blast-resistance alleles in 1883 rice varieties from major rice-producing areas across China. Of these, 361 varieties showed at least moderate resistance to natural infection by rice blast at rice blast nurseries in Enshi and Yichang, Hubei Province. We used sequence-based allele mining to amplify and sequence the allelic variants of the major rice blast-resistance genes at the Pi2/Pi9 locus of chromosome 6 from the 361 blast-resistant varieties, and the full-length coding region of this gene could be amplified from 107 varieties. Thirteen novel Pi9 alleles (named Pi9-Type1 to Pi9-Type13) were identified in these 107 varieties based on comparison to the Pi9 referenced sequence. Based on the sequencing results, the Pi2/Pi9 locus of the 107 varieties was divided into 15 genotypes (including three different genotypes of Pi9-Type5). Fifteen varieties, each representing one genotype, were evaluated for resistance to 34 M. oryzae isolates. The alleles from seven varieties with the highest resistance and widest resistance spectra were selected for transformation into the susceptible variety J23B to construct near-isogenic lines (NILs). These NILs showed resistance in a field test in Enshi and Yichang, indicating that the seven novel rice blast-resistance tandem-repeat regions at the Pi2/Pi9 locus of chromosome 6 could potentially serve as a genetic resource for molecular breeding of resistance to rice blast. Conclusions The thirteen novel Pi9 alleles identified in this study expand the list of available of blast-resistance alleles. Seven tandem-repeat regions of the Pi2/Pi9 locus from different donors were characterized as broad-spectrum rice blast-resistance fragments; these donors enrich the genetic resources available for rice blast-resistance breeding programs.

Identification of Novel Loci and Candidate Genes for Cucumber Downy Mildew Resistance Using GWAS

Downy mildew (DM) is one of the most serious diseases in cucumber. Multiple quantitative trait loci (QTLs) for DM resistance have been detected in a limited number of cucumber accessions. In this study we applied genome-wide association analysis (GWAS) to detected genetic loci for DM resistance in a core germplasm (CG) of cucumber lines that represent diverse origins and ecotypes. Phenotypic data on responses to DM infection were collected in four field trials across three years, 2014, 2015, and 2016. With the resequencing data of these CG lines, GWAS for DM resistance was performed and detected 18 loci that were distributed on all the seven cucumber chromosomes. Of these 18 loci, only six (dmG1.4, dmG4.1, dmG4.3, dmG5.2, dmG7.1, and dmG7.2) were detected in two experiments, and were considered as loci with a stable effect on DM resistance. Further, 16 out of the 18 loci colocalized with the QTLs that were reported in previous studies and two loci, dmG2.1 and dmG7.1, were novel ones identified only in this study. Based on the annotation of homologous genes in Arabidopsis and pairwise LD correlation analysis, several candidate genes were identified as potential causal genes underlying the stable and novel loci, including Csa1G575030 for dmG1.4, Csa2G060360 for dmG2.1, Csa4G064680 for dmG4.1, Csa5G606470 for dmG5.2, and Csa7G004020 for dmG7.1. This study shows that the CG germplasm is a very valuable resource carrying known and novel QTLs for DM resistance. The potential of using these CG lines for future allele-mining of candidate genes was discussed in the context of breeding cucumber with resistance to DM.