A multitiered haplotype strategy to enhance phased assembly and fine mapping of a disease resistance locus.

Fine mapping of quantitative trait loci (QTL) to dissect the genetic basis of traits of interest is essential to modern breeding practice. Here, we employed a multitiered haplotypic marker system to increase fine mapping accuracy by constructing a chromosome-level, haplotype-resolved parental genome, accurate detection of recombination sites, and allele-specific characterization of the transcriptome. In the first tier of this system, we applied the preexisting panel of 2,000 rhAmpSeq core genome markers that is transferable across the entire Vitis genus and provides a genomic resolution of 200 kb to 1 Mb. The second tier consisted of high-density haplotypic markers generated from Illumina skim sequencing data for samples enriched for relevant recombinations, increasing the potential resolution to hundreds of base pairs. We used this approach to dissect a novel Resistance to Plasmopara viticola-33 (RPV33) locus conferring resistance to grapevine downy mildew, narrowing the candidate region to only 0.46 Mb. In the third tier, we used allele-specific RNA-seq analysis to identify a cluster of 3 putative disease resistance RPP13-like protein 2 genes located tandemly in a nonsyntenic insertion as candidates for the disease resistance trait. In addition, combining the rhAmpSeq core genome haplotype markers and skim sequencing-derived high-density haplotype markers enabled chromosomal-level scaffolding and phasing of the grape Vitis × doaniana 'PI 588149' assembly, initially built solely from Pacific Biosciences (PacBio) high-fidelity (HiFi) reads, leading to the correction of 16 large-scale phasing errors. Our mapping strategy integrates high-density, phased genetic information with individual reference genomes to pinpoint the genetic basis of QTLs and will likely be widely adopted in highly heterozygous species.

Diverse Chromosomal Locations of Quantitative Trait Loci for Tolerance to Maize chlorotic mottle virus in Five Maize Populations.

The recent rapid emergence of maize lethal necrosis (MLN), caused by coinfection of maize with Maize chlorotic mottle virus (MCMV) and a second virus usually from the family Potyviridae, is causing extensive losses for farmers in East Africa, Southeast Asia, and South America. Although the genetic basis of resistance to potyviruses is well understood in maize, little was known about resistance to MCMV. The responses of five maize inbred lines (KS23-5, KS23-6, N211, DR, and Oh1VI) to inoculation with MCMV, Sugarcane mosaic virus, and MLN were characterized. All five lines developed fewer symptoms than susceptible controls after inoculation with MCMV; however, the virus was detected in systemic leaf tissue from each of the lines similarly to susceptible controls, indicating that the lines were tolerant of MCMV rather than resistant to it. Except for KS23-5, the inbred lines also developed fewer symptoms after inoculation with MLN than susceptible controls. To identify genetic loci associated with MCMV tolerance, large F2 or recombinant inbred populations were evaluated for their phenotypic responses to MCMV, and the most resistant and susceptible plants were genotyped by sequencing. One to four quantitative trait loci (QTL) were identified in each tolerant population using recombination frequency and positional mapping strategies. In contrast to previous studies of virus resistance in maize, the chromosomal positions and genetic character of the QTL were unique to each population. The results suggest that different, genotype-specific mechanisms are associated with MCMV tolerance in maize. These results will allow for the development of markers for marker-assisted selection of MCMV- and MLN-tolerant maize hybrids for disease control.

Genotyping-by-sequencing highlights original diversity patterns within a European collection of 1191 maize flint lines, as compared to the maize USDA genebank.

KEY MESSAGE: Genotyping by sequencing is suitable for analysis of global diversity in maize. We showed the distinctiveness of flint maize inbred lines of interest to enrich the diversity of breeding programs. Genotyping-by-sequencing (GBS) is a highly cost-effective procedure that permits the analysis of large collections of inbred lines. We used it to characterize diversity in 1191 maize flint inbred lines from the INRA collection, the European Cornfed association panel, and lines recently derived from landraces. We analyzed the properties of GBS data obtained with different imputation methods, through comparison with a 50 K SNP array. We identified seven ancestral groups within the Flint collection (dent, Northern flint, Italy, Pyrenees-Galicia, Argentina, Lacaune, Popcorn) in agreement with breeding knowledge. Analysis highlighted many crosses between different origins and the improvement of flint germplasm with dent germplasm. We performed association studies on different agronomic traits, revealing SNPs associated with cob color, kernel color, and male flowering time variation. We compared the diversity of both our collection and the USDA collection which has been previously analyzed by GBS. The population structure of the 4001 inbred lines confirmed the influence of the historical inbred lines (B73, A632, Oh43, Mo17, W182E, PH207, and Wf9) within the dent group. It showed distinctly different tropical and popcorn groups, a sweet-Northern flint group and a flint group sub-structured in Italian and European flint (Pyrenees-Galicia and Lacaune) groups. Interestingly, we identified several selective sweeps between dent, flint, and tropical inbred lines that co-localized with SNPs associated with flowering time variation. The joint analysis of collections by GBS offers opportunities for a global diversity analysis of maize inbred lines.

Switchgrass genomic diversity, ploidy, and evolution: novel insights from a network-based SNP discovery protocol.

Switchgrass (Panicum virgatum L.) is a perennial grass that has been designated as an herbaceous model biofuel crop for the United States of America. To facilitate accelerated breeding programs of switchgrass, we developed both an association panel and linkage populations for genome-wide association study (GWAS) and genomic selection (GS). All of the 840 individuals were then genotyped using genotyping by sequencing (GBS), generating 350 GB of sequence in total. As a highly heterozygous polyploid (tetraploid and octoploid) species lacking a reference genome, switchgrass is highly intractable with earlier methodologies of single nucleotide polymorphism (SNP) discovery. To access the genetic diversity of species like switchgrass, we developed a SNP discovery pipeline based on a network approach called the Universal Network-Enabled Analysis Kit (UNEAK). Complexities that hinder single nucleotide polymorphism discovery, such as repeats, paralogs, and sequencing errors, are easily resolved with UNEAK. Here, 1.2 million putative SNPs were discovered in a diverse collection of primarily upland, northern-adapted switchgrass populations. Further analysis of this data set revealed the fundamentally diploid nature of tetraploid switchgrass. Taking advantage of the high conservation of genome structure between switchgrass and foxtail millet (Setaria italica (L.) P. Beauv.), two parent-specific, synteny-based, ultra high-density linkage maps containing a total of 88,217 SNPs were constructed. Also, our results showed clear patterns of isolation-by-distance and isolation-by-ploidy in natural populations of switchgrass. Phylogenetic analysis supported a general south-to-north migration path of switchgrass. In addition, this analysis suggested that upland tetraploid arose from upland octoploid. All together, this study provides unparalleled insights into the diversity, genomic complexity, population structure, phylogeny, phylogeography, ploidy, and evolutionary dynamics of switchgrass.

Extensive intraspecific gene order and gene structural variations between Mo17 and other maize genomes.

Maize is an important crop with a high level of genome diversity and heterosis. The genome sequence of a typical female line, B73, was previously released. Here, we report a de novo genome assembly of a corresponding male representative line, Mo17. More than 96.4% of the 2,183 Mb assembled genome can be accounted for by 362 scaffolds in ten pseudochromosomes with 38,620 annotated protein-coding genes. Comparative analysis revealed large gene-order and gene structural variations: approximately 10% of the annotated genes were mutually nonsyntenic, and more than 20% of the predicted genes had either large-effect mutations or large structural variations, which might cause considerable protein divergence between the two inbred lines. Our study provides a high-quality reference-genome sequence of an important maize germplasm, and the intraspecific gene order and gene structural variations identified should have implications for heterosis and genome evolution.