BackgroundWith its simple library preparation and robust approach to genome reduction, genotyping-by-sequencing (GBS) is a flexible and cost-effective strategy for SNP discovery and genotyping, provided an appropriate reference genome is available. For resource-limited curation, research, and breeding programs of underutilized plant genetic resources, however, even low-depth references may not be within reach, despite declining sequencing costs. Such programs would find value in an open-source bioinformatics pipeline that can maximize GBS data usage and perform high-density SNP genotyping in the absence of a reference.ResultsThe GBS SNP-Calling Reference Optional Pipeline (GBS-SNP-CROP) developed and presented here adopts a clustering strategy to build a population-tailored “Mock Reference” from the same GBS data used for downstream SNP calling and genotyping. Designed for libraries of paired-end (PE) reads, GBS-SNP-CROP maximizes data usage by eliminating unnecessary data culling due to imposed read-length uniformity requirements. Using 150 bp PE reads from a GBS library of 48 accessions of tetraploid kiwiberry (Actinidia arguta), GBS-SNP-CROP yielded on average three times as many SNPs as TASSEL-GBS analyses (32 and 64 bp tag lengths) and over 18 times as many as TASSEL-UNEAK, with fewer genotyping errors in all cases, as evidenced by comparing the genotypic characterizations of biological replicates. Using the published reference genome of a related diploid species (A. chinensis), the reference-based version of GBS-SNP-CROP behaved similarly to TASSEL-GBS in terms of the number of SNPs called but had an improved read depth distribution and fewer genotyping errors. Our results also indicate that the sets of SNPs detected by the different pipelines above are largely orthogonal to one another; thus GBS-SNP-CROP may be used to augment the results of alternative analyses, whether or not a reference is available.ConclusionsBy achieving high-density SNP genotyping in populations for which no reference genome is available, GBS-SNP-CROP is worth consideration by curators, researchers, and breeders of under-researched plant genetic resources. In cases where a reference is available, especially if from a related species or when the target population is particularly diverse, GBS-SNP-CROP may complement other reference-based pipelines by extracting more information per sequencing dollar spent. The current version of GBS-SNP-CROP is available at https://github.com/halelab/GBS-SNP-CROP.gitElectronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-0879-y) contains supplementary material, which is available to authorized users.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a re‐emerging disease, posing a threat to wheat production. In Spain, stem rust has been rarely detected since the 1970s, but infection was observed in wheat fields in 2018. We analysed six stem rust samples collected in Rota, Cádiz province and one from Monteagudo del Castillo, Teruel province. All the samples from Rota were typed as race TKTTF, whereas the sample from Monteagudo del Castillo, collected in a wheat field adjacent to barberry bushes, was typed as race TKHBK. This race has a unique and significant virulence combination that includes virulence to Sr31, Sr33, Sr53 and Sr59, and is avirulent to Rusty, a durum line developed for universal susceptibility to the wheat stem rust pathogen. TKHBK is the first race outside the Ug99 race group with virulence to Sr31 and the first known race with virulence to Sr59. Genotyping studies indicate that race TKHBK does not belong to the Ug99 or TKTTF race groups and constitutes a previously unknown lineage. Two hundred bread and durum wheat cultivars and breeding lines from Spain were evaluated against TKHBK, TKTTF, and six additional races. Resistance was observed to all the races evaluated. Molecular markers confirmed the presence of Sr7a, Sr24, Sr31, Sr38 and Sr57 in bread wheat, and Sr13 in durum wheat. The re‐emergence of wheat stem rust in Spain and the occurrence of unique virulences underscore the need to continue surveying and monitoring this disease.

Stem rust, caused by Puccinia graminis, is a destructive group of diseases. The pathogen uses Berberis species as alternate hosts to complete its life cycle. B. vulgaris and the endemic species B. hispanica and B. garciae are present in Spain. The objective of this study was to investigate the functionality of the indigenous barberry as alternate hosts. Field surveys were conducted in 2018 and 2019 in Huesca, Teruel and Albacete provinces of Spain. Aecial samples on barberry were analysed via infection assays and DNA analysis. B. garciae was predominant in Huesca and Teruel provinces, often found in the field margins of cereal crops. Aecial infections on B. garciae were observed in May and uredinial infections on cereal crops in June. Scattered B. hispanica bushes were occasionally found near cereal crops in Albacete, where aecial infections on B. hispanica were observed in June when most cereal crops were mature. Infection assays using aeciospores resulted in stem rust infections on susceptible genotypes of wheat, barley, rye and oat, indicating the presence of the sexual cycle for P. graminisf. sp. tritici, f. sp. secalis and f. sp. avenae. Sequence analyses from aecial samples supported this finding as well as the presence of Puccinia brachypodii. This study provides the first evidence that indigenous Berberis species play an active role in the sexual cycle of P. graminis under natural conditions in Spain.

This study demonstrates for the first time the feasibility of dissecting the genetic mechanism of non-host resistance to the stem rust pathogen in its ancestral host genus Berberis.

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