GBS-SNP-CROP: a reference-optional pipeline for SNP discovery and plant germplasm characterization using variable length, paired-end genotyping-by-sequencing data

Background: With 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. Results: The 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. Conclusions: By 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.gi

Barberry plays an active role as an alternate host of Puccinia graminis in Spain

Stem rust, caused by Puccinia graminis, is a destructive group of diseases. The patho - gen 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 ob- served 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. graminis f. sp. tritici, f. sp. secalis and f. sp. avenae. Sequence analyses from aecial samples sup- ported 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 SpainThe research was supported by the United States Department of Agriculture‐Agricultural Research Service, the Bill and Melinda Gates Foundation and the United Kingdom Department for International Development managed by Cornell University, USA, grant number: DGGW‐OPP1133199, and the State Research Agency (AEI), Spain, project PID2020‐118650RR‐C31. Thanks to Dr Fanny Álvaro for the initial observation and sampling of aecial infection on barberry. The assistance of Dr Joan Pedrol and Dr Rosario Fanlo (University of Lleida, Spain) in grass species identification is gratefully acknowledged. We thank the assistance of Amparo López Olmeda (Teruel Plant Health Service), Gloria Herrero Sánchez (Cereales Teruel Sociedad Cooperativa) and Vicente Lozano Herrera (farmer cereal producer). Meteorological data from the El Ballestero site (Albacete province) were kindly provided by Manuel Fernández. Data from surface cropping area of Aragón have been provided by Sección de estadística del Departamento de Agricultura, Ganadería y Medio Ambiente del Gobierno de Aragón (Silvia Quílez‐Domingo). The support of Daniel Gómez and Alberto Pastoriza (Herbarium curators in the Pyrenean Institute of Ecology, IPE‐CSIC, Jaca) is thankfully acknowledged. Technical assistance of Melissa Carter, Jerry Johnson and Kim‐Phuong Nguyen from USDA‐ARS is acknowledged. Dr Villegas acknowledges the contribution of the CERCA program (Generalitat de Catalunya, Spain)

Barberry plays an active role as an alternate host of Puccinia graminis in Spain

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. graminis f. 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.info:eu-repo/semantics/publishedVersio

Barberry plays an active role as an alternate host of Puccinia graminis in Spain

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. graminis f. 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

Genetic Dissection of Non-host Resistance to the Wheat Stem Rust Pathogen, Using an Interspecific Barberry Hybrid

Stem rust, caused by the macrocyclic fungal pathogen P. graminis (Pg), is one of the most devastating diseases of wheat and other small grains globally; and the emergence of new stem rust races virulent on deployed resistance genes brings urgency to the discovery of more durable sources of genetic resistance. Given its intrinsic durability and effectiveness across a broad range of pathogens, non-host resistance (NHR) presents a compelling strategy for achieving long-term rust control in wheat. However, NHR to Pg (Pg-NHR) remains largely unexplored as a protection strategy in wheat, in part due to the challenge of developing a genetically tractable system in which Pg-NHR segregates. In this dissertation, an investigation of Pg-NHR is undertaken via the pathogen's alternate (sexual) host, barberry ( Berberis spp.). Within the highly diverse Berberis genus, numerous species function as alternate hosts to Pg but others are non-hosts. European barberry (B. vulgaris L.), for example, is susceptible to Pg infection but Japanese barberry (B. thunbergii DC.) is a non-host. In this study, the nothospecies B. ×ottawensis C.K. Scheid, an inter-specific hybrid between Pg-susceptible B. vulgaris and Pg-resistant B. thunbergii, is explored as a possible means of mapping the gene(s) underlying the apparent Pg-NHR exhibited by B. thunbergii. The overall goal of this research is to contribute to the global search for novel sources of potentially durable stem rust resistance genes. The first chapter describes a field study conducted in western Massachusetts, in which a natural population of B. ×ottawensis was characterized to determine if the hybrid can be used to genetically dissect the Pg-NHR exhibited by B. thunbergii. A population of 63 B. ×ottawensis individuals were clonally propagated, phenotyped for disease response to Pg via controlled inoculation using overwintered telia of Pg found on naturally infected E. repens, and genotyped using the de novo genotyping-by-sequencing (GBS) pipeline GBS-SNP-CROP. Controlled inoculation of a subset of 53 B. ×ottawensis accessions, verified via GBS to be true, first-generation hybrids, revealed 51% susceptible, 33% resistant, and 16% intermediate phenotypes. Although such variation in disease response within a natural population of F1 hybrids could be explained by non-nuclear (cytoplasmic) inheritance of resistance, a similar pattern of segregation was observed in a population of B. ×ottawensis full-sibs, developed via controlled crosses. The results of this first chapter demonstrate not only that the Pg-NHR observed in B. thunbergii segregates among F1 interspecific hybrids with Pg-susceptible B. vulgaris but that the resistance is likely nuclearly inherited. Therefore, at least in principle, the gene(s) underlying Pg-NHR in B. thunbergii should be mappable in an F1 population derived from the controlled hybridization of the two parental species. Building on the results of first chapter, the second chapter of this dissertation details the generation and use of a bi-parental B. ×ottawensis mapping population to develop genetic linkage maps for both parental species and begin mapping the gene(s) underlying Pg-NHR in B. thunbergii. Using 162 full-sib F1 hybrids and a total of 15,411 sequence variants (SNPs and indels) identified between the parents via GBS, genetic linkage maps with 1,757 and 706 markers were constructed for B. thunbergii accession 'BtUCONN1' and B. vulgaris accession 'Wagon Hill', respectively. In each map, the markers segregated into 14 linkage groups, in agreement with the 14 chromosomes present in these Berberis spp. The total lengths of the linkage maps were 1474 cM (B. thunbergii) and 1714 cM (B. vulgaris), with average distances between markers of 2.6 cM and 5.5 cM. QTL analysis for Pg resistance led to the identification of a single QTL, dubbed QPgr-3S, on the short arm of chromosome 3 of B. thunbergii. The peak LOD score of QPgr-3S is 28.2, and the QTL spans 13 cM, bounded by the distal SNP marker M411 and proximal SNP marker M969. To gain further insight into the QPgr-3S region, a chromosome-level 1.2 Gb draft genome for B. thunbergii was assembled using long PacBio reads and Hi-C data. By anchoring the B. thunbergii linkage map to the draft genome, the 13 cM Q Pgr-3S region was found to correspond to ~3.4 Mbp, represented by 10 contigs. Using a 189.3 Mb transcriptome assembled from a multiple tissue library of RNA-seq data, the QPgr-3S region was found to contain 99 genes. To help narrow this list to candidate genes of highest priority for subsequent investigation, a combination of approaches was taken. Specifically, annotation of the QTL region and differential gene expression analysis led to the identification of 12 candidate genes within the region. (Abstract shortened by ProQuest.

Additional file 3: of GBS-SNP-CROP: a reference-optional pipeline for SNP discovery and plant germplasm characterization using variable length, paired-end genotyping-by-sequencing data

The distribution of pre-filtered SNPs across three different depth classes: Low (<4), Acceptable (4–200), and Over-represented (>200). The bar plot in AdditionalFile3.pdf compares the distributions of average read depths for the pre-filtered SNPs called by the five evaluated pipelines. (PDF 43 kb

Additional file 2: of GBS-SNP-CROP: a reference-optional pipeline for SNP discovery and plant germplasm characterization using variable length, paired-end genotyping-by-sequencing data

Bubble plot showing the pair-wise percentage of shared markers called by the different pipelines. AdditionalFile2.pdf complements the visualization in Fig. 3, showing all pair-wise proportions of shared SNPs among the five evaluated pipelines. (PDF 732 kb

Additional file 4: of GBS-SNP-CROP: a reference-optional pipeline for SNP discovery and plant germplasm characterization using variable length, paired-end genotyping-by-sequencing data

List of the 48 A. arguta genotypes used for GBS-SNP-CROP development and analysis. AdditionalFile4.pdf presents the names of the genotypes from the USDA National Clonal Germplasm Repository used in this study, along with their barcodes and the number of parsed GBS reads obtained for each. (PDF 50 kb

A unique race of the wheat stem rust pathogen with virulence on Sr31 identified in Spain and reaction of wheat and durum cultivars to this race

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.info:eu-repo/semantics/acceptedVersio