Nested association mapping of stem rust resistance in wheat using genotyping by sequencing

We combined the recently developed genotyping by sequencing (GBS) method with joint mapping (also known as nested association mapping) to dissect and understand the genetic architecture controlling stem rust resistance in wheat (Triticum aestivum). Ten stem rust resistant wheat varieties were crossed to the susceptible line LMPG-6 to generate F6 recombinant inbred lines. The recombinant inbred line populations were phenotyped in Kenya, South Africa, and St. Paul, Minnesota, USA. By joint mapping of the 10 populations, we identified 59 minor and medium-effect QTL (explained phenotypic variance range of 1%- 20%) on 20 chromosomes that contributed towards adult plant resistance to North American Pgt races as well as the highly virulent Ug99 race group. Fifteen of the 59 QTL were detected in multiple environments. No epistatic relationship was detected among the QTL. While these numerous small- to medium-effect QTL are shared among the families, the founder parents were found to have different allelic effects for the QTL. Fourteen QTL identified by joint mapping were also detected in single-population mapping. As these QTL were mapped using SNP markers with known locations on the physical chromosomes, the genomic regions identified with QTL could be explored more in depth to discover candidate genes for stem rust resistance. The use of GBS-derived de novo SNPs in mapping resistance to stem rust shown in this study could be used as a model to conduct similar markertrait association studies in other plant species.This is an open access article, free of all copiright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication

Transcriptome characterization and polymorphism detection between subspecies of big sagebrush (Artemisia tridentata)

<p>Abstract</p> <p>Background</p> <p>Big sagebrush (<it>Artemisia tridentata</it>) is one of the most widely distributed and ecologically important shrub species in western North America. This species serves as a critical habitat and food resource for many animals and invertebrates. Habitat loss due to a combination of disturbances followed by establishment of invasive plant species is a serious threat to big sagebrush ecosystem sustainability. Lack of genomic data has limited our understanding of the evolutionary history and ecological adaptation in this species. Here, we report on the sequencing of expressed sequence tags (ESTs) and detection of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in subspecies of big sagebrush.</p> <p>Results</p> <p>cDNA of <it>A. tridentata </it>sspp. <it>tridentata </it>and <it>vaseyana </it>were normalized and sequenced using the 454 GS FLX Titanium pyrosequencing technology. Assembly of the reads resulted in 20,357 contig consensus sequences in ssp. <it>tridentata </it>and 20,250 contigs in ssp. <it>vaseyana</it>. A BLASTx search against the non-redundant (NR) protein database using 29,541 consensus sequences obtained from a combined assembly resulted in 21,436 sequences with significant blast alignments (≤ 1e^-15). A total of 20,952 SNPs and 119 polymorphic SSRs were detected between the two subspecies. SNPs were validated through various methods including sequence capture. Validation of SNPs in different individuals uncovered a high level of nucleotide variation in EST sequences. EST sequences of a third, tetraploid subspecies (ssp. <it>wyomingensis</it>) obtained by Illumina sequencing were mapped to the consensus sequences of the combined 454 EST assembly. Approximately one-third of the SNPs between sspp. <it>tridentata </it>and <it>vaseyana </it>identified in the combined assembly were also polymorphic within the two geographically distant ssp. <it>wyomingensis </it>samples.</p> <p>Conclusion</p> <p>We have produced a large EST dataset for <it>Artemisia tridentata</it>, which contains a large sample of the big sagebrush leaf transcriptome. SNP mapping among the three subspecies suggest the origin of ssp. <it>wyomingensis </it>via mixed ancestry. A large number of SNP and SSR markers provide the foundation for future research to address questions in big sagebrush evolution, ecological genetics, and conservation using genomic approaches.</p

Characterization of Genetic Resistance to Fusarium Head Blight and Bacterial Leaf Streak in Intermediate Wheatgrass (Thinopyrum intermedium)

Intermediate wheatgrass (IWG, Thinopyrum intermedium, (Host) Barkworth &amp; D.R. Dewey subsp. intermedium, 2n = 6x = 42) is a novel perennial crop currently undergoing domestication efforts. It offers remarkable ecosystem services and yields higher relative to other perennial grain crops. While IWG is mostly resistant to Fusarium head blight (FHB), identifying genomic regions associated with resistance will help protect the crop from potential disease epidemics. An IWG biparental population of 108 individuals was developed by crossing parents differing in their response to FHB and bacterial leaf streak (BLS). The population was screened for disease reaction over three years using isolates collected from IWG plants in St. Paul, Minnesota, USA. Linkage maps representing the 21 IWG chromosomes were constructed from 4622 Single Nucleotide Polymorphism (SNP) markers, with one SNP at every 0.74 cM. Interval mapping identified 15 quantitative trait loci (QTL) associated with FHB resistance and 11 with BLS resistance. Models with two or three QTL combinations reduced FHB disease severity by up to 15%, and BLS by up to 17%. When markers associated with FHB resistance were used as cofactors in genomic selection models, trait predictive ability improved by 24&ndash;125%. These genomic regions and genetic markers associated with FHB and BLS resistance can also be used to safeguard annual cereal grains through gene introgression and selective breeding

Multi-Allelic Haplotype-Based Association Analysis Identifies Genomic Regions Controlling Domestication Traits in Intermediate Wheatgrass

Intermediate wheatgrass (IWG) is a perennial forage grass undergoing a rigorous domestication as a grain crop. As a young grain crop, several agronomic and domestication traits need improvement for IWG to be relevant in current agricultural landscapes. This study genetically maps six domestication traits in the fourth cycle IWG breeding population at the University of Minnesota: height, seed length, seed width, shattering, threshability, and seed mass. A weak population structure was observed and linkage disequilibrium (r2) declined rapidly: 0.23 mega base pairs at conventional r2 value of 0.2. Broad-sense heritabilities were overall high and ranged from 0.71–0.92. Association analysis was carried out using 25,909 single SNP markers and 5379 haplotype blocks. Thirty-one SNP markers and 17 haplotype blocks were significantly associated with the domestication traits. These associations were of moderate effect as they explained 4–6% of the observed phenotypic variation. Ten SNP markers were also detected by the haplotype association analysis. One SNP marker on Chromosome 8, also discovered in haplotype block analysis, was common between seed length and seed mass. Increasing the frequency of favorable alleles in IWG populations via marker-assisted selection and genomic selection is an effective approach to improve IWG’s domestication traits

Data from: Deep sequencing of amplicons reveals widespread intraspecific hybridization and multiple origins of polyploidy in big sagebrush (Artemisia tridentata)

PREMISE OF THE STUDY: Hybridization has played an important role in the evolution and ecological adaptation in diploid and polyploid plants. Artemisia tridentata (Asteraceae) tetraploids are extremely widespread and of great ecological importance. These tetraploids are often taxonomically identified as A. tridentata ssp. wyomingensis, or as autotetraploids of diploid subspecies tridentata and vaseyana. Few details are available as to how these tetraploids are formed or how they are related to diploid subspecies. METHODS: We used amplicon sequencing to assess phylogenetic relationships among three recognized subspecies: tridentata, vaseyana and wyomingensis. DNA sequence data from putative genes were pyrosequenced and assembled from 329 samples. Nucleotide diversity and putative haplotypes were estimated from the high-read coverage. Phylogenies were constructed from Bayesian coalescence and neighbor-net network analyses. KEY RESULTS: Analyses support distinct diploid subspecies of tridentata and vaseyana in spite of known hybridization in ecotones. Nucleotide diversity estimates of populations compared to the total diversity indicate the relationships are predominately driven by a small proportion of the amplicons. Tetraploids, including subspecies wyomingenesis, are polyphyletic occurring within and between diploid subspecies groups. CONCLUSIONS: Artemisia tridentata is a species comprised of phylogenetically distinct diploid progenitors and a tetraploid complex with varying degrees of phylogenetic and morphological affinities to the diploid subspecies. These analyses suggest tetraploids are formed locally or regionally from diploid tridentata and vaseyana populations via autotetraploidy, followed by introgression between tetraploid groups. Understanding the phylogenetic versus ecological relationships of A. tridentata subspecies will have bearing on how to restore these desert ecosystems

Alignment of Artemisia tridentata consensus sequences

Concatenated consensus sequence of 25 amplicons from 49 populations including diploid and tetraploid populations. Readme file describes the populations

Recurrent parent genome recovery in the BC₂F₂ generation using 40,584 genome-wide SNPs.

Recurrent parent genome recovery in the BC2F2 generation using 40,584 genome-wide SNPs.</p

PCA plot of rye chromosome 2R using 15,116 SNPs from GBS reads.

A) Resistant parental lines (SLU238, SLU239, and TA5094) with chromosome 2R; B) BC2F2 population comprising recurrent parents carrying chromosome 2RL close to SLU238 and TA5094; C) BC2F2 population derived from recurrent parents carrying the chromosome 2RL segment; D) recurrent parents (BAJ #1, KACHU #1, and REEDLING #1) and the susceptible BC2F2 population without chromosome 2RL; E) lines CSph1bM and CSA.</p

Physical location of rye chromosome 2R based on GBS data and seedling responses to stem rust races TTTTF and TTKSK.

a) Seedling assay for race TTTTF in the BC2F3 population, where red denotes resistance and green susceptibility; b) physical positions of the 15,116 SNPs from GBS reads in the BC2F2 population, where red denotes the rye allele and dark blue the wheat allele; c) seedling assay for race TTKSK in the BC2F3 population, where red denotes resistance and green susceptibility.</p

Molecular characterization of genomic regions for adult plant resistance to stem rust in a spring wheat mapping population

Stem rust caused by Puccinia graminis Pers.:Pers. f. sp. tritici Erikss. & E. Henn. is an important disease of bread (Triticum aestivum L.) and durum (T. turgidum spp. durum Desf.) wheat in Ethiopia. The frequent emergence of new P. graminis f. sp. tritici virulent races remains a major constraint to wheat production. Wheat stem rust surveys are carried out annually during both the main (late August through October) and off-seasons (June and July) in Ethiopia. During the annual 2017 main season survey, a total of 60 stem rust samples were collected from farmer’s field in the major wheat growing areas of Oromia and Amhara Regions. Forty-eight single pustule isolates were derived from these samples at Ambo Plant Protection Research Center (Ethiopia) and multiplied on susceptible wheat cultivar McNair 701. Physiological race analysis was performed using the 20 North American stem rust differential lines following the standard protocol of Jin et al. (2008). Race analysis was repeated up to seven times until the race was confirmed. Ten isolates collected from different fields in four zones were determined to be race TTRTF (Supplementary Table S1). P. graminis f. sp. tritici race TTRTF was virulent to wheat differential lines containing Sr5, Sr21, Sr9e, Sr7b, Sr11, Sr6, Sr8a, Sr9g, Sr36, Sr9b, Sr17, Sr9a, Sr9d, Sr10, SrTmp, Sr38, SrMcN and avirulent on lines with Sr30, Sr24, Sr31 (Supplemental Table S2). Isolates belonging to this race were derived from stem rust samples from commercial wheat cultivars Digelu, Danda’a, Pavon 76, and Israel, as well as Triticale. Urediniospores of the 48 isolates were sent to the Cereal Disease Laboratory, St. Paul, MN for genotyping. DNA was extracted from spores (Olivera et al. 2015) and genotyped using 17 selected single nucleotide polymorphism markers (Szabo unpublished data). All isolates that were race typed as TTRTF were genotyped as MLG.04 (Clade III-B) and were identical to reference isolates (Supplemental Fig S1). P. graminis f. sp. tritici race TTRTF was first identified from wheat stem rust collections made in 2014 from Akhalkalaki, Georgia (Olivera et al. 2019). In addition to the virulences described above, P. graminis f. sp. tritici race TTRTF has virulence to several other important stem rust resistance genes but, these were not tested. Severe epidemics of wheat stem rust on durum wheat in Italy in 2016 and 2017 were caused by race P. graminis f. sp. tritici TTRTF (Patpour et al., 2018). The same race has also been reported in Hungary (Olivera et al. 2019) and Egypt (Samar and Szabo, 2018). However, this is the first confirmation of P. graminis f. sp. tritici race TTRTF in Ethiopia. These results indicate P. graminis f. sp. tritici TTRTF with complex virulence is spreading rapidly and has now become established in Ethiopia. Further spread is considered likely and close monitoring is required