Major Resistance Locus Research Articles

Broad-spectrum fungal resistance in sorghum is conferred through the complex regulation of an immune receptor gene embedded in a natural antisense transcript.

Sorghum (Sorghum bicolor), the fifth most widely grown cereal crop globally, provides food security for millions of people. Anthracnose caused by the fungus Colletotrichum sublineola is a major disease of sorghum worldwide. We discovered a major fungal resistance locus in sorghum composed of the nucleotide-binding leucine-rich repeat receptor gene ANTHRACNOSE RESISTANCE GENE1 (ARG1) that is completely nested in an intron of a cis-natural antisense transcript (NAT) gene designated CARRIER OF ARG1 (CARG). Susceptible genotypes express CARG and two alternatively spliced ARG1 transcripts encoding truncated proteins lacking the leucine-rich repeat domains. In resistant genotypes, elevated expression of an intact allele of ARG1, attributed to the loss of CARG transcription and the presence of miniature inverted-repeat transposable element sequences, resulted in broad-spectrum resistance to fungal pathogens with distinct virulence strategies. Increased ARG1 expression in resistant genotypes is also associated with higher histone H3K4 and H3K36 methylation. In susceptible genotypes, lower ARG1 expression is associated with reduced H3K4 and H3K36 methylation and increased expression of NATs of CARG. The repressive chromatin state associated with H3K9me2 is low in CARG-expressing genotypes within the CARG exon and higher in genotypes with low CARG expression. Thus, ARG1 is regulated by multiple mechanisms and confers broad-spectrum, strong resistance to fungal pathogens.

Transcriptome Analysis of Eucalyptus grandis Implicates Brassinosteroid Signaling in Defense Against Myrtle Rust (Austropuccinia psidii)

Eucalyptus grandis, in its native Australian range, varies in resistance to Austropuccinia psidii (syn. Puccinia psidii). The biotrophic rust fungus, A. psidii is the causal agent of myrtle rust and poses a serious threat to Australian biodiversity. The pathogen produces yellow pustules of urediniospores on young leaves and shoots, resulting in shoot tip dieback, stunted growth, and death. Dissecting the underlying mechanisms of resistance against this pathogen will contribute to improved breeding and control strategies to mitigate its devastating effects. The aim of this study was to determine the molecular dialogue between E. grandis and A. psidii, using an RNA-sequencing approach. Resistant and susceptible E. grandis seedlings grown from seed collected across its natural range were inoculated with the pandemic biotype of A. psidii. The leaf tissue was harvested at 12-h post inoculation (hpi), 1-day post inoculation (dpi), 2-dpi and 5-dpi and subjected to RNA-sequencing using Illumina 50 bp PE reads to a depth of 40 million reads per sample. Differential gene expression and gene ontology enrichment indicated that the resistant seedlings showed controlled, coordinated responses with a hypersensitive response, while the susceptible seedlings showed no systemic response against myrtle rust. Brassinosteroid signaling was apparent as an enriched term in the resistant interaction at 2-dpi, suggesting an important role of this phytohormone in defense against the pathogen. Brassinosteroid mediated signaling genes were also among the candidate genes within two major disease resistance loci (Puccinia psidii resistance), Ppr3 and Ppr5. While brassinosteroids have been tagged as positive regulators in other plant disease resistance interactions, this is the first report in the Eucalyptus – Austropuccinia psidii interaction. Furthermore, several putative resistance genes, underlying known resistance loci and implicated in the interaction have been identified and highlighted for future functional studies. This study provided further insights into the molecular interactions between E. grandis and A. psidii, contributing to our understanding of this pathosystem.

Construction of a high-density genetic map and detection of a major QTL of resistance to powdery mildew (Erysiphe necator Sch.) in Caucasian grapes (Vitis vinifera L.)

BackgroundVitis vinifera L. is the most cultivated grapevine species worldwide. Erysiphe necator Sch., the causal agent of grape powdery mildew, is one of the main pathogens affecting viticulture. V. vinifera has little or no genetic resistances against E. necator and the grape industry is highly dependent on agrochemicals. Some Caucasian V. vinifera accessions have been reported to be resistant to E. necator and to have no genetic relationships to known sources of resistance to powdery mildew. The main purpose of this work was the study and mapping of the resistance to E. necator in the Caucasian grapes ‘Shavtsitska’ and ‘Tskhvedianis tetra’.ResultsThe Caucasian varieties ‘Shavtsitska’ and ‘Tskhvedianis tetra’ showed a strong partial resistance to E. necator which segregated in two cross populations: the resistant genotypes delayed and limited the pathogen mycelium growth, sporulation intensity and number of conidia generated. A total of 184 seedlings of ‘Shavtsitska’ x ‘Glera’ population were genotyped through the Genotyping by Sequencing (GBS) technology and two high-density linkage maps were developed for the cross parents. The QTL analysis revealed a major resistance locus, explaining up to 80.15% of the phenotypic variance, on ‘Shavtsitska’ linkage group 13, which was associated with a reduced pathogen infection as well as an enhanced plant necrotic response. The genotyping of 105 Caucasian accessions with SSR markers flanking the QTL revealed that the resistant haplotype of ‘Shavtsitska’ was shared by ‘Tskhvedianis tetra’ and a total of 25 Caucasian grape varieties, suggesting a widespread presence of this resistance in the surveyed germplasm. The uncovered QTL was mapped in the region where the Ren1 locus of resistance to E. necator, identified in the V. vinifera ‘Kishmish vatkana’ and related grapes of Central Asia, is located. The genetic analysis conducted revealed that the Caucasian grapes in this study exhibit a resistant haplotype different from that of Central Asian grape accessions.ConclusionsThe QTL isolated in ‘Shavtsitska’ and present in the Caucasian V. vinifera varieties could be a new candidate gene of resistance to E. necator to use in breeding programmes. It co-localizes with the Ren1 locus but shows a different haplotype from that of grapevines of Central Asia. We therefore consider that the Caucasian resistance locus, named Ren1.2, contains a member of a cluster of R-genes, of which the region is rich, and to be linked with, or possibly allelic, to Ren1.

Registration of ‘White Pearl’ great northern common bean cultivar with upright plant architecture and high yield

AbstractGreat northern common bean (Phaseolus vulgaris L.) cultivar ‘White Pearl’ (Reg. no. CV‐343, PI 698189) was developed by the dry bean breeding program at the University of Nebraska and released in 2018. It was bred specifically for adaptation to western Nebraska growing conditions and for upright plant architecture, high yield, and enhanced resistance to bean rust, common bacterial blight (CBB), and Bean common mosaic virus (BCMV). Seed yield of White Pearl was comparable and slightly higher than other great northern cultivars in on‐station trials conducted in Nebraska and did not differ significantly in yield from ‘Aries’ and ‘Matterhorn’ in regional trials. Seed size (100‐seed weight) of White Pearl was larger than ‘Marquis’, ‘Orion’, ‘Gemini’, ‘Beryl‐R’, and Aries in trials grown in Nebraska. White Pearl carries the sequence characterized amplified region (SCAR) marker SAP6 linked to a major quantitative trait locus (QTL) for resistance to CBB. White Pearl had a CBB disease rating of 6.0 (intermediate) under field conditions at the Scottsbluff and Mitchell Ag Labs in 2020. White Pearl also has the Ur‐3 gene for resistance to bean rust and carries the SS68 Kompetitive Allele Specific polymerase chain reaction (KASP) and SK14 SCAR markers linked to the Ur‐3 locus. It also contains the single dominant hypersensitive I gene that confers resistance to all non‐necrotic strains of BCMV and the SW13 SCAR marker linked to the I gene. White Pearl has bright white seed, blooms 46 d after planting, and is a midseason bean, maturing 89 d after planting.

The Rlm13 Gene, a New Player of Brassica napus-Leptosphaeria maculans Interaction Maps on Chromosome C03 in Canola.

Canola exhibits an extensive genetic variation for resistance to blackleg disease, caused by the fungal pathogen Leptosphaeria maculans. Despite the identification of several Avr effectors and R (race-specific) genes, specific interactions between Avr-R genes are not yet fully understood in the Brassica napus–L. maculans pathosystem. In this study, we investigated the genetic basis of resistance in an F2:3 population derived from Australian canola varieties CB-Telfer (Rlm4)/ATR-Cobbler (Rlm4) using a single-spore isolate of L. maculans, PHW1223. A genetic linkage map of the CB-Telfer/ATR-Cobbler population was constructed using 7,932 genotyping-by-sequencing-based DArTseq markers and subsequently utilized for linkage and haplotype analyses. Genetic linkage between DArTseq markers and resistance to PHW1223 isolate was also validated using the B. napus 60K Illumina Infinium array. Our results revealed that a major locus for resistance, designated as Rlm13, maps on chromosome C03. To date, no R gene for resistance to blackleg has been reported on the C subgenome in B. napus. Twenty-four candidate R genes were predicted to reside within the quantitative trait locus (QTL) region. We further resequenced both the parental lines of the mapping population (CB-Telfer and ATR-Cobbler, > 80 × coverage) and identified several structural sequence variants in the form of single-nucleotide polymorphisms (SNPs), insertions/deletions (InDels), and presence/absence variations (PAVs) near Rlm13. Comparative mapping revealed that Rlm13 is located within the homoeologous A03/C03 region in ancestral karyotype block “R” of Brassicaceae. Our results provide a “target” for further understanding the Avr–Rlm13 gene interaction as well as a valuable tool for increasing resistance to blackleg in canola germplasm.

QTL mapping of lentil anthracnose (Colletotrichum lentis) resistance from Lens ervoides accession IG 72815 in an interspecific RIL population

Anthracnose, caused by Colletotrichum lentis, is one of the most damaging diseases of lentil (L. culinaris) in western Canada. Lens ervoides accession IG 72815 exhibits high levels of resistance to the pathogenic races 0 and 1. The objective of this study was to identify quantitative trait loci (QTL) associated with anthracnose resistance in lentil using a recombinant inbred line (RIL) population from the interspecific cross between IG 72815 and the susceptible cultivar Eston. A total of 168 RILs were genotyped and evaluated for anthracnose race 0 and race 1 reaction in the growth chamber and polyhouse. QTL analysis identified major resistance loci on chromosomes 3 and 7 for both races, accounting for 20.1–31.2% and 8.3–18.4%, respectively, of the total phenotypic variation for anthracnose resistance. Multiple classes of putative defense-related genes are located within both loci. Further characterization of these regions will facilitate the introgression of anthracnose resistance from Lens ervoides into elite lentil cultivars via marker-assisted selection.

Molecular mapping of a novel QTL for resistance to cereal cyst nematode in F4 wheat population

A wheat line selected from an off-type plant (Lnm136) was found to be resistant to Heterodera avenae (cereal cyst nematode, CCN). A F4 population developed by crossing this line with a CCN susceptible genotype, Ksu118, was used to study the map location of the CCN resistance. The results showed that the most significant QTLs for the cereal cyst nematode (CCN) score were identified on five chromosomes. One of the QTLs was located on chromosome 1B and linked closely to Xwmc85 based on composite interval mapping. A novel locus accounting for up to 70% of the resistance to CCN was mapped to chromosome 1B of Lnm136, by association with the microsatellite marker Xwmc85-1B. The selected CCN resistant F4 lines showed introgression for molecular markers identified to be linked with the CCN resistance locus Xwmc85-1B. This QTL on chromosome 1BS in the interval of Xwmc85–Xgpw4331 appears to be a major novel resistance locus. Multiple regression analysis showed the importance of the 1B region and marker-assisted selection for the favorable allele at this region should improve the resistance against CCN. The introgression lines can be used for marker-assisted transfer of Xwmc85-1B to elite wheat cultivars.

Conversion and Validation of Uniplex SNP Markers for Selection of Resistance to Cassava Mosaic Disease in Cassava Breeding Programs

Cassava mosaic disease (CMD) is a major viral disease adversely affecting cassava production in Africa and Asia. Genomic regions conferring resistance to the disease have been mapped in African cassava germplasm through biparental quantitative trait loci (QTL) mapping and genome-wide association studies. To facilitate the utilization of these markers in breeding pipelines to support selections, proof-of-concept technical and biological validation research was carried out using independent pre-breeding and breeding populations. Kompetitive Allele-Specific Polymerase Chain Reaction (KASP) assays were designed from three single nucleotide polymorphism (SNP) markers linked to a major resistance locus on chromosome 12 (S12_7926132, S12_7926163) and a minor locus on chromosome 14 (S14_4626854). The designed assays were robust and easy to score with >99% genotype call rate. The overall predictive accuracy (proportion of true positives and true negatives) of the markers (S12_7926132 and S14_4626854) was 0.80 and 0.78 in the pre-breeding and breeding population, respectively. On average, genotypes that carried at least one copy of the resistant allele at the major CMD2 locus had a significantly higher yield advantage. Nevertheless, variation was observed in prediction accuracies for the major locus (S12_7926132) among sub-families from the two populations, suggesting the need for context-specific utilization, for example, by screening for co-segregation of favorable SNP alleles with resistance in the parents being used for crosses. Availability of these validated SNP markers on the uniplex KASP genotyping platform represents an important step in translational genetics toward marker-assisted selection to accelerate introgression of favorable resistant alleles in breeding populations.

Analysis of the primary sources of quantitative adult plant resistance to stripe rust in U.S. soft red winter wheat germplasm.

Stripe rust, or yellow rust (Puccinia striiformis Westend. f. sp. tritic), is a disease of wheat (Triticum aestivum L.) historically causing significant economic losses in cooler growing regions. Novel isolates of stripe rust with increased tolerance for high temperatures were detected in the United States circa 2000. This increased heat tolerance puts geographic regions, such as the soft red winter wheat (SRWW) growing region of the southeastern United States, at greater risk of stripe rust induced losses. In order to identify sources of stripe rust resistance in contemporary germplasm, we conducted genome-wide association (GWA) studies on stripe rust severity measured in two panels. The first consisted of 273 older varieties, landraces, and some modern elite breeding lines and was evaluated in environments in the U.S. Pacific Northwest and the southeastern United States. The second panel consisted of 588 modern, elite SRWW breeding lines and was evaluated in four environments in Arkansas and Georgia. The analyses identified three major resistance loci on chromosomes: 2AS (presumably the 2NS:2AS alien introgression from Aegilops ventricosa Tausch; syn. Ae. caudata L.), 3BS, and 4BL. The 4BL locus explained a greater portion of variance in resistance than either the 2AS or 3BS loci in southeastern environments. However, its effects were unstable across different environments and sets of germplasm, possibly a result of its involvement in epistatic interactions. Relatively few lines carry resistance alleles at all three loci, suggesting that there is a pre-existing reservoir of enhanced stripe rust resistance that may be further exploited by regional breeding programs.

QRgls1.06, a major QTL conferring resistance to gray leaf spot disease in maize

Gray leaf spot (GLS) caused by Cercospora zeae-maydis and C. zeina is an extremely devastating leaf disease that limits maize production annually. The use of GLS-resistant maize hybrids is the most cost-effective approach for reducing losses. Resistance to GLS is quantitatively inherited in maize (Zea mays L.) and further sources of resistance remain to be analyzed. Here, we detected qRgls1.06, a major quantitative trait locus for GLS resistance in bin 1.06 that explained approximately 55% of the phenotype variance. Fine mapping over 2 consecutive years localized qRgls1.06 to a 2.38-Mb region. Homozygous qRgls1.06WGR/WGR plants in DZ01 background displayed higher GLS resistance and 100-grain weight than DZ01 plants. The GLS responses of several susceptible elite inbred lines were improved by the introduction of qRgls1.06 by marker-assisted backcrossing. Our findings extend the understanding of the genetic basis of resistance to GLS and provide a set of resistant germplasm for genetic improvement of resistance to GLS in maize.

QTL mapping for adult-plant resistance to powdery mildew in Chinese elite common wheat Chuanmai104

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a major fungal disease of wheat worldwide. The economic and environmentally friendly strategy is developing the wheat varieties with resistance to powdery mildew in wheat-breeding program. Chuanmai104 (CM104, Triticum aestivum L.), derived from synthetic hexaploid wheat, was an elite wheat variety with high adult-plant resistance to powdery mildew. The phenotype of powdery mildew resistance was conducted on a set of 234 recombinant inbred lines (RIL, F8) derived from the cross of CM104 and high susceptible wheat landrace Baimaomai (BMM) in five environments in this study. Three QTLs were identified on chromosomes 7D and 4A by the high-density wheat 50 K single-nucleotide polymorphism array, designed as QPm.saas-7DS, QPm.saas-4AL and QPm.saas-4AS, respectively. Of the three QTLs, QPm.saas-7DS derived from BMM and QPm.saas-4AL derived from CM104 were minor locus, QPm.saas-4AS derived from CM104 was detected as a new major locus for adult-plant resistance to powdery mildew in all environments and localized in a 1.06 Mb interval of the genomic region on chromosome arm 4AS referenced to Chinese Spring genome explained up to 26.5% of the phenotypic variances. The newly major locus QPm.saas-4AS would be supplied as a resistant source in wheat-breeding program.

Two-omics data revealed commonalities and differences between Rpv12- and Rpv3-mediated resistance in grapevine

Plasmopara viticola is the causal agent of grapevine downy mildew (DM). DM resistant varieties deploy effector-triggered immunity (ETI) to inhibit pathogen growth, which is activated by major resistance loci, the most common of which are Rpv3 and Rpv12. We previously showed that a quick metabolome response lies behind the ETI conferred by Rpv3 TIR-NB-LRR genes. Here we used a grape variety operating Rpv12-mediated ETI, which is conferred by an independent locus containing CC-NB-LRR genes, to investigate the defence response using GC/MS, UPLC, UHPLC and RNA-Seq analyses. Eighty-eight metabolites showed significantly different concentration and 432 genes showed differential expression between inoculated resistant leaves and controls. Most metabolite changes in sugars, fatty acids and phenols were similar in timing and direction to those observed in Rpv3-mediated ETI but some of them were stronger or more persistent. Activators, elicitors and signal transducers for the formation of reactive oxygen species were early observed in samples undergoing Rpv12-mediated ETI and were paralleled and followed by the upregulation of genes belonging to ontology categories associated with salicylic acid signalling, signal transduction, WRKY transcription factors and synthesis of PR-1, PR-2, PR-5 pathogenesis-related proteins.

An Africa-wide genomic evolution of insecticide resistance in the malaria vector Anopheles funestus involves selective sweeps, copy number variations, gene conversion and transposons.

Insecticide resistance in malaria vectors threatens to reverse recent gains in malaria control. Deciphering patterns of gene flow and resistance evolution in malaria vectors is crucial to improving control strategies and preventing malaria resurgence. A genome-wide survey of Anopheles funestus genetic diversity Africa-wide revealed evidences of a major division between southern Africa and elsewhere, associated with different population histories. Three genomic regions exhibited strong signatures of selective sweeps, each spanning major resistance loci (CYP6P9a/b, GSTe2 and CYP9K1). However, a sharp regional contrast was observed between populations correlating with gene flow barriers. Signatures of complex molecular evolution of resistance were detected with evidence of copy number variation, transposon insertion and a gene conversion between CYP6P9a/b paralog genes. Temporal analyses of samples before and after bed net scale up suggest that these genomic changes are driven by this control intervention. Multiple independent selective sweeps at the same locus in different parts of Africa suggests that local evolution of resistance in malaria vectors may be a greater threat than trans-regional spread of resistance haplotypes.

QGLS1.02, a novel major locus for resistance to gray leaf spot in maize

Gray leaf spot (GLS), caused by the fungal pathogen Cercospora zeae-maydis, is one of the most serious foliar diseases of maize worldwide. In the present study, a population of recombinant inbred lines (RILs) derived from a gray leaf spot–resistant inbred line Qi319 and a susceptible line Ye478 was used to map quantitative trait loci (QTL) for GLS resistance. For initial QTL analysis, genetic maps containing 199 simple sequence repeat (SSR) markers were constructed. One major QTL, qGLS1.02, was consistently detected between SSR markers umc2614 and bnlg1803, and explained 6.86–36.24% of the phenotypic variation in GLS resistance. The chromosome segment substitution lines harboring the qGLS1.02 from Qi319 were more resistant to GLS (P < 0.01). Using a recombinant-derived progeny test strategy, the interval containing qGLS1.02 was narrowed to ~ 314 kb according to the B73 (RefGen_v4 genome) including 12 candidate genes; only six of them have been annotated functionally.

Genetic mapping of quantitative trait loci and a major locus for resistance to grey leaf spot in maize.

The genetic basis of GLS resistance was dissected using two DH populations sharing a common resistant parent. A major QTL repeatedly detected in multiple developmental stages and environments was fine mapped in a backcross population. Grey leaf spot (GLS), caused by Cercospora zeae-maydis or Cercospora zeina, is a highly destructive foliar disease worldwide. However, the mechanism of resistance against GLS is not well understood. To study the inheritance of this resistance, we developed two doubled haploid (DH) populations sharing a common resistant parent. The two DH populations were grown in two locations representing the typical maize-growing mountain area in Southwest China for 2years. GLS disease severity was investigated 2 or 3 times until maturity in the 2years, and the area under the disease progress curve was calculated. Two high-density linkage maps were constructed by genotyping-by-sequencing. A total of 22 quantitative trait loci (QTLs) were detected for GLS resistance, with most QTLs being repeatedly detected in different stages, locations and years. The confidence intervals of two major QTLs (qGLS_Y2-2 and qGLS_Z2-1) on chromosome 2 from the two DH populations overlapped with each other and were integrated into one consensus QTL (qGLS_YZ2-1). Using highly resistant and highly susceptible plants from a BC3 population, we fine mapped this genetic locus to a genomic region of 2.4Mb. Using a panel of 255 inbred lines from breeding programmes, we detected associations between markers in the qGLS_YZ2-1 region and GLS resistance. The peak marker (ID-B1) will be very useful for marker-assisted breeding for GLS resistance.

Identification of Major Locus Bph35 Resistance to Brown Planthopper in Rice

An introgression line RBPH660, derived from wild rice Oryza rufipogon, showed stable resistance to brown planthopper (BPH). Segregation analysis indicated BPH resistance of RBPH660 was controlled by multiple genes/QTLs. By using the bulked segregant analysis (BSA)-seq method, two genomic regions harboring QTLs resistance to BPH were identified from 1.20 to 16.70 Mb on chromosome 4 and from 10.20 to 12.60 Mb on chromosome 9 in RBPH660, respectively. A major resistance locus, designated as Bph35 accounting for 51.27% of the phenotypic variation with a LOD score of 42.51, was mapped to the candidate region of chromosome 4 between InDel (insertion-deletion) markers PSM16 and R4M13. For fine mapping of Bph35, one simple sequence repeat and three newly developed InDel markers were used to screen the recombinants. Finally, the Bph35 locus was delimited in the region from 6.28 to 6.93 Mb and there were 18 predicted protein-encoding genes with a total of 114 non-synonymous single nucleotide polymorphism (SNP) variant sites between the resistant and susceptible parents. Out of these genes, Os04g0193950, encoding a putative NB-ARC (nucleotide- binding adaptor shared by APAF-1, R proteins and CED-4) and LRR (leucine-rich repeat) domain protein with nine non-synonymous SNP substitutions in its coding sequence regions, might be the candidate gene for Bph35. These findings would facilitate the map-based cloning of the Bph35 gene and development of resistant varieties against BPH in rice.

QTL mapping of resistance to tan spot induced by race 2 of Pyrenophora tritici-repentis in tetraploid wheat.

A total of 12 QTL conferring resistance to tan spot induced by a race 2 isolate, 86-124, were identified in three tetraploid wheat mapping populations. Durum is a tetraploid species of wheat and an important food crop. Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr), is a major foliar disease of both tetraploid durum wheat and hexaploid bread wheat. Understanding the Ptr-wheat interaction and identifying major QTL can facilitate the development of resistant cultivars and effectively mitigate the negative effect of this disease. Over 100 QTL have already been discovered in hexaploid bread wheat, whereas few mapping studies have been conducted in durum wheat. Utilizing resistant resources and identifying novel resistant loci in tetraploid wheat will be beneficial for the development of tan spot-resistant durum varieties. In this study, we evaluated four interconnected tetraploid wheat populations for their reactions to the race 2 isolate 86-124, which produces Ptr ToxA. Tsn1, the wheat gene that confers sensitivity to Ptr ToxA, was not associated with tan spot severity in any of the four populations. We found a total of 12 tan spot-resistant QTL among the three mapping populations. The QTL located on chromosomes 3A and 5A were detected in multiple populations and co-localized with race-nonspecific QTL identified in other mapping studies. Together, these QTL can confer high levels of resistance and can be used for the improvement in tan spot resistance in both hexaploid bread and durum wheat breeding. Two QTL on chromosomes 1B and 7A, respectively, were found in one population when inoculated with a ToxA knockout strain 86-124ΔToxA only, indicating that their association with tan spot was induced by other unidentified necrotrophic effectors, but under the absence of Ptr ToxA. In addition to removal of the known dominant susceptibility genes, integrating major race-nonspecific resistance loci like the QTL identified on chromosome 3A and 5A in this study could confer high and stable tan spot resistance in durum wheat.

Construction of genetic linkage map and identification of a novel major locus for resistance to pine wood nematode in Japanese black pine (Pinus thunbergii)

BackgroundPine wilt disease (PWD), which is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, is currently the greatest threat to pine forests in Europe and East Asian countries including Japan. Constructing a detailed linkage map of DNA markers and identifying PWD resistance genes/loci lead to improved resistance in Pinus thunbergii, as well as other Pinus species that are also susceptible to PWD.ResultsA total F1 mapping population of 188 individuals derived from a cross between the PWD-resistant P. thunbergii varieties ‘Tanabe 54’ (resistant rank 2 to PWD) and ‘Tosashimizu 63’ (resistant rank 4 to PWD) was inoculated with PWN, and was evaluated for disease symptoms. To perform linkage analysis for PWN resistance, a set of three maps was constructed; two parental maps generated using the integrated two-way pseudo-testcross method, and a consensus map with population-type cross-pollination. The linkage map of ‘Tanabe 54’ consisted of 167 loci, and covered 14 linkage groups (LGs), with a total genetic distance of 1214.6 cM. The linkage map of ‘Tosashimizu 63’ consisted of 252 loci, and covered 14 LGs, with a total genetic distance of 1422.1 cM. The integrated consensus map comprised 12 LGs with the basic chromosome number of P. thunbergii, and a total genetic distance of 1403.6 cM. Results from quantitative trait loci (QTL) analysis using phenotype data and linkage maps indicated that PWN resistance is controlled by a single dominant allele, which was derived from the ‘Tanabe 54’ female parent. This major QTL was located on linkage group 3 and was designated PWD1 for PINE WILT DISEASE 1.ConclusionsThe PWD1 locus is a major resistance QTL located on the Pinus consensus LG03 that acts in a dominant manner to confer pine wood nematode resistance. Information from the present study will be useful for P. thunbergii breeding programs to improve resistance to PWD, and also to help identify susceptibility genes in Pinus species.

Quantitative trait locus mapping of downy mildew and botrytis bunch rot resistance in a Vitis aestivalis-derived ‘Norton’-based population

Vitis aestivalis-derived ‘Norton’ is the official grape of the State of Missouri, grown in regions with high disease pressure and cold winter temperatures, where Vitis vinifera is not adapted. It reportedly offers an abundance of traits, including resistance to powdery and downy mildew as well as botrytis bunch rot, which can be used to naturally improve existing V. vinifera germplasm. To identify genetic determinants for resistance to downy mildew caused by Plasmopara viticola and botrytis bunch rot caused by Botrytis cinerea in V. aestivalis-derived ‘Norton’, a mapping population of 182 individuals was constructed from a cross between ‘Norton’ and V. vinifera ‘Cabernet Sauvignon’. A consensus genetic map was constructed with 411 simple sequence repeat (SSR) markers. In collaboration with VitisGen (http://www.vitisgen.org), approximately 43,320 single-nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing (GBS) were identified, and a consensus map of 3825 SNPs was developed. Of these, 1665 SNP and 407 SSR markers were clustered into 19 linkage groups, for a total of 2072 markers spanning a genetic distance of 2203.5 cM. In preparation for placing traits on this integrated high-resolution map, disease progression and resistance reaction in response to P. viticola and B. cinerea were evaluated in this population for 2 years. The quantitative trait loci (QTL) analysis indicated major resistance loci on linkage group 18 for downy mildew and linkage group 2 for botrytis bunch rot, respectively. The ultimate goal of this program is to use genetic markers to rapidly deploy favorable alleles and accelerate breeding cycles for new cultivar releases.

Comparative Subsequence Sets Analysis (CoSSA) is a robust approach to identify haplotype specific SNPs; mapping and pedigree analysis of a potato wart disease resistance gene Sen3

BackgroundStandard strategies to identify genomic regions involved in a specific trait variation are often limited by time and resource consuming genotyping methods. Other limiting pre-requisites are the phenotyping of large segregating populations or of diversity panels and the availability and quality of a closely related reference genome. To overcome these limitations, we designed efficient Comparative Subsequence Sets Analysis (CoSSA) workflows to identify haplotype specific SNPs linked to a trait of interest from Whole Genome Sequencing data.ResultsAs a model, we used the resistance to Synchytrium endobioticum pathotypes 2, 6 and 18 that co-segregated in a tetraploid full sib population. Genomic DNA from both parents, pedigree genotypes, unrelated potato varieties lacking the wart resistance traits and pools of resistant and susceptible siblings were sequenced. Set algebra and depth filtering of subsequences (k-mers) were used to delete unlinked and common SNPs and to enrich for SNPs from the haplotype(s) harboring the resistance gene(s). Using CoSSA, we identified a major and a minor effect locus. Upon comparison to the reference genome, it was inferred that the major resistance locus, referred to as Sen3, was located on the north arm of chromosome 11 between 1,259,552 and 1,519,485 bp. Furthermore, we could anchor the unanchored superscaffold DMB734 from the potato reference genome to a synthenous interval. CoSSA was also successful in identifying Sen3 in a reference genome independent way thanks to the de novo assembly of paired end reads matching haplotype specific k-mers. The de novo assembly provided more R haplotype specific polymorphisms than the reference genome corresponding region. CoSSA also offers possibilities for pedigree analysis. The origin of Sen3 was traced back until Ora. Finally, the diagnostic power of the haplotype specific markers was shown using a panel of 56 tetraploid varieties.ConclusionsCoSSA is an efficient, robust and versatile set of workflows for the genetic analysis of a trait of interest using WGS data. Because the WGS data are used without intermediate reads mapping, CoSSA does not require the use of a reference genome. This approach allowed the identification of Sen3 and the design of haplotype specific, diagnostic markers.