Comprehensive genetic analyses reveal differential expression of spot blotch resistance in four populations of barley

Spot blotch, caused by Cochliobolus sativus, is an important disease of barley in the Upper Midwest region of the United States. The resistance of six-rowed malting cultivars like Morex has remained effective for over 40 years and is considered durable. Previous research on Steptoe/Morex (S/M), a 6×6-rowed doubled haploid (DH) population, showed that seedling resistance is controlled by a single gene (Rcs5) on chromosome 1(7H) and adult plant resistance by two quantitative trait loci (QTL): one of the major effect on chromosome 5(1H) explaining 62% of the phenotypic variance and a second of minor effect on chromosome 1(7H) explaining 9% of the phenotypic variance. To corroborate these results in a 2×6-rowed DH population, composite interval mapping (CIM) was performed on Harrington/Morex (H/M). As in the S/M population, a single major gene (presumably Rcs5) on chromosome 1(7H) conferred resistance at the seedling stage. However, at the adult plant stage, the results were markedly different as no chromosome 5(1H) effect whatsoever was detected. Instead, a QTL at or near Rcs5 on chromosome 1(7H) explained nearly all of the phenotypic variance (75%) for disease severity. To determine whether this result might be due to the genetic background of the two-rowed susceptible parent Harrington, we analyzed another DH population that included the same resistance donor (Morex) and another six-rowed susceptible cultivar Dicktoo (D/M). Three QTL conferred seedling resistance in the D/M population: one near Rcs5 on chromosome 1(7H) explaining 30%, a second near the centromere of chromosome 1(7H) explaining 9%, and a third on the short arm of chromosome 3(3H) explaining 19% of the phenotypic variation. As in the H/M population, no chromosome 5(1H) QTL was detected for adult plant resistance in the D/M population. Instead, three QTL on other chromosomes explained most of the variation: one on the short arm of chromosome 3(3H) explaining 36%, a second on the long arm of chromosome 3(3H) explaining 11%, and a third at or near Rcs5 on chromosome 1(7H) explaining 20% of the phenotypic variation. These data demonstrate the complexity of expression of spot blotch resistance in different populations and have important implications in breeding for durable resistance.     

Mapping quantitative trait loci associated with spot blotch and net blotch resistance in a doubled-haploid barley population

Spot blotch and net blotch are important foliar barley (Hordeum vulgare L.) diseases in Canada and elsewhere. These diseases result in significant yield reduction and, more importantly, loss of grain quality, downgrading barley from malt to feed. Combining resistance to these diseases is a breeding priority but is a significant challenge using conventional breeding methodology. In the present investigation, an evaluation of the inheritance of resistance to spot and net blotch was conducted in a doubled-haploid barley population from the cross CDC Bold (susceptible)?×?TR251 (resistant). The population was screened at the seedling stage in the Phytotron and at the adult-plant stage in the field for several years. Chi-squared analysis indicated one- to four-gene segregation depending on disease, isolate, plant development stage, location and year. A major seedling and adult-plant resistance quantitative trait locus (QTL), designated QRpt6, was re-confirmed for net-form net blotch resistance, explaining 32?C61% of phenotypic variation in different experiments. Additional QTL for seedling and adult-plant resistance to net blotch were identified. For spot blotch resistance, a major seedling resistance QTL (QRcss1) was detected on chromosome 1H for isolate WRS1909, explaining 79% of the phenotypic variation. A highly significant QTL on 3H (QRcs3) was identified for seedling resistance to isolate WRS1908 and adult-plant resistance at Brandon, MB, Canada in 2008. The identification of QTL at only one location or from 1?year suggests spot blotch resistance is complex and highly influenced by the environment. Efforts are being made to combine spot and net blotch resistance in elite barley lines using molecular marker-assisted selection.     

Association mapping of spot blotch resistance in wild barley

Spot blotch, caused by Cochliobolus sativus, is an important foliar disease of barley. The disease has been controlled for over 40 years through the deployment of cultivars with durable resistance derived from the line NDB112. Pathotypes of C. sativus with virulence for the NDB112 resistance have been detected in Canada; thus, many commercial cultivars are vulnerable to spot blotch epidemics. To increase the diversity of spot blotch resistance in cultivated barley, we evaluated 318 diverse wild barley accessions comprising the Wild Barley Diversity Collection (WBDC) for reaction to C. sativus at the seedling stage and utilized an association mapping (AM) approach to identify and map resistance loci. A high frequency of resistance was found in the WBDC as 95% (302/318) of the accessions exhibited low infection responses. The WBDC was genotyped with 558 Diversity Array Technology (DArT^®) and 2,878 single nucleotide polymorphism (SNP) markers and subjected to structure analysis before running the AM procedure. Thirteen QTL for spot blotch resistance were identified with DArT and SNP markers. These QTL were found on chromosomes 1H, 2H, 3H, 5H, and 7H and explained from 2.3 to 3.9% of the phenotypic variance. Nearly half of the identified QTL mapped to chromosome bins where spot blotch resistance loci were previously reported, offering some validation for the AM approach. The other QTL mapped to unique genomic regions and may represent new spot blotch resistance loci. This study demonstrates that AM is an effective technique for identifying and mapping QTL for disease resistance in a wild crop progenitor.     

Mapping quantitative trait loci associated with barley net blotch resistance

Net blotch of barley, caused by Pyrenophora teres Drechs., is an important foliar disease worldwide. Deployment of resistant cultivars is the most economic and eco-friendly control method. This report describes mapping of quantitative trait loci (QTL) associated with net blotch resistance in a doubled-haploid (DH) barley population using diversity arrays technology (DArT) markers. One hundred and fifty DH lines from the cross CDC Dolly (susceptible)/TR251 (resistant) were screened as seedlings in controlled environments with net-form net blotch (NFNB) isolates WRS858 and WRS1607 and spot-form net blotch (SFNB) isolate WRS857. The population was also screened at the adult-plant stage for NFNB resistance in the field in 2005 and 2006. A high-density genetic linkage map of 90 DH lines was constructed using 457 DArT and 11 SSR markers. A major NFNB seedling resistance QTL, designated QRpt6, was mapped to chromosome 6H for isolates WRS858 and WRS1607. QRpt6 was associated with adult-plant resistance in the 2005 and 2006 field trials. Additional QTL for NFNB seedling resistance to the more virulent isolate WRS858 were identified on chromosomes 2H, 4H, and 5H. A seedling resistance QTL (QRpts4) for the SFNB isolate WRS857 was detected on chromosome 4H as was a significant QTL (QRpt7) on chromosome 7H. Three QTL (QRpt6, QRpts4, QRpt7) were associated with resistance to both net blotch forms and lines with one or more of these demonstrated improved resistance. Simple sequence repeat (SSR) markers tightly linked to QRpt6 and QRpts4 were identified and validated in an unrelated barley population. The major 6H QTL, QRpt6, may provide adequate NFNB field resistance in western Canada and could be routinely selected for using molecular markers in a practical breeding program.     

Mapping resistance to spot blotch in a CIMMYT synthetic-derived bread wheat

Spot blotch, caused by Cochliobolus sativus, is an important foliar disease of wheat in warmer wheat-growing regions leading to significant reductions in grain yield and quality. Although inoculum levels can be reduced by planting disease-free seed, treatment of plants with fungicides and crop rotation, genetic resistance is likely to be a robust, economical and environmentally friendly tool in the control of spot blotch. The spot blotch resistant synthetic derivative ‘SYN1’ was developed from a cross between two resistance sources, Mayoor and the primary synthetic bread wheat Tksn1081/Ae. squarrosa (222) that are likely to form an important component of resistance in many elite CIMMYT bread wheats. In order to map the loci underlying the resistance of ‘SYN1’, a doubled-haploid population produced from a cross between ‘SYN1’ and the susceptible CIMMYT-derived variety Ocoroni-86 was evaluated in artificially inoculated field nurseries in the 2010–2011 and 2011–2012 crop seasons at CIMMYT’s research station in Agua Fría, Mexico. Disease assessment was performed on three or four occasions and subsequently area under disease progress curve (AUDPC) calculated. Genotyping was with genotyping by sequencing and simple sequence repeat markers. Using inclusive composite interval mapping, three genomic regions were found to have a significant effect on spot blotch AUDPC in each of the 2 years of trials with phenotypic variation explained by QSb.cim-1B of 8.5 %, 17.6 % by QSb.cim-3B and 12.3 % by QSb.cim-5A. The quantitative trait loci (QTL) mapping results showed that the favorable alleles of QSb.cim-1B, QSb.cim-3B and QSb.cim-5A were derived from the synthetic-derived bread wheat SYN1. Genotypes of the parents of SYN1 indicated that the favorable alleles at these three QTLs were all inherited from Mayoor.     

Mapping of resistance to spot blotch disease caused by Bipolaris sorokiniana in spring wheat

Spot blotch caused by Bipolaris sorokiniana is a destructive disease of wheat in warm and humid wheat growing regions of the world. The development of disease resistant cultivars is considered as the most effective control strategy for spot blotch. An intervarietal mapping population in the form of recombinant inbred lines (RILs) was developed from a cross ‘Yangmai 6’ (a Chinese source of resistance) × ‘Sonalika’ (a spot blotch susceptible cultivar). The 139 single seed descent (SSD) derived F₆, F₇, F₈ lines of ‘Yangmai 6’ × ‘Sonalika’ were evaluated for resistance to spot blotch in three blocks in each of the 3 years. Joint and/or single year analysis by composite interval mapping (CIM) and likelihood of odd ratio (LOD) >2.2, identified four quantitative trait loci (QTL) on the chromosomes 2AL, 2BS, 5BL and 6DL. These QTLs were designated as QSb.bhu-2A, QSb.bhu-2B, QSb.bhu-5B and QSb.bhu-6D, respectively. A total of 63.10% of phenotypic variation was explained by these QTLs based on the mean over years. Two QTLs on chromosomes 2B and 5B with major effects were consistent over 3 years. All QTL alleles for resistance were derived from the resistant parent ‘Yangmai 6’.     

Identification of 50 K Illumina-chip SNPs associated with resistance to spot blotch in barley

Background

Spot blotch, caused by Cochliobolus sativus, is one of the most widespread and harmful diseases in barley. Identification of genetic loci associated with resistance to C. sativus is of importance for future marker-assisted selection. The goal of the current study was to identify loci conferring seedling resistance to two different pathotypes of C. sativus in the Siberian spring barley core collection.

Results

A total of 96 spring barley cultivars and lines were phenotyped at the seedling stage with two C. sativus isolates (Kr2 and Ch3). According to the Fetch-Steffenson rating scale 16%/17% of genotypes were resistant and 26%/30% were moderate-resistant to the Kr2/Ch3 isolates respectively. A total of 94 genotypes were analyzed with the barley 50 K Illumina Infinium iSELECT assay. From 44,040 SNPs, 40,703 were scorable, from which 39,140 were polymorphic. 27,319 SNPs passed filtering threshold and were used for association mapping. Data analysis by GLM revealed 48 and 41 SNPs for Kr2 and Ch3 isolates, respectively. After application of 5% Bonferroni multiple test correction, only 3 and 27 SNPs were identified, respectively. A total of three genomic regions were associated with the resistance. The region on chromosome 3H associated with Ch3-resistance was expanded between markers SCRI_RS_97417 and JHI-Hv50k-2016-158003 and included 11 SNPs, from which JHI-Hv50k-2016-157070, JHI-Hv50k-2016-156842 had the lowest p-values. These two SNPs were also significant in case of Kr2 isolate. The region on chromosome 2H included 16 loci (7 of them with the lowest p-values were tightly linked to BOPA2_12_11504). Three loci corresponding to this region had suggestive p-values in case of Kr2 tests, so the locus on chromosome 2H may also contribute to resistance to Kr2 isolate. The third region with significant p-value in case of Kr2 tests was identified on chromosome 1H at the locus JHI-Hv50k-2016-33568.

Conclusions

Three genomic regions associated with the resistance to one or both isolates of C. sativus were identified via screening of the Siberian spring barley core collection. Comparison of their location with QTLs revealed previously either with biparental mapping populations studies or with GWAS of distinct germplasm and other isolates, demonstrated that resistance to isolates Kr2 and Ch3 is conferred by known spot blotch resistance loci. Information on SNPs related can be used further for development of DNA-markers convenient for diagnostics of resistance-associated alleles in barley breeding programs.

     

Genome-wide association mapping reveals genetic architecture of durable spot blotch resistance in US barley breeding germplasm

Spot blotch, an economically important disease of both barley and wheat, is caused by Cochliobolus sativus (anamorph: Bipolaris sorokiniana). The disease has been reported in many regions of the world, but is particularly severe on barley in the Upper Midwest region of the USA and adjacent areas of Canada. For over 50 years, spot blotch has been effectively controlled through the deployment of durable resistance in six-rowed malting cultivars. To characterize loci conferring spot blotch resistance in US barley germplasm, we employed an association mapping approach using 3,840 breeding lines and cultivars. Three quantitative trait loci (QTL), Rcs-qtl-1H-11_10764, Rcs-qtl-3H-11_10565 and Rcs-qtl-7H-11_20162, were found to confer both seedling and adult plant resistance. Together, these three QTL comprise the Midwest Six-rowed Durable Resistant Haplotype (MSDRH), which is present in all Midwest six-rowed cultivars released since the 1960s. Each QTL alone only partially reduced disease levels, but combining all three together reduced the seedling infection response and adult plant disease severity by 47 and 83 %, respectively. The identified MSDRH will be valuable for marker-assisted selection of breeding lines to deploy spot blotch resistance and can also be incorporated into genomic selection as one of the disease resistance traits.     

Inheritance and RAPD tagging of multiple genes for resistance to net blotch in barley.

A doubled haploid barley (Hordeum vulgare L.) population that was created from a cross between cultivars 'Léger' and 'CI 9831' was characterized by RAPD (random amplified polymorphic DNA) markers for resistance to isolate WRS857 of Pyrenophora teres Drechs. f. sp. maculata Smedeg., the causal agent of the spot form of net blotch. Resistance, which initially appeared to be conferred by a single gene from the approximate 1:1 (resistant : susceptible) segregation ratio of the doubled-haploid (DH) progeny, was found to be associated with three different genomic regions by RAPD analysis. Of 500 RAPD random primers that were screened against the parents, 195 revealed polymorphic bands, seven showed an association to the resistance in bulks, and these seven markers were mapped to three unlinked genomic regions. Two of these regions, one of which was mapped to chromosome 2, have major resistance genes. The third region has some homology to the chromosome 2 region. This study demonstrates the simultaneous location of markers for more than one gene governing a trait by using RAPD and bulked segregant analysis (BSA).     

Field screening for resistance to barley net blotch

Inoculation with barley net blotch from infested straw debris was compared with that from diseased plants after sowing infected grains. The straw debris had a high, uniform inoculation potential which gave an early, continuing infection and easily reproducible results that were effective for screening barley cultivars in the field for resistance against a natural population of the pathogen. Further, it minimises an eventual influence from other leaf pathogens coming from the surroundings. Irrigation was decisive for the success of the method - especially in the initial phase. Ten m separation with an immune crop was insufficient to completely prevent infection in the uninoculated plots. The tested 25 cvs were differentiated in six categories of resistant and susceptible on the basis of disease development and final level of attack. None of them was free of symptoms. The most resistant cvs kept a constant, low level of attack during the whole growing season, whereas the most susceptible cvs showed an early and rapidly increasing attack. Intermediate cvs were characterised with more or less slow increase of the attack. The size and proportion of the brown necrotic spots and the surrounding yellow halo varied greatly from one cultivar to the other. The grain yield reduction was due solely to an effect on the thousand grain weight which decreased linearly with the squared point score for net blotch. Further, the disease affected the quality of the grains as less nitrogen was transported from straw to grain in the severely diseased plants.     

Genetics of seedling and adult plant resistance to net blotch (Pyrenophora teres f. teres) and spot blotch (Cochliobolus sativus) in barley

Net blotch (caused by Pyrenophora teres f. teres) and spot blotch (Cochliobolus sativus) are important foliar diseases of barley in the midwestern region of the USA. To determine the number and chromosomal location of Mendelian and quantitative trait loci (QTL) controlling resistance to these diseases, a doubled haploid population (Steptoe/Morex) was evaluated to the pathogens at the seedling stage in the greenhouse and at the adult plant stage in the field. Alleles at two or three unlinked loci were found to confer resistance to the net blotch pathogen at the seedling stage depending on how progeny exhibiting an intermediate infection response were classified. This result was corroborated in the quantitative analysis of the raw infection response data as 2 major QTL were identified on chromosomes 4 and 6M. A third QTL was also identified on chromosome 6P. Seven QTL were identified for net blotch resistance at the adult plant stage and mapped to chromosomes 1P, 2P, 3P, 3M, 4, 6P, and 7P. The 7 QTL collectively accounted for 67.6% of the phenotypic variance under a multiple QTL model. Resistance to the spot blotch pathogen was conferred by a single gene at the seedling stage. This gene was mapped to the distal region of chromosome 1P on the basis of both qualitative and quantitative data analyses. Two QTL were identified for spot blotch resistance at the adult plant stage: the largest QTL effect mapped to chromosome 5P and the other mapped to chromosome 1P near the seedling resistance locus. Together, the 2 QTL explained 70.1% of the phenotypic variance under a multiple QTL model. On the basis of the chromosomal locations of resistance alleles detected in this study, it should be feasible to combine high levels of resistance to both P. teres f. teres and C. sativus in barley cultivars.     

Mapping resistance to cereal aphids in barley

 A set of 150 doubled-haploid (DH) barley (Hordeum vulgare L.) lines derived from the cross of Harrington/TR306 was used to determine the number and chromosomal location of quantitative trait loci (QTLs) controlling resistance to cereal aphids. The experiments were conducted under natural infestation in the field during two growing seasons: 1994 and 1995. Aphid resistance was measured by counting the number of aphids per plot. Counts were made on a weekly basis. Each year at the time of maximum aphid density there was an obvious difference in reaction between the parental genotypes. The DH lines showed continuous variation for aphid density. Simple interval mapping and simplified composite interval mapping revealed that the principal QTL determining cereal aphid resistance is on the distal region of the short arm of chromosome 1. This aphid-resistance QTL is linked with a heading-date QTL. At the time of highest aphid infestation, this QTL accounted for 31% and 22% of the total variance of aphid density in 1994 and 1995, respectively. A QTL on chromosome 5 was also detected but only by simplified composite interval mapping. However, the largest consistent effect was due to the QTL on the short arm of chromosome 1. This QTL may be a useful target for marker-assisted selection for adult plant cereal aphid resistance in barley. Received: 10 September 1996/Accepted: 11 October 1996     

A region of barley chromosome 6H harbors multiple major genes associated with net type net blotch resistance

Net type net blotch (NTNB), caused by Pyrenophora teres f. teres Drechs., is prevalent in barley growing regions worldwide. A population of 118 doubled haploid (DH) lines developed from a cross between barley cultivars ‘Rika’ and ‘Kombar’ were used to evaluate resistance to NTNB due to their differential reaction to various isolates of P. teres f. teres. Rika was resistant to P. teres f. teres isolate 15A and susceptible to isolate 6A. Conversely, Kombar was resistant to 6A, but susceptible to 15A. A progeny isolate of a 15A × 6A cross identified as 15A × 6A#4 was virulent on both parental lines. The Rika/Kombar (RK) DH population was evaluated for disease reactions to the three isolates. Isolate 15A induced a resistant:susceptible ratio of 78:40 (R:S) whereas isolate 6A induced a resistant:susceptible ratio of 40:78. All but two lines had opposite disease reactions indicating two major resistance genes linked in repulsion. Progeny isolate 15A × 6A#4 showed a resistant:susceptible ratio of 1:117 with the one resistant line also being the single line that was resistant to both 15A and 6A. An RK F₂ population segregated in a 1:3 (R:S) ratio for both 15A and 6A indicating that resistance is recessive. Molecular markers were used to identify a region on chromosome 6H that harbors the two NTNB resistance genes. This work shows that multiple NTNB resistance genes exist at the locus on chromosome 6H, and the recombinant DH line harboring the resistance alleles from both parents will be useful for the development of NTNB-resistant barley germplasm.     

Mapping of the ADP-glucose pyrophosphorylase genes in barley

cDNA probes encoding the barley endosperm ADP-glucose pyrophosphorylase (AGP) small subunit (bepsF2), large subunit (bepl10), and leaf AGP large subunit (blpl) were hybridized with barley genomic DNA blots to determine copy number and polymorphism. Probes showing polymorphism were mapped on a barley RFLP map. Probes that were not polymorphic were assigned to chromosome arms using wheat-barley telosomic addition lines. The data suggested the presence of a single-copy gene corresponding to each of the cDNA probes. In addition to the major bands, several weaker cross-hybridizing bands indicated the presence of other, related sequences. The weaker bands were specific to each probe and were not due to cross-hybridization with the other probes examined here. The endosperm AGP small subunit (bepsF2) majorband locus was associated with chromosome 1P and designated Aga1. The endosperm AGP large subunit (bepl10) major-band locus was mapped to chromosome 5M and designated Aga7. The endosperm AGP large-subunit minor bands were not mapped. The leaf AGP large-subunit major band was associated with chromosome 7M and designated Aga5. One of the leaf AGP large-subunit minor bands was mapped to chromosome 5P and designated Aga6. A clone for the wheat endosperm AGP large-subunit (pAga7) hybridized to the same barley genomic DNA bands as the corresponding barley probe indicating a high degree of identity between the two probes.     

Esterase genes in parallel composite cross barley populations

The California population of Composite Cross V of barley was used as the source of three subpopulations that were started from generations 10, 20 and 30, respectively, and were grown in parallel environmental conditions in Cambridge for eight generations. Outcrossing rates (0.2%) were even lower than in the California material, and heterozygotes were correspondingly rare, so that the populations were essentially mixtures of homozygous lines. Four esterase loci that were polymorphic in the base Composite Cross V remained so in all the derived populations, but showed considerable changes in allelic frequency over time, particularly at two of the genes. Multilocus analysis showed that strong directional changes occurred in all three populations, but they were not consistent. One particular genotype became predominant in the population derived from generation 10, whereas in the other two populations it was a genotype with different alleles at the Est1 and Est3 loci that rose to frequencies of more than 50%. Strong directional selection undoubtedly occurred in these populations, but did not cause parallel changes in esterase gene frequencies. These data do not facilitate a discrimination between the alternative explanations of hitchhiking or multilocus selection at these loci.     

Identification and mapping of a gene conferring resistance to the spot form of net blotch (Pyrenophora teres f maculata) in barley

 Spot form of net blotch (SFNB) (Pyrenophora teres f maculata) is an economically damaging foliar disease of barley in many of the world’s cereal growing areas. The development of SFNB-resistant cultivars may be accelerated through the use of molecular markers. A screen for SFNB resistance in 96 lines identified four new sources of resistance, including a feed variety, ‘Galleon’, for which a fully mapped doubled haploid population was available. Segregation data indicated SFNB resistance was conferred by a single gene in the ‘Galleon’בHaruna Nijo’ cross, positioned on the long arm of chromosome 7H. This gene is designated Rpt4 and is flanked by the RFLP loci Xpsr117(D) and Xcdo673 at distances of 6.9 cM and 25.9 cM, respectively. The marker Xpsr117(D) was validated using another population segregating for Rpt4, correctly predicting SFNB resistance with more than 90% accuracy. Received: 24 September 1998 / Accepted: 19 December 1998     

Effect of chitosan and vanillin-modified chitosan on wheat resistance to spot blotch

The effect of chitosan- and vanillin-based immune modulators on the development of the phytopathogen Cochliobolus sativus (S. Ito & Kurib.) Drechsler ex Dastur, which induces dark-brown blotch (helminthosporiosis) in wheat, has been studied. It was shown that treatment with these substances led to a decreased injured area in leaves and an increase in the biotrophic period of pathogen development. It was found that vanillin-modified chitosan effectively provided wheat resistance to hemibiotrophic pathogen C. sativus. Changes in leaf peroxidase activity correlated with the manifestation of disease symptoms.