Mapping spot blotch resistance genes in four barley populations

Bipolaris sorokiniana (teleomorph: Cochliobolus sativus) is the fungal pathogen responsible for spot blotch in barley (Hordeum vulgare L.) and occurs worldwide in warmer, humid growing conditions. Current Australian barley varieties are largely susceptible to this disease and attempts are being made to introduce sources of resistance from North America. In this study we have compared chromosomal locations of spot blotch resistance reactions in four North American two-rowed barley lines; the North Dakota lines ND11231-12 and ND11231-11 and the Canadian lines TR251 and WPG8412-9-2-1. Diversity arrays technology-based PCR, expressed sequence tag and SSR markers have been mapped across four populations derived from crosses between susceptible parental lines and these four resistant parents to determine the location of resistance loci. Quantitative trait loci (QTL) conferring resistance to spot blotch in adult plants (APR) were detected on chromosomes 3HS and 7HS. In contrast, seedling resistance (SLR) was controlled solely by a locus on chromosome 7HS. The phenotypic variance explained by the APR QTL on 3HS was between 16 and 25% and the phenotypic variance explained by the 7HS APR QTL was between 8 and 42% across the four populations. The SLR QTL on 7HS explained between 52 and 64% of the phenotypic variance. An examination of the pedigrees of these resistance sources supports the common identity of resistance in these lines and indicates that only a limited number of major resistance loci are available in current two-rowed germplasm.     

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.     

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.     

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.     

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.

     

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 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.     

Gene chip analyses reveal differential genetic responses to iron deficiency in rat duodenum and jejunum

Previous studies revealed novel genetic changes in the duodenal mucosa of iron-deprived rats during postnatal development. These observations are now extended to compare the genetic response to iron deficiency in the duodenum versus jejunum of 12-wk-old rats. cRNA samples were prepared from the duodenal and jejunal mucosa of three groups each of control and iron-deficient rats and hybridized with RAE 230A and 230B gene chips (Affymetrix). Stringent data reduction strategies were employed. Results showed that several genes were similarly induced in both gut segments, including DMT1, Dcytb, transferrin receptor 1, heme oxygenase 1, metallothionein, the Menkes copper ATPase (ATP7A), tripartitie motif protein 27, and the sodium-dependent vitamin C transporter. However, a subset of genes showed regulation in only one or the other gut segment. In duodenum only, gastrokine 1, trefoil factor 1 and claudin 2 were induced by iron-deficiency. Other genes previously identified were only regulated in the duodenum. Overall, these studies demonstrate similarities and distinct differences in the genetic response to iron deprivation in the duodenum versus jejunum and provide evidence that more distal gut segments also may play a role in increasing iron absorption in iron-deficiency anemia.     

Spatial genetic analyses reveal strong genetic structure in two populations of the outcrossing tree fern Alsophila firma (Cyatheaceae)

The development of spatial genetic structure (SGS) in seed plants has been linked to several biological attributes of species, such as breeding system and life form. However, little is known about SGS in ferns, which together with lycopods are unique among land plants in having two free‐living life stages. We combined spatial aggregation statistics and spatial genetic autocorrelation analyses using five plastid microsatellites and one nuclear gene to investigate SGS in two populations of the outcrossing tree fern Alsophila firma (Cyatheaceae). We assessed how the observed patterns compare with those estimated for other ferns and seed plants. Populations of A. firma exhibited strong SGS, spatial clustering of individuals, substantial clonal diversity and no inbreeding. SGS in ferns appears to be higher than in most seed plants analysed to date. Contrary to our expectations, an outcrossing breeding system, wind dispersal and an arborescent life form did not translate into weak or no SGS. In ferns, SGS is probably being affected by the life cycle with two free‐living life stages. The reproductive biology of ferns appears to be more complex than previously thought. This implies that SGS in ferns is affected by some factors that cannot be inferred from the study of flowering plants. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 439–449.     

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.     

Microsatellites reveal genetic diversity in Rotylenchulus reniformis populations

Rotylenchulus reniformis is the predominant parasitic nematode of cotton in the Mid South area of the United States. Although variable levels of infection and morphological differences have been reported for this nematode, genetic variability has been more elusive. We developed microsatellite-enriched libraries for R. reniformis, produced 1152 clones, assembled 694 contigs, detected 783 simple sequence repeats (SSR) and designed 192 SSR-markers. The markers were tested on six R. reniformis cultures from four states, Texas, Louisiana, Mississippi and Georgia, in the USA. Based on performance we selected 156 SSR markers for R. reniformis from which 88 were polymorphic across the six reniform nematode populations, showing as the most frequent motif the dinucleotide AG. The polymorphic information content of the markers ranged from 0.00 to 0.82, and the percentage of multiallelic loci of the isolates was between 40.9 and 45.1%. An interesting finding in this study was the genetic variability detected among the three Mississippi isolates, for which 22 SSR markers were polymorphic. We also tested the level of infection of these isolates on six cotton genotypes, where significant differences were found between the Texas and Georgia isolates. Coincidentally, 62 polymorphic markers were able to distinguish these two populations. Further studies will be necessary to establish possible connections, if any, between markers and level of pathogenicity of the nematode. The SSR markers developed here will be useful in the assessment of the genetic diversity of this nematode, could assist in management practices for control of reniform nematode, be used in breeding programs for crop resistance, and help in detecting the origin and spread of this nematode in the United States.     

Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus

Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multi-phenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and five genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9-Mb chromosome and six plasmids. There were three single nucleotide mutations and five transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature-resistant strain grown at an unviable high-temperature (42°C). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92-kb deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria.     

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 and mapping of net form of net blotch resistance in South African barley

Net form of net blotch (NFNB) caused by the fungus Pyrenophora teres f. teres is an economically important foliar disease of barley (Hordeum vulgare) in southern and eastern Africa. Little attention has been given to disease resistance breeding, and knowledge about the presence of NFNB resistance in breeding lines is limited. Deploying resistance into varieties used in this region is important for future control of the disease. We have identified NFNB disease resistance in existing South African breeders’ lines and have mapped the resistance in line UVC8. Six different trials, three conducted in South Africa and another three in Australia, were used to identify resistance QTL. A major QTL was identified on chromosome 6H having a LOD score of 40.5 and 55% of the phenotypic variance explained. Kompetitive Allele Specific PCR (KASP?) markers were designed for this QTL region. These and microsatellite markers can now be used to routinely select for NFNB resistance.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.