Genome-Wide Association Study of Spot Form of Net Blotch Resistance in the Upper Midwest Barley Breeding Programs.

Pyrenophora teres f. maculata, the causal agent of spot form of net blotch (SFNB), is an emerging pathogen of barley in the United States and Australia. Compared with net form of net blotch (NFNB), less is known in the U.S. Upper Midwest barley breeding programs about host resistance and quantitative trait loci (QTL) associated with SFNB in breeding lines. The main objective of this study was to identify QTL associated with SFNB resistance in the Upper Midwest two-rowed and six-rowed barley breeding programs using a genome-wide association study approach. A total of 376 breeding lines of barley were evaluated for SFNB resistance at the seedling stage in the greenhouse in Fargo in 2009. The lines were genotyped with 3,072 single nucleotide polymorphism (SNP) markers. Phenotypic evaluation showed a wide range of variability among populations from the four breeding programs and the two barley-row types. The two-rowed barley lines were more susceptible to SFNB than the six-rowed lines. Continuous distributions of SFNB severity indicate the quantitative nature of SFNB resistance. The mixed linear model (MLM) analysis, which included both population structure and kinship matrices, was used to identify significant SNP-SFNB associations. Principal component analysis was used to control false marker-trait association. The linkage disequilibrium (LD) estimates varied among chromosomes (10 to 20 cM). The MLM analysis identified 10 potential QTL in barley: SFNB-2H-8-10, SFNB-2H-38.03, SFNB-3H-58.64, SFNB-3H-78.53, SFNB-3H-91.88, SFNB-3H-117.1, SFNB-5H-155.3, SFNB-6H-5.4, SFNB-6H-33.74, and SFNB-7H-34.82. Among them, four QTL (SFNB-2H-8-10, SFNB-2H-38.03 SFNB-3H-78.53, and SFNB-3H-117.1) have not previously been published. Identification of SFNB resistant lines and QTL associated with SFNB resistance in this study will be useful in the development of barley genotypes with better SFNB resistance.

Markers for seedling and adult plant crown rot resistance in four partially resistant bread wheat sources.

KEY MESSAGE: QTL identified for seedling and adult plant crown rot resistance in four partially resistant hexaploid wheat sources. PCR-based markers identified for use in marker-assisted selection. Crown rot, caused by Fusarium pseudograminearum, is an important disease of wheat in many wheat-growing regions globally. Complete resistance to infection by F. pseudograminearum has not been observed in a wheat host, but germplasm with partial resistance to this pathogen has been identified. The partially resistant wheat hexaploid germplasm sources 2-49, Sunco, IRN497 and CPI133817 were investigated in both seedling and adult plant field trials to identify markers associated with the resistance which could be used in marker-assisted selection programs. Thirteen different quantitative trait loci (QTL) conditioning crown rot resistance were identified in the four different sources. Some QTL were only observed in seedling trials whereas others appeared to be adult plant specific. For example while the QTL on chromosomes 1AS, 1BS, and 4BS contributed by 2-49 and on 2BS contributed by Sunco were detected in both seedling and field trials, the QTL on 1DL present in 2-49 and the QTL on 3BL in IRN497 were only detected in seedling trials. Genetic correlations between field trials of the same population were strong, as were correlations between seedling trials of the same population. Low to moderate correlations were observed between seedling and field trials. Flanking markers, most of which are less than 10 cM apart, have now been identified for each of the regions associated with crown rot resistance.

Green and brown bridges between weeds and crops reveal novel Diaporthe species in Australia.

Diaporthe (syn. Phomopsis) species are well-known saprobes, endophytes or pathogens on a range of plants. Several species have wide host ranges and multiple species may sometimes colonise the same host species. This study describes eight novel Diaporthe species isolated from live and/or dead tissue from the broad acre crops lupin, maize, mungbean, soybean and sunflower, and associated weed species in Queensland and New South Wales, as well as the environmental weed bitou bush (Chrysanthemoides monilifera subsp. rotundata) in eastern Australia. The new taxa are differentiated on the basis of morphology and DNA sequence analyses based on the nuclear ribosomal internal transcribed spacer region, and part of the translation elongation factor-1α and ß-tubulin genes. The possible agricultural significance of live weeds and crop residues ('green bridges') as well as dead weeds and crop residues ('brown bridges') in aiding survival of the newly described Diaporthe species is discussed.

Development of Weather-Based Predictive Models for Fusarium Head Blight and Deoxynivalenol Accumulation for Spring Malting Barley.

The associations between Fusarium head blight (FHB), caused by Gibberella zeae, and deoxynivalenol (DON) accumulation in spring malting barley (Hordeum vulgare) and hourly weather conditions predictive of DON accumulation were examined using data from six growing seasons in the U.S. Northern Great Plains. Three commonly grown cultivars were planted throughout the region, and FHB disease and DON concentration were recorded. Nine predictor variables were calculated using hourly temperature and relative humidity during the 10 days preceding full head spike emergence. Simple logistic regression models were developed using these predictor variables based on a binary threshold for DON of 0.5 mg/kg. Four of the nine models had sensitivity greater than 80%, and specificity of these models ranged from 67 to 84% (n = 150). The most useful predictor was the joint effect of average hourly temperature and a weighted duration of uninterrupted hours (h) with relative humidity greater than or equal to 90%. The results of this study confirm that FHB incidence is significantly associated with DON accumulation in the grain and that weather conditions prior to full head emergence could be used to accurately predict the risk of economically significant DON accumulation for spring malting barley.

Stem cankers on sunflower (Helianthus annuus) in Australia reveal a complex of pathogenic Diaporthe (Phomopsis) species.

The identification of Diaporthe (anamorph Phomopsis) species associated with stem canker of sunflower (Helianthus annuus) in Australia was studied using morphology, DNA sequence analysis and pathology. Phylogenetic analysis revealed three clades that did not correspond with known taxa, and these are believed to represent novel species. Diaporthe gulyae sp. nov. is described for isolates that caused a severe stem canker, specifically pale brown to dark brown, irregularly shaped lesions centred at the stem nodes with pith deterioration and mid-stem lodging. This pathogenicity of D. gulyae was confirmed by satisfying Koch's Postulates. These symptoms are almost identical to those of sunflower stem canker caused by D. helianthi that can cause yield reductions of up to 40 % in Europe and the USA, although it has not been found in Australia. We show that there has been broad misapplication of the name D. helianthi to many isolates of Diaporthe (Phomopsis) found causing, or associated with, stem cankers on sunflower. In GenBank, a number of isolates had been identified as D. helianthi, which were accommodated in several clades by molecular phylogenetic analysis. Two less damaging species, D. kochmanii sp. nov. and D. kongii sp. nov., are also described from cankers on sunflower in Australia.

A novel QTL for Septoria speckled leaf blotch resistance in barley (Hordeum vulgare L.) accession PI 643302 by whole-genome QTL mapping.

Septoria speckled leaf blotch (SSLB), caused by Septoria passerinii, is one of the most important foliar diseases of barley (Hordeum vulgare L.) in North America. The primary problem caused by this disease is substantial yield loss. The objective of this study was to determine the chromosomal location of SSLB resistance genes in the barley accession PI 643302. A recombinant inbred line population was developed from the cross Zhenongda 7/PI 643302. PI 643302 is resistant while Zhenongda 7 is susceptible to SSLB. The population was phenotyped for SSLB resistance in five experiments in the greenhouse. A linkage map comprising 113 molecular markers was constructed and simplified composite interval mapping was performed. Two QTLs, designated QrSp-1H and QrSP-2H, were found. QrSp-1H was found on the short arm of chromosome 1H (1HS) in all five experiments and showed a large effect against SSLB. Based on the location of QrSp-1H, it is likely the SSLB resistance gene Rsp2. The QTL QrSp-2H mapped to the distal region on the long arm of chromosome 2H (2HL), had a smaller effect than QrSp-1H, and was also detected consistently in all five experiments. A QTL for SSLB resistance in the same region on chromosome 2H has not been reported previously in either cultivated or wild barley; thus, QrSp-2H is a new QTL for SSLB resistance in barley.

A native QTL for Fusarium head blight resistance in North American barley (Hordeum vulgare L.) independent of height, maturity, and spike type loci.

Fusarium head blight (FHB), caused by Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schwein.) Petch), is one of the major diseases of barley (Hordeum vulgare L.) in eastern China, the Upper Midwest of the USA, and the eastern Prairie Provinces of Canada. To identify quantitative trait loci (QTL) controlling FHB resistance, a recombinant inbred line population (F6:7) was developed from the cross Zhenongda 7/PI 643302. The population was phenotyped for resistance to FHB in two experiments in China and four experiments in North Dakota. Accumulation of the mycotoxin deoxynivalenol was determined in one experiment in China and two in North Dakota. Simplified composite interval mapping was performed on the whole genome level using the software MQTL. The QTL FHB-2 from PI 643302 for FHB resistance was found on the distal portion of chromosome 2HL in all six FHB screening environments. This QTL accounted for 14% of phenotypic variation over six environments and was not associated with heading date or plant height. The FHB resistance QTL FHB-2 detected near the end of chromosome 2HL is in a different location from those found previously and is therefore probably unique. Because the QTL was not contributed by the Chinese cultivar Zhenongda 7, it is likely a native QTL present in North American barley. The QTL FHB-2 represents the first reported QTL for native FHB resistance in North American germ plasm and has been given the provisional name Qrgz-2H-14. This QTL should be considered for pyramiding with other FHB QTL previously mapped.

Genetic relationships among populations of Gibberella zeae from barley, wheat, potato, and sugar beet in the upper Midwest of the United States.

Gibberella zeae, a causal agent of Fusarium head blight (FHB) in wheat and barley, is one of the most economically harmful pathogens of cereals in the United States. In recent years, the known host range of G. zeae has also expanded to noncereal crops. However, there is a lack of information on the population genetic structure of G. zeae associated with noncereal crops and across wheat cultivars. To test the hypothesis that G. zeae populations sampled from barley, wheat, potato, and sugar beet in the Upper Midwest of the United States are not mixtures of species or G. zeae clades, we analyzed sequence data of G. zeae, and confirmed that all populations studied were present in the same clade of G. zeae. Ten variable number tandem repeat (VNTR) markers were used to determine the genetic structure of G. zeae from the four crop populations. To examine the effect of wheat cultivars on the pathogen populations, 227 strains were sampled from 10 subpopulations according to wheat cultivar types. The VNTR markers also were used to analyze the genetic structure of these subpopulations. In all populations, gene (H = 0.453 to 0.612) and genotype diversity (GD = or >0.984) were high. There was little or no indication of linkage disequilibrium (LD) in all G. zeae populations and subpopulations. In addition, high gene flow (Nm) values were observed between cereal and noncereal populations (Nm = 10.69) and between FHB resistant and susceptible wheat cultivar subpopulations (Nm = 16.072), suggesting low population differentiation of G. zeae in this region. Analysis of molecular variance also revealed high genetic variation (>80%) among individuals within populations and subpopulations. However, low genetic variation (<5%) was observed between cereal and noncereal populations and between resistant and susceptible wheat subpopulations. Overall, these results suggest that the populations or subpopulations are likely a single large population of G. zeae affecting crops in the upper Midwest of the United States.

Intraspecific variation of Rhizoctonia solani AG 3 isolates recovered from potato fields in Central Iran and South Australia.

Pectic zymogram, RFLP and PCR analyses were used to characterize Rhizoctonia solani AG 3 isolates collected from diseased potatoes in South Australia. The pectic zymogram data were compared with those obtained for isolates collected from central Iran. Analyses of bands corresponding to pectin esterase and polygalacturonase revealed three zymogram subgroups (ZG) in AG 3. In addition to the previously reported ZG7 (here renamed ZG7-1), two new zymogram subgroups, ZG7-2 and ZG7-3, were identified. Of the 446 isolates tested, 50% of the South Australian and 46% of the Iranian isolates were ZG7-1. The majority of the isolates originating from stem and root cankers were ZG7-1, whereas most of the isolates designated ZG7-2 and ZG7-3 originated from tuber-borne sclerotia. Pathogenicity tests revealed that ZG7-1 generally produced fewer sclerotia and more severe cankers of underground parts of the potato plants than the other two ZGs. Two random DNA clones, one originating from an AG 3 isolate and the other from an AG 4 isolate, were used as probes for RFLP analyses of Australian isolates. The AG 3 probe, previously identified to be specific to this group, detected a high level of genetic diversity, with 11 genotypes identified amongst 50 isolates analysed. The low-copy AG 4 probe resolved three genotypes amongst 24 isolates. For 23 isolates analysed with both markers, the combined data distinguished a total of six genotypes and similarity analysis resolved the isolates into two main groups with 50% homology. PCR, using primers for the plant intron splice junction region (R1), also revealed variation. No obvious relationship among pectic zymogram groups, RFLP and PCR genotypes was observed.

Population Genetic Structure of Septoria passerinii in Northern Great Plains Barley.

ABSTRACT The genetic structure of Septoria passerinii from nine field populations was examined at several scales (within lesions, among lesions in a leaf, among leaves in a field, and among fields in North Dakota and western Minnesota) by using amplified fragment length polymorphism (AFLP) markers. A total of 390 isolates were sampled from seven barley fields located in North Dakota and two barley fields located nearby in western Minnesota in 2003 and 2004. Based on 57 polymorphic AFLP markers, AFLP DNA fingerprints identified 176 different genotypes among 390 (non-clone-corrected) isolates in nine different fields. In two intensively sampled sites, ND16 (Williston, ND) and ND17 (Langdon, ND), only one to four different genotypes were found within a lesion. A higher level of genetic and genotypic diversity was found within a leaf in which six to nine different genotypes were found from lesions on a leaf. The genetic diversity within a leaf was similar to the genetic diversity within a field. The average genetic diversity (H) within a field across all AFLP loci was approximately 0.3, except at site ND12 (Carrington, ND) where it was 0.16. Genotypic diversity was high in all populations, and with the exception of ND15 (Rothsay, MN), very low multilocus linkage disequilibrium values ( r(d)) were found in all populations. The population differentiation, G(ST), was relatively high (G(ST) = 0.238) among the nine populations due to the high G(ST) in ND12, ND14 (Twin Valley, MN), and ND15. Population differentiation without those three populations was 0.09. A lack of correlation between geographical distance and genetic distance was found, suggesting the potential for a high level of gene flow between different geographical regions. The population genetic structure described in this study for S. passerinii in North Dakota and western Minnesota is consistent with that of a sexually reproducing fungus.

Molecular mapping of rsp1, rsp2, and rsp3 genes conferring resistance to septoria speckled leaf blotch in barley.

ABSTRACT Septoria speckled leaf blotch (SSLB) caused by Septoria passerinii is a common disease in barley. SSLB resistance genes Rsp1, Rsp2, and Rsp3 have previously been identified in the United States Department of Agriculture National Small Grains collection accessions CIho 14300, CIho 4780, and CIho 10644, respectively. Populations of 100 to 120 F(2) individuals were evaluated for SSLB resistance in the greenhouse. Inheritance was evaluated in F(2:3)-derived families in the field. Partial molecular maps for three Rsp genes were constructed on F(2) and F(2:3) families derived from crosses between Robust and the resistant accessions CIho 14300, CIho 4780, and CIho 10644. The resistant locus Rsp1 was mapped to the short arm of chromosome 3H with two flanking diversity arrays technology (DArT) markers, bPb-6978 (8.9 cM) and bPb-9945 (16.3 cM), and two random amplified polymorphic DNA (RAPD) markers, OPC2(441R) (3.0 cM) and UBC285(158R) (4.3 cM). The genes Rsp2 and Rsp3 were positioned on the short arm of barley chromosome 1H with two restriction fragment length polymorphism (RFLP), six DArT, and three RAPD markers. An RFLP marker, MWG938, and an RAPD marker, OPAH5(545C), were tightly associated with Rsp2 at a distance of 0 cM. Five DArT markers spanning the short arm of 1H surrounded Rsp3 at a distance of 2.3 and 5.8 cM, while two RAPD markers-OPBA12(314C) (2.4 cM) in coupling and OPB17(451R) (3.5 cM) in repulsion-flanked Rsp3. Molecular marker data associated with Rsp2 and Rsp3 indicated that the two genes are closely linked on chromosome 1HS. A total of 17 of 154 simple sequence repeats (SSRs) tested were associated with Rsp genes on chromosome 1H and 3H, and they were also integrated into genetic linkage maps of the three F(2) Robust populations. Knowledge about the map position of Rsp genes on barley chromosomes will be useful for breeding for SSLB resistance in barley and eventual gene cloning.

Sequence tagged site markers to rsp1, rsp2, and rsp3 genes for resistance to septoria speckled leaf blotch in barley.

ABSTRACT Five random amplified polymorphic DNA markers, two in coupling (OPAH5(545C), and OPBA12(314C)) and three in repulsion phase (UBC285(158R), OPC2(441R), and OPB17(451R)), closely linked to Rsp genes conferring resistance to Septoria speckled leaf blotch (SSLB), were identified using bulked segregant analysis in three F(2) populations, each containing a Rsp gene. These markers were converted into the sequence tagged site (STS) markers SUBC285, SOPC2, SOPAH5, and SOPBA12. Another STS marker (MWG938) linked to Rsp2 in coupling phase was also identified in an F(2) population from the cross Robust/CIho 4780. The STS markers were tested on a set of 42 resistant and susceptible barley germplasm lines and 98 landraces. The expected sizes of marker fragments associated with each allele at Rsp loci were present in resistant or susceptible accessions. Efficiency of marker-assisted selection (MAS) for Rsp1, Rsp2, and Rsp3 using STS markers were evaluated in three F(23) populations in the greenhouse and the field. Results of testing F(23) progeny demonstrated that the accuracy of MAS was, with one exception, greater than 97% in the greenhouse and in two field locations (90% in the Osnabrock, ND trial for Rsp2). The STS markers closely linked to Rsp genes also identified the SSLB resistance corresponding to Rsp1, Rsp2, or Rsp3 in gene pyramiding F(2) populations. The STS markers tightly linked to Rsp genes may be useful for M and for pyramiding with other genes in barley breeding for SSLB resistance.

Widespread Occurrence of Ergot in Upper Midwestern U.S. Barley, 2005.

Ergot, caused by Claviceps purpurea (Fr.) Tul., occurs every year on cereals and grasses in North Dakota, but the occurrence on barley (Hordeum vugare L) is generally sporadic with a very low incidence of sclerotia. Disease surveys conducted during the 2005 growing season revealed an unusually widespread occurrence. This is of concern since barley production in North Dakota was estimated at 1.25 million metric tons, 27% of the total 2005 U.S. production. Barley samples (n = 304, ~0.50 kg) collected in all crop-reporting districts of North Dakota, northwestern Minnesota, and northeastern Montana, as part of an annual regional survey of barley crop quality (4), were examined for sclerotia. All barley samples were cleaned for dockage, and ergot (% [w/w]) was estimated on subsamples of ~100 g from a sample divider. Of all barley samples collected, 62% contained ergots. The regional average for ergot infested kernels was 0.06%, and samples ranged from <0.01 to 1.19%. Approximately 15% of all samples were in excess of 0.10% ergots and would have been downgraded to ergoty barley under the Official United States Standards for Grain. Occurrence of ergot was most common in northwestern Minnesota and the three eastern and north-central districts of North Dakota. Ergot was less frequent in the south-central and three western districts of North Dakota and was not detected in samples from northeastern Montana. Floret infection occurs during and up to 15 days after anthesis (2), and in the three eastern and north-central districts of North Dakota that occurred around the last week in June and first week in July. Between 22 June and 4 July, the North Dakota Agricultural Weather Network Stations in that region recorded average daily temperatures of 99% of the 30-year norm, but multiple precipitation events amounted to 227% of the 30-year norm. Rain splash and associated high relative humidity favor conidiation and spread of the fungus (1) and may have contributed to the high disease incidence. Average sclerotia weight for a sample ranged from <10 to 70 mg. However, large sclerotia (37 to 180 mg) often were removed by the no. 6 riddle of the dockage tester and were not counted in the ergot estimates as per U.S. Grading Standards. Samples containing 1.19, 0.81, 0.22, 0.14, 0.05, and 0.02% ergots were analyzed for ergopeptine alkaloids (3). These were found to contain 27.9, 25.4, 2.4, 1.1, 1.7, and 5.7 µg/g ergopeptine alkaloids, respectively. The average ratio of ergosine/ergotamine/ergocornine/ergocryptine/ergocristine was approximately 1:2:2:3:9. There also was widespread occurrence of the Fusarium mycotoxin deoxynivalenol (DON) on North Dakota barley in 2005. While there was no apparent relationship between the level of the DON and the amount of ergot in the samples (r = 0.042), more than 90% of samples with ergot had detectable levels (0.1 to 69 µg/g) of DON. While only DON is routinely measured in the crop survey (4), other tricothecenes and zearalenone have also been detected. This should be of concern to livestock producers and grain processors since the potential interactions of multiple mycotoxins are not well known. References: (1) G. M. Marshall. Ann. Appl. Biol. 48:19, 1960. (2) S. B. Puranik and D. E. Mathre. Phytopathology 61:1075, 1971. (3) G. E. Rottinghaus et al. J. Vet. Diag. Invest. 5:242 1993. (4) P. B. Schwarz et al. J. Am. Soc. Brew. Chem. 64:1, 2006.