H. W. Ohm

Inheritance of resistance to Fusarium graminearum in wheat.

Abstract To study the inheritance of resistance in wheat to Fusarium graminearum, six resistant cultivars from China were crossed to two susceptible cultivars. The parents and their progenies were evaluated in the greenhouse for resistance to the spread of scab within a spike. A central floret was inoculated by injecting a droplet of inoculum at the time of anthesis. Inoculated plants were kept in a moist chamber for three subsequent nights. The proportion of scabbed spikelets was recorded six-times from 3-days to 21-days after inoculation, and the area under the disease progress curve (AUDPC) was calculated from these proportions. One to three genes, depending on the cultivar, conditioned resistance to scab as reflected by the AUDPC. A simple additive-dominance effect model fitted the segregation data for 8 of the 11 crosses. Dominance and epistatic effects were significant in a few crosses. These effects increased resistance in some crosses but decreased resistance in others. However, relative to additive effects, dominant and epistatic effects accounted for only a small portion of the genetic effects in the populations evaluated. The importance of additive effects means that it should be possible to accumulate different genes to enhance resistance to scab in wheat.

Detection of DNA sequence polymorphisms among wheat varieties.

SummaryA DNA marker detection strategy that allows the rapid, efficient resolution of high levels of polymorphism among closely related lines of common wheat (Triticum aestivum) has been developed to circumvent the apparent lack of restriction fragment length polymorphism in many important self-pollinated crop species. The technique of randomly amplified polymorphic DNA (RAPD) was combined with a denaturing gradient gel electrophoresis system (DGGE) to explore DNA sequence polymorphisms among different genotypes of wheat. Of the 65 primer combinations used for the polymerase chain reaction (PCR) amplifications, over 38% of them produced readily detectable and reproducible DNA polymorphisms between a spring wheat line, SO852, and a winter wheat variety, ‘Clark’. A high level of polymorphism was observed among a number of commercial varieties and breeding lines of wheat. This procedure was also used to detect polymorphisms in a recombinant inbred population to test the feasibility of its application in genome mapping. This DNA polymorphism detection system provides an opportunity for pedigree analysis and fingerprinting of developed wheat lines as well as construction of a high density genetic map of wheat. Without the need for 32P and sophisticated DNA extraction procedures, this approach should make it feasible to utilize marker-based selection in a plant breeding program.

Comparison of genetic and cytogenetic maps of hexaploid wheat (Triticum aestivum L.) using SSR and DArT markers

A number of technologies are available to increase the abundance of DNA markers and contribute to developing high resolution genetic maps suitable for genetic analysis. The aim of this study was to expand the number of Diversity Array Technology (DArT) markers on the wheat array that can be mapped in the wheat genome, and to determine their chromosomal location with respect to simple sequence repeat (SSR) markers and their position on the cytogenetic map. A total of 749 and 512 individual DArT and SSR markers, respectively, were identified on at least one of four genetic maps derived from recombinant inbred line (RIL) or doubled haploid (DH) populations. A number of clustered DArT markers were observed in each genetic map, in which 20–34% of markers were redundant. Segregation distortion of DArT and SSR markers was also observed in each mapping population. Only 14% of markers on the Version 2.0 wheat array were assigned to chromosomal bins by deletion mapping using aneuploid lines. In this regard, methylation effects need to be considered when applying DArT marker in genetic mapping. However, deletion mapping of DArT markers provides a reference to align genetic and cytogenetic maps and estimate the coverage of DNA markers across the wheat genome.

Identification of RAPD markers for 11 Hessian fly resistance genes in wheat

Abstract The pyramiding of genes that confer race- or biotype-specific resistance has become increasingly attractive as a breeding strategy now that DNA-based marker-assisted selection is feasible. Our objective here was to identify DNA markers closely linked to genes in wheat (Triticum aestivum L.) that condition resistance to Hessian fly [Mayetiola destructor (Say)]. We used a set of near-isogenic wheat lines, each carrying a resistance gene at 1 of 11 loci (H3, H5, H6, H9, H10, H11, H12, H13, H14, H16 or H17) and developed by backcrossing to the Hessian fly-susceptible wheat cultivar ‘Newton’. Using genomic DNA of these 11 lines and ‘Newton’, we have identified 18 randomly amplified polymorphic DNA (RAPD) markers linked to the 11 resistance genes. Seven of these markers were identified by denaturing gradient gel electrophoresis and the others by agarose gel electrophoresis. We confirmed linkage to the Hessian fly resistance loci by cosegregation analysis in F2 populations of 50–120 plants for each different gene. Several of the DNA markers were used to determine the presence/absence of specific Hessian fly resistance genes in resistant wheat lines that have 1 or possibly multiple genes for resistance. The use of RAPD markers presents a valuable strategy for selection of single and combined Hessian fly resistance genes in wheat improvement.

Pedigree assessment using RAPD-DGGE in cereal crop species

SummaryThe introduction of molecular biology methodologies to plant improvement programs offers an invaluable opportunity for extensive germplasm characterization. However, the detection of adequate DNA polymorphism in self-pollinating species remains on obstacle. We have optimized a denaturing-gradient-gel electrophoresis (DGGE) system which, when used in combination with random amplified polymorphic DNA (RAPD) analysis, greatly facilitates the detection of reproducible DNA polymorphism among closely related plant lines. We have used this approach to estimate pedigree relationships among a spectrum of plant materials in wheat, barley and oat. Based on analysis with one or two primers, we were able to distinguish soft from hard winter wheat, and 2-rowed from 6-rowed barley. Further analysis with additional primers allowed resolution of polymorpisms even among closely related lines in highly selected populations. We placed 17 cultivars of oat into two distinct clusters that differed significantly from previous oat pedigree assessments. We believe that DGGE-RAPD is a superior method for detecting DNA polymorphism when compared to RFLP, agarose-RAPD, or polyacrylamide-RAPD methods.

Identification of RAPD markers linked to the gene PM 1 for resistance to powdery mildew in wheat

Abstract Powdery mildew caused by Blumeria graminis DC. f. sp. triticiÉm. Marchal is an important disease of wheat (Triticum aestivum L. em Thell). We report here the identification of three random amplified polymorphic DNA (RAPD) markers closely linked to a gene for resistance to B. graminis in wheat. RAPD-PCR (polymerase chain reaction) analysis was conducted using bulked segregant analysis of closely related lines developed from a segregating F5 family. The F5 family was derived from a cross between the susceptible cultivar Clark and the resistant line Zhengzhou 871124. Genetic analysis indicated that resistance of Zhengzhou 871124 to powdery mildew is conferred by the gene Pm1. After performing RAPD-PCR analysis with 1300 arbitrary 10-mer primers and agarose-gel electrophoresis, two RAPD markers, UBC320420 and UBC638550, were identified to be co-segregating with the disease resistance. No recombinants were observed between either of the RAPD markers and the gene for resistance to powdery mildew after analysis of 244 F2 plants. The third RAPD marker, OPF12650, was identified with denaturing gradient-gel electrophoresis (DGGE), and was determined to be 5.4±1.9 cM from the resistance gene. UBC320420 and UBC638550 were present in wheat powdery mildew differential lines carrying the gene Pm1, suggesting linkage between these markers and the Pm1 resistance gene. Co-segregation between Pm1 and the two markers UBC320420 and UBC638550 was confirmed in a segregating population derived from a cross with CI14114, the wheat differential line carrying Pm1. The method of deriving closely related lines from inbred families that are segregating for a trait of interest should find wide application in the identification of DNA markers linked to important plant genes. The RAPD marker UBC638550 was converted to a sequence tagged site (STS). RAPD markers tightly linked to target genes may facilitate selection and enable gene pyramiding for powdery mildew resistance in wheat breeding programs.

Fusarium head blight resistance in hexaploid wheat (Triticum aestivum)-Lophopyrum genetic lines and tagging of the alien chromatin by PCR markers

The objective of this research was to identify Fusarium head blight (FHB) resistance in wheat (Triticum aestivum)-Lophopyrum genetic lines that might complement FHB resistance in common wheat; and to identify DNA markers that can be used to tag the resistance gene in the alien chromatin (E or el2 genome) for the development of improved wheat cultivars. FHB resistance was evaluated in 19 Chinese Spring-Lophopyrum elongatum (EE) substitution lines, two Thatcher-L. ponticum (el1 and el2) substitution lines, and four Thatcher-L. ponticum translocation lines. Significant resistance was identified in the substitution lines 7E(7A), 7E(7B), and 7E(7D). The homoeologous chromosome, 7el2,also showed resistance in the Thatcher genetic background. Both the Thatcher-7el1 substitution and translocation lines were susceptible, like Thatcher, indicating that there is no resistance gene on the 7el1 chromosome. Simple sequence repeat (SSR) and cleaved amplified polymorphic sequences (CAPS) in homoeologous group 7 chromosomes were used to identify DNA markers located on 7E and 7el2. As expected, the transferability of wheat SSR markers to Lophopyrum is low. Of the 52 SSR markers that we tested, only five were found to be co-dominant on 7E of L. elongatum versus 7A, 7B, and 7D, one of which is also positive on 7el2. A CAPS marker, derived from the RFLP probe PSR129, can serve as a dominant marker for 7el2 chromatin.

A novel wheat gene encoding a putative chitin-binding lectin is associated with resistance against Hessian fly.

SUMMARY The gene-for-gene interaction triggering resistance of wheat against first-instar Hessian fly larvae utilizes specialized defence response genes not previously identified in other interactions with pests or pathogens. We characterized the expression of Hfr-3, a novel gene encoding a lectin-like protein with 68-70% identity to the wheat germ agglutinins. Within each of the four predicted chitin-binding hevein domains, the HFR-3 translated protein sequence contained five conserved saccharide-binding amino acids. Quantification of Hfr-3 mRNA levels confirmed a rapid response and gradual increase, up to 3000-fold above the uninfested control in the incompatible interaction 3 days after egg hatch. Hfr-3 mRNA abundance was influenced by the number of larvae per plant, suggesting that resistance is localized rather than systemic. In addition, Hfr-3 was responsive to another sucking insect, the bird cherry-oat aphid, but not to fall armyworm attack, wounding or exogenous application of methyl jasmonate, salicylic acid or abscisic acid. Western blot analysis demonstrated that HFR-3 protein increased in parallel to mRNA levels in crown tissues during incompatible interactions. HFR-3 protein was detected in both virulent and avirulent larvae, indicating ingestion. Anti-nutritional proteins, such as lectins, may be responsible for the apparent starvation of avirulent first-instar Hessian fly larvae during the initial few days of incompatible interactions with resistant wheat plants.

Phenotypic assessment and mapped markers for H31, a new wheat gene conferring resistance to Hessian fly (Diptera: Cecidomyiidae).

A new source of resistance to the highly virulent and widespread biotype L of the Hessian fly, Mayetiola destructor (Say), was identified in an accession of tetraploid durum wheat, Triticum turgidum Desf., and was introgressed into hexaploid common wheat, Triticum aestivum L. Genetic analysis and deletion mapping revealed that the common wheat line contained a single locus for resistance, H31, residing at the terminus of chromosome 5BS. H31 is the first Hessian fly-resistance gene to be placed on 5BS, making it unique from all previously reported sources of resistance. AFLP analysis identified two markers linked to the resistance locus. These markers were converted to highly specific sequence-tagged site markers. The markers are being applied to the development of cultivars carrying multiple genes for resistance to Hessian fly biotype L in order to test gene pyramiding as a strategy for extending the durability of deployed resistance.

DNA markers associated with low Fusarium head blight incidence and narrow flower opening in wheat

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum, is an important fungal disease in many wheat-growing areas of the world. The objectives of this study were to determine the relationship between width and duration of flower opening and incidence of FHB in wheat, and to identify DNA markers associated with narrow flower opening and low FHB incidence. It was hypothesized that wheat lines whose flowers open briefly and narrowly have a reduced risk of infection. To test the hypothesis, we crossed wheat cultivars Patterson and Goldfield to generate a population of 100 random F2-derived recombinant inbred lines (RILs). Florets of Patterson open wide; florets of Goldfield tend to stay closed. The population of RILs was characterized for FHB incidence and flower opening width (FOW) and duration in the F7:9 and F7:10 generations. Of the 305 simple sequence repeat primer pairs screened on the parents, 79 amplified polymorphic DNA bands. Pooled DNA from each of the two bulks was tested with these 79 SSR primer pairs. Four markers were found to have significant marker-trait association with low FHB incidence and narrow flower opening. The major QTL effect associated with narrow flower opening and low FHB incidence was found between the map interval Xbarc200–Xgwm210, explaining 29% of the phenotypic variation for FHB incidence averaged over six replicated tests in Indiana in 2002 and 2003. This adds credence to the hypothesis that narrow flower opening is responsible for low FHB incidence in this population. Breeding wheat lines for both morphological avoidance, such as narrow flower opening, and physiological resistance to FHB may be valuable in future breeding research to reduce crop production and grain quality losses in wheat due to FHB.