Frank Ordon

Comparative analysis on the genetic relatedness of Sorghum bicolor accessions from Southern Africa by RAPDs, AFLPs and SSRs

Abstract In order to get an overview on the genetic relatedness of sorghum (Sorghum bicolor) landraces and cultivars grown in low-input conditions of small-scale farming systems, 46 sorghum accessions derived from Southern Africa were evaluated on the basis of amplified fragment length polymorphism (AFLPs), random amplified polymorphic DNAs (RAPDs) and simple sequence repeats (SSRs). By this approach all sorghum accessions were uniquely fingerprinted by all marker systems. Mean genetic similarity was estimated at 0.88 based on RAPDs, 0.85 using AFLPs and 0.31 based on SSRs. In addition to this, genetic distance based on SSR data was estimated at 57 according to a stepwise mutation model (Δμ-SSR). All UPGMA-clusters showed a good fit to the similarity estimates (AFLPs: r = 0.92; RAPDs: r = 0.88; SSRs: r = 0.87; Δμ-SSRs: r = 0.85). By UPGMA-clustering two main clusters were built on all marker systems comprising landraces on the one hand and newly developed varieties on the other hand. Further sub-groupings were not unequivocal. Genetic diversity (H, DI) was estimated on a similar level within landraces and breeding varieties. Comparing the three approaches to each other, RAPD and AFLP similarity indices were highly correlated (r = 0.81), while the Spearmans rank correlation coefficient between SSRs and AFLPs was r = 0.57 and r = 0.51 between RAPDs and SSRs. Applying a stepwise mutation model on the SSR data resulted in an intermediate correlation coefficient between Δμ-SSRs and AFLPs (r = 0.66) and RAPDs (r = 0.67), respectively, while SSRs and Δμ-SSRs showed a lower correlation coefficient (r = 0.52). The highest bootstrap probabilities were found using AFLPs (56% on average) while SSR, Δμ-SSR and RAPD-based similarity estimates had low mean bootstrap probabilities (24%, 27%, 30%, respectively). The coefficient of variation (CV) of the estimated genetic similarity decreased with an increasing number of bands and was lowest using AFLPs.

Molecular mapping and genetic fine-structure of the rym5 locus encoding resistance to different strains of the Barley Yellow Mosaic Virus Complex

Abstract The genetic structure of the rym5 locus was studied in a population comprising 391 doubled-haploid lines that were evaluated for resistance to two strains of Barley Yellow Mosaic Virus (BaYMV-1, 2) and to Barley Mild Mosaic Virus (BaMMV). The absence of recombinants that are able to differentiate between the reaction to these different bymoviruses provides evidence that rym5 is a complex locus, which is either composed of several closely linked genes or of an allelic series of a single gene. For marker-assisted introgression of this locus into adapted barley germplasm, a CAPS (cleaved amplified polymorphic sequence) and a microsatellite marker were developed that flank the gene at distances of 0.8 and 1.3% recombination, respectively.

QTL analysis of tolerance to a German strain of BYDV-PAV in barley (Hordeum vulgare L.)

Abstract  One hundred and forty six barley doubled-haploid lines (DH lines) were tested for variation in grain yield, yield components, plant height, and heading date after artificial infection with a German isolate of barley yellow dwarf virus (BYDV-PAV-Braunschweig). Of these 146 lines 76 were derived from the cross of the barley yellow dwarf virus (BYDV) tolerant cultivar ’Post’ to cv ’Vixen’ (Ryd2) and 70 from the cross of Post to cv ’Nixe’. Phenotypic measurements were gathered on both non-infected plants and plants artificially inoculated with BYDV-PAV by viruliferous aphids in pot and field experiments for three years at two locations. For all traits a continuous variation was observed suggesting a quantitative mode of inheritance for tolerance against BYDV-PAV. Using skeleton maps constructed using SSRs, AFLPs and RAPDs, two QTLs for relative grain yield per plant after BYDV infection, explaining about 47% of the phenotypic variance, were identified in Post × Vixen at the telomeric region of chromosome 2HL and at a region containing the Ryd2 gene on chromosome 3HL. In Post × Nixe, a QTL was found in exactly the same chromosome 2HL marker interval. In this cross, additional QTL were mapped on chromosomes 7H and 4H and together these explained about 40% of the phenotypic variance. QTL for effects of BYDV infection on yield components, plant height, and heading date generally mapped to the same marker intervals, or in the vicinity of the QTL for relative grain yield, on chromosomes 2HL and 3HL, suggesting that these regions are of special importance for tolerance to the Braunschweig isolate of BYDV-PAV. Possible applications of marker-assisted selection for BYDV tolerance based on these results are discussed.

Molecular mapping of novel resistance genes against Barley Mild Mosaic Virus (BaMMV)

Abstract In the present study three novel genes from barley accessions 10247 (ym8), Bulgarian 347 (ym9), and Russia 57 (ym11), which confer resistance to Barley Mild Mosaic Virus (BaMMV), were mapped using molecular markers. Bulked segregant analysis of four progenies segregating for resistance to BaMMV was followed by fine-scale mapping of the resistance genes using individual F2 or BC1F2 plants. The resistance genes are inherited recessively and are located on the long arm of barley chromosome 4HL. A series of closely linked molecular markers are available for marker-assisted breeding programs. A marker (MWG2134) linked with resistance gene ym11 from Russia 57 was identified, which is diagnostic for the resistance gene.

A novel major gene on chromosome 6H for resistance of barley against the barley yellow dwarf virus

In a mapping population derived from the Ethiopian barley line L94 × Vada, natural infection by barley yellow dwarf virus (BYDV) occurred. While line L94 hardly showed symptoms, Vada was severely affected. The 103 recombinant inbred lines segregated bimodally. The major gene responsible for this resistance mapped to chromosome 6H. We propose to name the locus Ryd3. A subset of recombinant inbred lines, L94, and Vada were planted in a subsequent field test which confirmed the previous field observations. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) indicated that the epidemic was due to a combination of the serotypes BYDV-PAV and BYDV-MAV. In the accessions with the least BYDV symptoms no virus was detected, justifying the consideration of the gene as conferring true resistance rather than tolerance to these viruses. In a laboratory/gauze house trial a near-isogenic line carrying the Vada chromosome 6H fragment in an L94 background was affected as much as Vada. The effect of Ryd3 was quantified, and compared with that of the only other known major gene for resistance to BYDV, Ryd2, which is also of Ethiopian origin and is located on chromosome 3H. Both genes seemed to reduce the chance of the viral isolate used in this study to establish infection. In plants in which it became established, the virus concentration reached a similar level as in susceptible accessions, but with less dramatic symptom development. Inoculated plants in which the virus failed to multiply tended to show an increase in the number of ears per plant, resulting in higher grain yield per plant. Ryd3 co-segregates with several PCR-based molecular markers that may serve for marker assisted selection.

Effects of Introgression and Recombination on Haplotype Structure and Linkage Disequilibrium Surrounding a Locus Encoding Bymovirus Resistance in Barley

We present a detailed analysis of linkage disequilibrium (LD) in the physical and genetic context of the barley gene Hv-eIF4E, which confers resistance to the barley yellow mosaic virus (BYMV) complex. Eighty-three SNPs distributed over 132 kb of Hv-eIF4E and six additional fragments genetically mapped to its flanking region were used to derive haplotypes from 131 accessions. Three haplogroups were recognized, discriminating between the alleles rym4 and rym5, which each encode for a spectrum of resistance to BYMV. With increasing map distance, haplotypes of susceptible genotypes displayed diverse patterns driven mainly by recombination, whereas haplotype diversity within the subgroups of resistant genotypes was limited. We conclude that the breakdown of LD within 1 cM of the resistance gene was generated mainly by susceptible genotypes. Despite the LD decay, a significant association between haplotype and resistance to BYMV was detected up to a distance of 5.5 cM from the resistance gene. The LD pattern and the haplotype structure of the target chromosomal region are the result of interplay between low recombination and recent breeding history.

Molecular assessment of genetic diversity in winter barley and its use in breeding

SummaryDuring the last decades extensive progress has been achieved in winter barley breeding with respect to both, yield and resistance to fungal and viral diseases. This progress is mainly due to the efficient use of the genetic diversity present within high yielding adapted cultivars and – with respect to resistance – to the extensive evaluation of genetic resources followed by genetic analyses and introgression of respective genes by sexual recombination. Detailed knowledge on genetic diversity present on the molecular level regarding specific traits as well as on the whole genome level may enhance barley breeding today by facilitating efficient selection of parental lines and marker assisted selection procedures. In the present paper the state of the art with respect to virus diseases, i.e. Barley mild mosaic virus, Barley yellow mosaic virus, and Barley yellow dwarf virus is briefly reviewed and first results on a project aiming on a genome wide estimation of genetic diversity which in combination with data on yield and additional agronomic traits may facilitate the detection of marker trait associations and a more efficient selection of parental genotypes are presented. By field tests of 49 two-rowed and 64 six-rowed winter barley cultivars the genetic gain in yield for the period 1970–2003 was estimated at 54.6 kg ha−1 year−1 (r2 = 0.567) for the six-rowed cultivars and at 37.5 kg ha−1 year−1 (r2 = 0.621) for the two-rowed cultivars. Analysis of 30 SSRs revealed a non-homogenous allele distribution between two and six-rowed cultivars and changes of allele frequencies in relation to the time of release. By PCoA a separation between two and six-rowed cultivars was observed but no clear cut differentiation in relation to the time of release. In the two-rowed cultivars an increase in genetic diversity (DI) from older to newly released cultivars was detected.

Mode of inheritance and genetic diversity of BaMMV resistance of exotic barley germplasms carrying genes different from 'ym4'.

SummaryIn order to obtain information about the mode of inheritance of BaMMV resistance in germplasms carrying genes different from the “German” gene ‘ym4’ f1-tests for resistance as well as F2-segregation analyses of crosses to susceptible German cultivars were carried out by mechanical inoculation in the greenhouse. In the f1 the majority of plants of each combination tested reacted susceptible to BaMMV while in the F2 a good fit to a segregation of 1r:3s or 7r:9s was observed. Therefore, the results of these tests revealed that the BaMMV resistance of all the varieties tested is inherited either by a single or by two recessive genes. By testing intercrosses of these resistant varieties segregation of BaMMV-susceptile plants was observed in the majority of combinations, revealing a high degree of genetic diversity in the barley gene pool.

High-resolution mapping of the Rym4/Rym5 locus conferring resistance to the barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV-2) in barley (Hordeum vulgare ssp. vulgare L.)

Soil-borne barley yellow mosaic virus disease – caused by a complex of at least three viruses, i.e. Barley mild mosaic virus (BaMMV), Barley yellow mosaic virus (BaYMV) and BaYMV-2 – is one of the most important diseases of winter barley in Europe. The two genes rym4, effective against BaMMV and BaYMV, and rym5, additionally effective against BaYMV-2, comprise a complex locus on chromosome 3HL, which is of special importance to European barley breeding. To provide the genetic basis for positional cloning of the Rym4/Rym5 locus, two high-resolution maps were constructed based on co-dominant flanking markers (MWG838/Y57c10 - MWG010/Bmac29). Mapping at a resolution of about 0.05% rec., rym4 has been located 1.07% recombination distal of marker MWG838 and 1.21% recombination proximal to marker MWG010. Based on a population size of 3,884 F2 plants (0.013% recombination) the interval harbouring rym5 was delimited to 1.49±0.14% recombination. By testing segmental recombinant inbred lines (RILs) for reaction to the different viruses at a resolution of 0.05% rec. (rym4) and 0.019% rec. (rym5), no segregation concerning the reaction to the different viruses could be observed. AFLP-based marker saturation for rym4, using 932 PstI+2/MseI+3 primer combinations only resulted in three markers with the closest one linked at 0.9% recombination to the gene. Two of these markers detected epialleles arising from the differential cytosine methylation of PstI sites. Regarding rym5, profiling of 1,200 RAPD primers (about 18,000 loci) and 2,048 EcoRI+3/MseI+3 AFLP primer combinations (about 205,000 loci) resulted in one RAPD marker and seven AFLP markers tightly linked to the resistance gene. Flanking markers with the closest linkage to rym5 (0.05% and 0.88% recombination) were converted into STS markers. These markers provide a starting point for chromosomal walking and may be exploited in marker-assisted selection for virus resistance based on rym5.

Assessment of the genetic relatedness of barley accessions (Hordeum vulgare s.l.) resistant to soil-borne mosaic-inducing viruses (BaMMV, BaYMV, BaYMV-2) using RAPDs

Abstract Thirty-six Hordeum vulgare varieties and 12 H. spontaneum germplasms originating from different parts of the world and showing different reactions to the barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV-2) were analyzed for genetic similarity using RAPDs. On the basis of an analysis of 20 selected RAPD-primers corresponding to 544 bands genetic similarity according to Nei and Li (1979) was estimated to be between 0.685 and 0.964. Associations between the 48 genotypes were calculated using UPGMA-clustering and principal coordinate analysis. By applying these methods we were able to separate H. spontaneum accessions from H. vulgare varieties, and within these groups all the genotypes were clustered correctly according to their origin. Consequently, RAPD analysis can be considered a very useful and efficient tool for the fast estimation of genetic relationships in barley. The correlation between genetic similarity with respect to German varieties and adaptation of exotic barley varieties to German growing conditions is discussed.