Temperature-dependent resistance to crown rust infection in perennial ryegrass, Lolium perenne

Differences were found in the expression of resistance of perennial ryegrass, Lolium perenne, cultivars to crown rust, Puccinia coronata Corda, when plants were incubated at either 10°C or 25°C. In an extreme example, all the genotypes of ‘Carrick’ were resistant at 10°C but susceptible at 25°C. The variation between cultivars was due both to a general increase in expression of susceptibility and the proportion of plants which became susceptible. A BC₁ line containing a stay‐green gene derived from Festuca pratensis was insensitive to temperature, while a sister line was sensitive.     

Breeding perennial ryegrass with better crown rust resistance

Improving rust resistance is one of the main goals in our breeding programme with perennial ryegrasses. We have studied the behaviour of our breeding material over several years in several locations under natural and artificial infection conditions. The narrow sense heritability of crown rust resistance is fairly high (0.46), providing scope for improving resistance, but this optimism is reduced by the negative correlation with yield. The most resistant material yielded 4% less than the most susceptible material. No decline in digestibility is expected when improving the disease resistance. The incidence of crown rust is very variable between years, locations and between spontaneous and artificial infections, creating constraints in selection. Observations for several years and at several locations appear to be the best way to improve rust resistance in perennial ryegrass.     

Evidence of pathotypes among Australian isolates of crown rust infecting perennial ryegrass

Fifty‐three genotypes of perennial ryegrass selected from a range of cultivars and ecotypes were evaluated for their reaction to crown rust isolates collected from Western Australia, Victoria, Queensland, South Australia or New South Wales in Australia. The reactions of the clones to inoculation provided evidence for the development of a differential set of perennial ryegrass genotypes, but also demonstrated that a large number of genotypes displayed universal reactions to infection with any of the crown rust isolates.     

A maternal factor conferring resistance to crown rust in Lolium multiflorum cv.'Axis'

The genetic basis for crown rust resistance was analysed in the progeny of reciprocal crosses between Lolium multiflorum plants that were either resistant (cv. ‘Axis’) or susceptible to crown rust. The progeny harvested from the resistant parent was two‐ to seven‐fold more resistant than the progeny harvested from the susceptible parent. These data indicate the presence of a maternal factor which increases resistance to crown rust in some of the ryegrass plants used as parents.     

Introgression of crown rust resistance from Festuca spp. into Lolium multiflorum

Crown rust, Puccinia coronata Corda, causes one of the most damaging foliar diseases in Italian ryegrass, Lolium multiflorum Lam. For introgression of crown rust resistance, highly resistant hybrids of the crosses Festuca arundinacea (2n = 6x = 42) ×L. multiflorum (2n = 4x = 28) and reciprocally, L. multiflorum (2n = 2x = 14) ×Festuca pratensis (2n = 4x = 28) and subsequently resistant recombinant individuals were used as female parents and susceptible cultivars of Italian ryegrass as male parents in three successive backcrosses. The BC₃ plants were selfed and crossed mutually. Uredospores of seven different crown rust isolates collected from plants of L. multiflorum, Lolium perenne, F. pratensis, F. arundinacea and L. multiflorum × F. pratensis hybrids were applied to identify the resistance or susceptibility of the parental species, backcrossed, selfed and intercrossed progenies. The various crown rust populations revealed a species-specific capability to infect plants of the Lolium-Festuca complex corresponding to the host species from which the spores originated. Selected BC⁹ plants, however, were found to be completely resistant to all crown rust populations tested. Successful introgression of the resistance was achieved from F. arundinacea as well as from F. pratensis. The resistance represents a dominant character, apparently based on a strong heterologous incompatibility between host and pathogen. In phenotype, bivalent formation during meiosis and in fertility, the novel germplasms are comparable with the L. multiflorum cultivars.     

Preliminary evaluation of turfgrass (perennial ryegrass and red fescue) cultivars for resistance to red thread disease using artificial infection

Selection of main cool‐season turfgrasses (perennial ryegrass and red fescue) for resistance to red thread disease caused by Laetisaria fuciformis has not progressed during the last 30 years because of low reproducibility of experiments carried out under natural infection. In this study, evaluation of a set of the turfgrass cultivars, including non‐host fodder grasses as control, was performed in a climatic chamber, a cold house and in the field using three types of artificial inoculum, and correlations between trials were analysed. In general, cultivars of Festuca rubra were more susceptible to the disease but differentiated better than cultivars of Lolium perenne. Significant interactions between cultivars of L. perenne and F. rubra and isolates of L. fuciformis were found. The fungus was also able to colonize leaves of sow thistle and wheat seedlings. In field conditions and in the glass cold‐house Dactylis glomerata was found to be relatively resistant to red thread disease. A possible selection programme is proposed.     

Isozyme and quantitative traits polymorphisms in European provenances of perennial ryegrass (Lolium perenne L)

Twenty populations of Lolium perenne originating from a range of habitats in Europe were compared for isoenzyme polymorphisms and agronomically important quantitative traits in order to establish relationships of the levels of diversity with the origin of each population and to assess their suitability to be included in the European core collection of Lolium germplasm. Forty genotypes from each of the twenty populations and each genotype represented by three clonal propagules, were field planted in a fully randomized spaced plant design and fifteen quantitative characters including yield, persistency, reproductive and disease resistence characters were evaluated over a period of two years. Seven putative isozyme loci were assayed to compare the allozyme divergence of populations. The results of the isozyme survey indicate that 71–100% of the loci were polymorphic, 2.3–3.0 alleles/locus and the gene diversity was varying from 0.234–0.410. Of the total allelic diversity 94% remained within populations (H_s) whilst only 6% was distributed among populations (D_st). The differences between populations were determined on the basis of allele frequencies and multivariate analyses of quantitative characters. Populations significantly differed in their allele frequencies at all loci analysed. Random mating was predominant in all populations at most of the loci. The study revealed that the German accession BA 10998 was clearly distinct from the rest both in quantitative characters and allele frequencies. German accession BA 11015 with the lowest gene diversity showed the highest genetic variation for quantitative characters. However, no strict relationship was found between the genetic distance and the geograpical distribution of the populations. Among the quantitative characters, flowering time showed a strong relationship with the type of management practised at the collection site than their place of origin. This revised version was published online in July 2006 with corrections to the Cover Date.     

QTL analysis on plant height and flowering time in Brassica napus

To identify quantitative trait loci (QTL) controlling plant height (PH) and flowering time (FT), a high stalk/later bloom Brassica napus line '2091' was crossed with a dwarf/early bloom mutant '99CDAM'. A segregating population with 145 F_2:3 lines was constructed and grown in field for 2 years. Based on this population, a linkage map consisting of 199 amplified fragment length polymorphism and 42 simple sequence repeat loci was constructed. The LOD threshold of 3.0 was used for declaring the existence of putative QTL. In total, seven QTL related to PH were identified, accounting for 8.5–28.6% of phenotypic variation, respectively, of which two QTL were identified in both years. Six QTL for FT were detected, accounting for 8.1–30.4% of phenotypic variation individually, of which two QTL were identified in both years. The main QTL of PH and FT were both mapped on linkage group 13, with overlapped creditable regions. QTL ph17 and ft17 existed at the same location. These might be the genetic base for the significant correlation observed between PH and FT.     

Identification and mapping of adult‐plant stripe rust resistance in soft red winter wheat cultivar ‘USG 3555’

Little is known about the extent or diversity of resistance in soft red winter wheat (Triticum aestivum L.) to stripe rust, caused by the fungal pathogen Puccinia striiformis f.sp. tritici. The soft red winter (SRW) wheat cultivar ‘USG 3555’ has effective adult‐plant resistance to stripe rust, which was characterized in a population derived from ‘USG 3555’/‘Neuse’. The mapping population consisted of 99 recombinant inbred lines, which were evaluated for stripe rust infection type (IT) and severity to race PST‐100 in field trials in North Carolina in 2010 and 2011. Genome‐wide molecular‐marker screenings with 119 simple sequence repeats and 560 Diversity Arrays Technology (DArT) markers were employed to identify quantitative trait loci (QTL) for stripe rust resistance. QTL on chromosomes 1AS, 4BL and 7D of ‘USG 3555’ explained 12.8, 73.0 and 13.6% of the variation in stripe rust IT, and 13.5, 72.3 and 10.5% of the variation in stripe rust severity, respectively. Use of these and additional diagnostic markers for these QTL will facilitate the introgression of this source of stripe rust resistance into SRW wheat lines via marker‐assisted selection.     

Discovery and genotyping of high-throughput SNP markers for crown rust resistance gene Pc94 in cultivated oat

Crown rust caused by Puccinia coronata f. sp. avenae Eriks is a serious problem for oat production worldwide and pyramiding multiple resistance genes into new cultivars is a key objective of breeders. Many race specific resistance genes have been mapped and markers that are closely linked to them have been identified. However, the use of these markers in oat breeding practice has been limited due to the economics of marker assisted selection (MAS) deployment. Single nucleotide polymorphism (SNP) markers have been demonstrated to have a high-throughput capability with relatively low cost and numerous semi-automated SNP scoring platforms exist. Gene Pc94 has remained highly effective since it was first tested on the Canadian crown rust populations in 1993 and is one of the few effective genes available in Western Canada. In the present study, PCR products were amplified using primers derived from sequences of amplified fragment length polymorphism bands which have been shown to be linked to Pc94 . Genomic DNA from genotypes, with and without the Pc94 gene, were used as the PCR templates. By comparative sequence alignment amongst the PCR fragments, many putative SNP sites were identified. From these sites, four SNP sites were selected and validated by the single base extension method. One SNP site, Pc94 -SNP1a, was tested on two F_2:3 populations segregating for the resistance gene. The map distances between the SNP marker and Pc94 were 2.1 and 5.4 cM in the two different populations. Various oat cultivars and germplasm lines were also tested for a wider application of the SNP marker. Fluorescent technology and capillary electrophoresis allowed for the semi-automated, fairly high-throughput scoring of the SNP markers.     

Identification and comparative analysis of quantitative trait loci associated with parthenocarpy in processing cucumber

Parthenocarpy (seedless fruit) is an economically important yield‐related trait in cucumber (Cucumis sativus L.; 2n = 2x = 14). However, the genomic locations of factors controlling parthenocarpic fruit development in this species are not known. Therefore, an F_{2 : 3} mating design was utilized to map quantitative trait loci (QTL) for parthenocarpy using a narrow cross employing two gynoecious, indeterminate and normal leaf lines [2A (parthenocarpic) and Gy8 (non‐parthenocarpic)]. QTL detection was performed employing 2A‐ and Gy8‐coupling phase data using the parthenocarpic yield of 126 F₃ families grown at two locations at Hancock, WI in 2000. The QTLs detected in this study were compared with the map locations of QTLs conditioning first‐harvest yield of seeded cucumber characterized in a previous study. There were 10 QTLs for parthenocarpy detected defining four genomic regions, in which three QTLs also mapped to the same genomic regions as QTLs detected for fruit yield at first‐harvest as reported in a previous study. The eight fluorescence amplified fragment length polymorphism (AFLP) markers linked to parthenocarpy through QTL mapping defined herein (four each in linkage groups 1 and 4) are candidates for use in marker‐assisted selection programmes where breeding for increased levels of parthenocarpy is an objective in the elite‐processing cucumber populations.     

Association of agronomic traits with isozyme loci in perennial ryegrass (Lolium perenne L.)

Summary Inbred lines possessing different genotypes at three isozyme loci, PGI-2, GOT-3 and ACP-1, were produced by two generations of selfing from two contrasting cultivars of perennial ryegrass. Four separate F₂ populations were obtained by selfing or crossing F₁ plants from crosses between cultivar inbred lines. F₂ plants were scored for genotype at the three isozyme loci and for eight agronomic traits. The three isozyme loci showed independent segregation confirming that they belonged to separate linkage groups. A consistent association was observed between water soluble carbohydrate content and genotype at the PGI-2 locus. Some association was also detected between genotype at the ACP-1 locus and heading date. The relevance of these observations to ryegrass breeding programmes is discussed.     

Comparison and integration of genetic maps generated from F2 and BC1-type mapping populations in perennial ryegrass

Linkage maps of perennial ryegrass were constructed from F₂ and BC₁‐type populations using, predominantly, restriction fragment length polymorphism data based on heterologous probes used in mapping other grass species. The maps identified seven linkage groups, which covered a total of 515 cM (F₂) and 565 cM (BC₁). They were aligned using 38 loci identified in both populations (common loci) and a possible marker order for all mapped loci in either population was identified in an integrated map. The estimated recombination frequencies and map distances between adjacent common loci were compared between the two data sets and regions of heterogeneity identified. Overall, the common markers identified a map distance of 446 cM in the F₂ population and 327 cM in the BC₁ population, reflecting a higher recombination frequency in the former, although the difference was not evenly spread over the seven linkage groups.     

Cold stress tolerance of soybeans during flowering: QTL mapping and efficient selection strategies under controlled conditions

Breeding soybeans for higher latitudes requires cultivars with an increased chilling stress tolerance, especially when flowering occurs. Phenotyping in climate chambers to select for this trait is labour‐intensive and requires an optimal allocation of resources due to limited space. We screened a diversity panel of 35 early maturity cultivars and a biparental population of 103 RILs for their cold stress tolerance at flowering stage. Pod number under control and stress conditions is highly heritable and showed only a weak correlation between the two treatments. Based on different testing scenarios, we could show that testing more genotypes with less replicates yields much higher responses to selection and hence should be pursued in such climate‐controlled experiments. We identified quantitative trait loci (QTL) for pod number under both conditions (chromosomes 7 and 13) and a cold tolerance‐specific QTL (chromosome 11). Furthermore, we performed genomic predictions using different test set scenarios and prediction models, showing that genomic prediction is a promising tool to select for cold stress tolerance, particularly if known QTL can be used as fixed effects in the model.     

Development of STS markers and QTL validation for common bacterial blight resistance in common bean

Common bacterial blight (CBB) of common bean ( Phaseolus vulgaris L.), is one of the major diseases that decrease yield and quality. A major quantitative trait locus (QTL) for CBB resistance from line XAN 159 was transferred into two bean lines, HR45 and HR67. Previous studies identified that two markers are linked to this QTL but the chromosome location was not consistent. To identify more tightly linked markers and to verify the chromosome location, 65 additional markers were mapped using 81 recombinant inbred lines (RILs) derived from a cross HR67 × OAC95-4. The QTL was mapped to a 13 cM region on chromosome 1 and defined by eight molecular markers that explained 25–52% of the phenotypic variation. Six tightly linked amplified fragment length polymorphism markers (0.6–9.7 cM from the QTL peak) were converted into seven sequence tagged site markers, three of which were mapped to this QTL. Five tightly linked markers were used to screen 907 F₂ plants derived from a cross HR45 × 'OAC Rex' and four of them were linked to each other within 4.2 cM. These markers may be useful in marker-assisted selection and map-based cloning of this major QTL.     

Rapid phenotyping of adult plant resistance in barley (Hordeum vulgare) to leaf rust under controlled conditions

Breeding for adult plant resistance (APR) is currently impeded by the low frequency of annual field‐based testing and variable environmental conditions. We developed and implemented a greenhouse‐based methodology for the rapid phenotyping of APR to leaf rust in barley to improve the efficacy of gene discovery and cloning. We assessed the effects of temperature (18 and 23°C) and growth stage (1–5 weeks) on the expression of APR in the greenhouse using 28 barley genotypes with both known and uncharacterized APR. All lines were susceptible in week 1, while lines carrying Rph20 and several with uncharacterized resistance expressed resistance as early as week 2. In contrast, lines lacking Rph20 and carrying either Rph23 and/or Rph24 expressed resistance from week 4. Resistant phenotypes were clearest at 18°C. A subset of 16 of the 28 lines were assessed for leaf rust across multiple national and international field sites. The greenhouse screening data reported in this study were highly correlated to most of the field sites, indicating that they provide comparable data on APR phenotypes for screening purposes.     

Detection of loci controlling seed glucosinolate content and their association with Sclerotinia resistance in Brassica napus

A genetic linkage map of Brassica napus constructed from a cross between a low glucosinolate cultivar ‘H5200’ and a high glucosinolate line ‘NingRS‐1’ was used to identify loci associated with seed glucosinolate content and to understand the association between specific glucosinolate components and Sclerotinia resistance. Seed glucosinolate content was assessed by standard High pressure Liquid Chromatogram (HPLC) protocol. Seven components of seed glucosinolate, including four types of aliphatic glucosinolate, two types of indolyl glucosinolates and one aromatic glucosinolate were detected in the seeds. Three quantitative trait loci (QTLs) were identified for seed total glucosinolate content. From three to 15 loci were found to be responsible for different types of glucosinolates, and by comparing the overlapped intervals, eight genomic regions were defined. One of the nine loci associated with aliphatic glucosinolate content was found to be associated with Sclerotinia resistance on the leaf at the seedling stage, and one locus, responsible for 3‐indolyl‐methyl glucosinolate content, was probably linked with Sclerotinia resistance on the stem of the maturing plant. The association between seed glucosinolate content and Sclerotinia resistance is discussed.     

Genetic variation in barley of crossability with wheat and its quantitative trait loci analysis

To study genetic variation in crossability, 80 barley accessions of diverse geographic origin consisting of 50 wild barleys (H. vulgare ssp. spontaneum or ssp. agriocrithon) and 30 cultivated barleys (H. vulgare ssp. vulgare) were crossed as the male parent with a highly crossable wheat variety, Shinchunaga. Crossabilities, expressed as the percentage of pollinated florets giving embryo-containing caryopses, ranged from 0% to 68.6%. Barley accessions from East Asia had generally a low crossability, while barley accessions from other regions exhibited a wider range of crossability including highly crossable genotypes. No significant difference in mean crossability was found between wild and cultivated barleys. To estimate the number and location of barley genes controlling the crossability, doubled haploid lines derived from the cross between the barley varieties Steptoe and Morex were crossed as the male parent with wheat. Quantitative trait loci (QTL) analysis using molecular markers identified four QTL. These were mapped to the centromeric regions of chromosomes 2H, 3H and 5H and the short arm of chromosome 7H. The QTL on chromosomes 3H and 5H had larger effects than those on chromosomes 2H and 7H. The four QTL collectively explained 35.4% of the total variance under a multiple QTL model. Relationships of the QTL identified in the present study with previously reported crossability genes of barley and wheat are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Association analysis of five candidate genes with plant height and dry matter yield in perennial ryegrass

Five candidate genes LpIAA1, LpRUB1, LpBRI1, LpSHOOT1 and LpTB1 with putative function in plant architecture were allele sequenced in a panel of 96 diploid perennial ryegrass genotypes of diverse origin. The total length of the non‐redundant genomic DNA alignment was 5425 bp and revealed 270 polymorphic sites in total. A negative significant Tajima's D value was detected in LpTB1 gene, suggesting selection pressure for low‐frequency alleles in this gene. All 96 genotypes were evaluated for plant height and dry matter yield over two years. Marker–trait associations were calculated between polymorphic sites and both phenotypic traits. Three indels and three single nucleotide substitutions in LpTB1 gene were significantly (P < 0.05, q < 0.05) associated with plant height, while one indel was associated with dry matter yield. The results suggest putative role of LpTB1 gene in plant height determination in perennial ryegrass and provide means for target allele selection.