Production of doubled haploid lines in frequencies sufficient for barley breeding programs

Winter barley (Hordeum vulgare L.) anthers were cultured on different liquid and on starch-solidified media. The optimal embryo and callus formation with different F₁-lines and the cv. Igri was obtained on a liquid medium with 20% Ficoll, 20 g/l maltose and barley starch. But the influence of the growth conditions of the donor plants and the genotypical differences are still enormous. The procedure has been optimized to such an extent that it can be used economically.     

Identification of novel QTLs for seedling and adult plant leaf rust resistance in a wheat doubled haploid population

Pyramiding of genes that confer partial resistance is a method for developing wheat (Triticum aestivum L.) cultivars with durable resistance to leaf rust caused by Puccinia triticina. In this research, a doubled haploid population derived from the cross between the synthetic hexaploid wheat (SHW) (×Aegilotriticum spp.) line TA4152-60 and the North Dakota breeding line ND495 was used for identifying genes conferring partial resistance to leaf rust in both the adult plant and seedling stages. Five QTLs located on chromosome arms 3AL, 3BL, 4DL, 5BL and 6BL were associated with adult plant resistance with the latter four representing novel leaf rust resistance QTLs. Resistance effects of the 4DL QTL were contributed by ND495 and the effects of the other QTLs were contributed by the SHW line. The QTL on chromosome arm 3AL had large effects and also conferred seedling resistance to leaf rust races MJBJ, TDBG and MFPS. The other major QTL, which was on chromosome arm 3BL, conferred seedling resistance to race MFPS and was involved in a significant interaction with a locus on chromosome arm 5DS. The QTLs and the associated molecular markers identified in this research can be used to develop wheat cultivars with potentially durable leaf rust resistance.     

Effects of Fusarium culmorum head blight on mycotoxin accumulation and yield traits in barley doubled haploids

The susceptibility of barley doubled haploids (DH) to Fusarium head blight (FHB) was investigated. Heads of 24 DH lines (11 two-rowed and 13 six-rowed) derived from F₁ Maresi (two-rowed) × Pomo (six-rowed) hybrids were inoculated with a conidial suspension of the single isolate IPO348-01 of Fusarium culmorum. The experiment was carried out in three consecutive years (1996–98) in one location. The number of kernels per ear, 1000-kernel weight and kernel weight per ear were recorded in inoculated and control plots. In the infected kernels nivalenol (NIV) content and deoxynivalenol (DON) content were determined. The effects of genotype, year and genotype-year interaction on reduction of yield traits were significant. For mycotoxin content only genotype and year effects were found to be important. The average NIV concentration in kernels of inoculated lines ranged from 0.15 mg/kg in the two-rowed line MP7 to 6.36 mg/kg in the six-rowed line MP113. A low accumulation of DON was observed in the studied population (from 0.01 to 0.20 mg/kg). Generally, no significant differences in mycotoxin content were found between two-rowed and six-rowed genotypes. The line MP7 was found to be superior — with the lowest mycotoxin accumulation, and reduction in yield traits. Environmental conditions (years) affected DON and NIV level in kernels; however, the tendency to a lower or higher accumulation of mycotoxin in individual lines was stable over the years.     

Comparison of frequency distributions of doubled haploid and single seed descent lines in barley

Summary Both doubled haploid (DH) and single seed descent (SSD) methods were used to derive homozygous lines from two crosses of barley. The frequency distributions of grain yield, heading date, and plant height of the DH and SSD lines were compared by the Mann-Whitney U test, Kolmogorov-Smirnov twosample test and Wald-Wolfowitz runs test. It was found that the DH lines distributed in the same manner as the SSD lines with respect to the three characters. The results indicated that although the SSD method had more opportunity for recombination than the DH method, it did not produce a sample of recombinants which differed significantly from the DH sample; thus both methods were equally efficient for use in deriving homozygous lines from F₁ hybrids in a relatively short time.Contribution no. 455 Charlottetown Research Station, Agriculture Canada, P.E.I. (Canada)     

Genetics study of resistance to yellow rust in CIMMYT origin wheat advanced lines at seedling and adult plant stages

To study genetically and evaluate resistance to yellow rust, 29 wheat advanced lines were evaluated in randomised complete blocks design with three replicates in seedling stage under greenhouse conditions using nine races 6E150A+, 198E150A+, 134E150A+, 6E158A+, 166E150A+, 198E130A+, 166E158A+, 230E158A+ and 70E0A+, separately. In the adult plant stage, the genotypes were evaluated in two regions of Iran, Zarghan and Gorgan. The components of resistance including latent period and infection type were recorded under greenhouse conditions. Cluster analysis in all races showed that the genotypes 11, 28 and 29 were completely resistant to all races. Under Zarghan and Gorgan races, 27 and 73% of genotypes were resistant in the adult plant stage, respectively. Seven percent of genotypes were resistant in both stages, seedling and adult plant. All resistant lines can be used in plant breeding programme.     

Genome-wide association studies of net form of net blotch resistance at seedling and adult plant stages in spring barley collection

Net form of net blotch (NFNB) of barley (Hordeum vulgare L.), caused by Pyrenophora teres f. teres (Ptt) Drechsler (anamorph: Drechslera teres [Sacc.] Shoem.), is considered one of the major constraints of successful barley production in major barley growing regions of the world. Resistance to NFNB was evaluated in a barley collection of 336 genotypes (AM-2014), at seedling stage using isolates LGDPtt.19 and TD10 in the USA, and adult stage in seven hotspot environments in Morocco. The AM-2014 panel was genotyped with 9K SNP markers and genome-wide association studies (GWAS) were carried out using mixed linear model (MLM: Q?+?K) accounting for population structure (Q) and kinship (K) as covariates. Significant (P?<?0.001) marker trait associations were corrected for false discovery rate (FDR) at the q?<?0.05. Four genotypes showed an average infection response (IRs ≤ 2) to both isolates, LGDPttt.19 and TD10, at the seedling stage, and 30 genotypes showed resistance in all environments in the field while three genotypes exhibited the highest resistance at both stages. The GWAS of NFNB identified 31 distinct QTLs on all seven barley chromosomes, of which 8 with resistance at seedling stage, 21 were associated with resistance at the adult stage, and two QTLs, QRptt.2H-132.15 and QPtt.6H-54-55, conferred resistance at both stages. Of 31 resistance QTLs reported in this study, 10 QTLs coincided with previously mapped QTL while 21 are novel, thereby validating the GWAS approach used in this study. The resistance sources identified in AM-2014 and QTL mapped in this study are valuable resources for marker-assisted breeding for NFNB resistance in the future.     

The relationship between in vitro performance of haploid embryos and the agronomic performance of the derived doubled haploid lines in barley

Summary The relationship between in vitro performance of haploid embryos and the agronomic performance of the derived doubled haploid (DH) lines during the stages of field evaluation was investigated. The results showed a positive correlation between coleoptile height of haploid plantlets in culture and final plant height of their DH progeny lines in the field. These results illustrate that in vitro selection for plant height and other linked quantitative or pleiotropic characters can be carried out at the initial stages of a DH breeding programme.     

Genome wide association studies (GWAS) of spot blotch resistance at the seedling and the adult plant stages in a collection of spring barley

Spot blotch (SB) in barley is caused by the fungal pathogen Cochliobolus sativus and considered one of the major constraints to successful barley production. Resistance to C. sativus was evaluated, using a barley collection of 336 genotypes (AM-2014), at the seedling and adult stages. Seedling resistance was evaluated by using a mixture of 19 virulent isolates in Morocco. Virulent isolates prevalent in Uttar Pradesh were used for phenotyping resistance at the adult stage in India. The AM-2014 panel was genotyped with 9-K single-nucleotide polymorphism (SNP) markers using iSelect Illumina Infinium. Genome wide association studies (GWAS) were carried out using SNP markers, infection responses, disease severity, and area under the disease progress curve (AUDPC). The mixed linear model was employed in TASSEL using principal component analysis (PCA) and Kinship matrix (K) as covariates. Higher SB severity, 82.3?±?13.5 (mean?±?SD), was recorded at the Banaras Hindu University (BHU) compared to 47.6?±?15.0 at the Narendra Dev University of Agriculture and Technology (NDUAT). Nine QTL, Rcs-qtl-1H-126.9, Rcs-qtl-2H-148.16, Rcs-qtl-3H-25.27, Rcs-qtl-5H-80.35, Rcs-qtl-6H-58.24, Rcs-qtl-7H-29.62, Rcs-qtl-7H-29.72, Rcs-qtl-7H-32.81, and Rcs-qtl-7H-34.74, were detected for SB resistance at the seedling stage. For SB severity at the adult stage, a QTL, Rcs-qtl-7H-32.81, was detected at BHU while seven QTL, Rcs-qtl-2H-91.09, Rcs-qtl-3H-145.64, Rcs-qtl-4H-14.43, Rcs-qtl-6H-6.49, Rcs-qtl-7H-114.43, Rcs-qtl-7H-151.66, and Rcs-qtl-7H-150.36, were found for SB severity at NDUAT. Three QTL, Rcs-qtl-4H-18.61, Rcs-qtl-4H-67.91, and Rcs-qtl-5H-110.25, were significant for AUDPC of SB at BHU. The QTLs reported in this study are important to advance marker-assisted selection and gene pyramiding of SB resistance in South Asia and North Africa in future.     

Resistance of cultivars and breeding lines of spring wheat to Fusarium culmorum and powdery mildew

Twelve Polish spring wheat cultivars and 18 spring wheat accessions from CIMMYT, Mexico, were examined for resistance to a highly pathogenic Fusarium culmorum strain KF846 and powdery mildew in 5-year field experiments. Resistant wheat cultivars (Sumai 3 and Frontana) served as controls. The mean percentage of Fusarium-damaged kernels (% FDK) for 5 years was lower in CIMMYT accessions (16.7%) than in Polish spring cultivars (28.3%). In all Polish spring cultivars, % FDK was higher than in the control cultivars Sumai 3 and Frontana (12-20%). The mean disease score (on a scale of 1-9) for powdery mildew (natural infection) for all examined cultivars and lines ranged from 0 to 7 and in the Polish spring cultivars was significantly lower (0-5). Cultivars Eta, Henika, Ismena, Jasna and Olimpia were found to be the least susceptible to powdery mildew in field experiments. The laboratory host-pathogen tests with Blumeria graminis f. sp. tritici isolates showed that only two cultivars were characterized by identical resistance patterns as the standard differential lines with documented resistance genes. Cultivar Alkora had the gene Pm3d, and Henika had Pm5. The gene Pm3d was identified in cultivars Jasna and Eta in combination with another unknown gene/genes. Cultivars Santa and Torka had the gene Pm5 in combination with another unknown gene/genes. Four cultivars: Banti, Ismena, Olimpia and Sigma, showed resistance to all mildew isolates employed in a laboratory test. The accession IPG-SW-14 was the least susceptible to both pathogens (F. culmorum and powdery mildew) in all 5 years of experiments. This line is the best candidate for deriving new cultivars with improved resistance to fungal diseases.     

A comparison of cereal root eelworm resistant and susceptible spring barley genotypes at two sites

A comparison between otherwise identical cereal root eelworm resistant and susceptible spring barley lines was made on two sites, one lightly infested the other heavily infested with the nematode. On the lightly infested site, the grain yields of the resistant and susceptible lines did not differ, but on the heavily infested site, the resistant lines out-yielded the susceptible lines by 20%. A similar comparison was also made between five reputedly eelworm-resistant spring-barley genotypes and three cultivars known to be susceptible. On the lightly infested site, the three susceptible cultivars out-yielded all other genotypes, while on the heavily infested site they were out-yielded by all other genotypes except one. Development of the nematode was compared in resistant and susceptible genotypes. Although the nematode was as numerous in resistant genotypes as in susceptible genotypes, either invasion or development of the nematode was retarded in resistant genotypes. Although not conclusive, there was evidence that the male was less retarded in development than the female, which was rarely found in the adult form on resistant plants.     

Genetic determination of variability of barley doubled haploids inoculated with Fusarium culmorum (W.G.Sm.) Sacc. with regard to mycotoxin accumulation and reduction in yield traits

The genetic determination of variability of barley doubled haploid (DH) lines in regard of their susceptibility to Fusarium head blight caused by Fusarium culmorum was studied. The susceptibility was evaluated in 3-year field experiment on the basis of reduction in yield traits and myotoxin accumulation in infected kernels. The following traits were analysed in inoculated and control plants: kernel number and weight per ear, 1000-kernel weight, percentage of plump kernels (>2.5 mm), deoxynivalenol (DON) content and nivalenol (NIV) content of kernels. On the basis of the obtained data, heritability coefficient (ratio of genotypic to phenotypic variance) was assessed, and genetic parameters as well as the number of effective factors were estimated. Heritability coefficients calculated from two-way analysis of variance, i.e.regarding the influence of years and year x genotype interaction, appeared to be exceptionally low and ranged from 5.2% for the reduction in plump kernels to 38.2% for the reduction in 1000-kernel weight. In the case of mycotoxin accumulation about 60% of the observed variability in NIV concentrations and 30% in DON concentration resulted form genetic differences among lines. Additive effects of genes were important for all the analysed traits. Significant effects of dominance and dominance x dominance were observed for 1000-kernel weight and percentage of plump kernels. Moreover, it was found that the observed variability in yield trait reduction resulted from segregation of 5-6 effective factors, DON contents from 4 factors, while NIV content from 5 factors.     

Mapping of two genes for resistance to clubroot (Plasmodiophora brassicae) in a population of doubled haploid lines of Brassica oleracea by means of RFLP and AFLP markers

A genetic map covering 615 cM in 12 linkage groups was assembled based on 92 RFLP and AFLP markers segregating in a population of 107 doubled haploid lines (DH lines) of Brassica oleracea. The DH-line population was obtained through microspore culture from the of two homozygous parents: DH-line Bi derived from the cabbage landrace Bindsachsener, and DH-line Gr from broccoli cv ‘Greenia’. Sixty-five percent of the loci, and in some cases complete linkage groups, displayed distorted segregation ratios, a frequency much higher than that observed in populations of the same species. DH-line Bi was resistant to clubroot, which is caused by a Dutch field isolate of Plasmodiophora brassicae. Resistance in the DH-line population was determined in two ways: by assigning symptom grades to each plant, and by measuring the fresh weights of the healthy and affected parts of the root system of each plant. Using a multiple QTL mapping approach to analyze the fresh weight data, we found two loci for clubroot resistance; these were designated pb-3 and pb-4. The additive effects of these loci were responsible for 68% of the difference between the parents and for 60% of the genetic variance among DH-line means. Also, indications for the presence of two additional, minor QTLs were found. Analysis of symptom grades revealed the two QTLs pb-3 and pb-4, as well as one of the two minor QTLs indicated by analysis of the fresh weight data. Received: 29 April 1996 / Accepted: 10 May 1996     

抗感白粉病青稞的内生真菌群落差异分析

种子内生微生物组与种子的健康发育有着密切的关系。本研究收集对白粉病抗病和易感共11个品种的青稞种子,利用扩增子测序技术对内生真菌的群落、丰度和多样性进行研究。从不同品种的青稞种子中共鉴定4个门、17个纲、35个目、66个科、107个属的内生真菌,其中子囊菌门Ascomycota、担子菌门Basidiomycota和被孢菌门Mortierellomycota是优势菌门;链格孢属Alternaria、微座孢属Microdochium、亚隔孢壳属Didymella、镰刀菌属Fusarium和球腔菌属Mycosphaerella为优势菌属。根据生物信息学注释结果,在鉴定到的107个属中,抗病品种的青稞种子中特有的属有20个,感病品种的青稞种子中特有的属有50个;通过对两组样本的分析,发现感病品种中真菌物种多样性高于抗病品种。本研究通过扩增子测序技术初步确定了青稞抗、感白粉病两组样品中的真菌群落结构和优势菌群,为进一步研究青稞内携真菌与抗/感病的关系提供了基础,从内生菌的角度为青稞白粉病的防控提供新的策略。     

Using molecular markers to map multiple quantitative trait loci: models for backcross,recombinant inbred,and doubled haploid progeny

Summary To maximize parameter estimation efficiency and statistical power and to estimate epistasis, the parameters of multiple quantitative trait loci (QTLs) must be simultaneously estimated. If multiple QTL affect a trait, then estimates of means of QTL genotypes from individual locus models are statistically biased. In this paper, I describe methods for estimating means of QTL genotypes and recombination frequencies between marker and quantitative trait loci using multilocus backcross, doubled haploid, recombinant inbred, and testcross progeny models. Expected values of marker genotype means were defined using no double or multiple crossover frequencies and flanking markers for linked and unlinked quantitative trait loci. The expected values for a particular model comprise a system of nonlinear equations that can be solved using an interative algorithm, e.g., the Gauss-Newton algorithm. The solutions are maximum likelihood estimates when the errors are normally distributed. A linear model for estimating the parameters of unlinked quantitative trait loci was found by transforming the nonlinear model. Recombination frequency estimators were defined using this linear model. Certain means of linked QTLs are less efficiently estimated than means of unlinked QTLs.     

RFLP mapping of isozymes,RAPD and QTLs for grain shape,brown planthopper resistance in a doubled haploid rice population

We have developed an RFLP framework map with 146 RFLP markers based on a doubled haploid population derived from a cross between an indica variety IR64 and a japonica variety Azucena. The population carries 50.2% of IR64 loci and 49.8% of Azucena loci, indicating an equal amount of genetic materials from each parent has been transmitted to the progenies through anther culture. However, some markers show segregation distortion. These distorted marker loci are located on 10 chromosomal segments. Using this map we were able to place 8 isozymes, 14 RAPDs, 12 cloned genes, 1 gene for brown planthopper (BPH) resistance, and 12 QTLs for grain length, grain width and length/width ratio onto rice chromosomes. The major gene for BPH resistance was mapped on chromosome 12 near RG463 and isozyme Sdh-1. Most of the QTLs identified for the three grain characters were closely linked on chromosomes 1, 2, 3 and 10. We concluded that the RFLP framework map presented here will be useful for mapping other genes segregating in this doubled haploid population. Thus rapid generation of doubled haploid lines and their unbiased segregation make it very attractive for gene mapping.     

Screening of certain barley lines for resistance to root rot disease caused by Fusarium graminearum

This investigation was conducted in 2005/2006 and 2006/2007 to test 235 barley lines plus two varieties Giza 127 and Giza 128 for resistance and susceptibility to Fusarium graminearum. All screened barley lines showed varied significant degrees of infestation to root rot pathogen. A screening system is described for identifying barley lines which are effective in controlling resistant or susceptible lines. By detecting small but consistent differences in root rot severity, the bioassay proved effective in large-scale screening for partial resistance: already 335 barley lines and two varieties have been screened. We found five groups (7.12%), 22 barley lines and both varieties are resistant (R) (8.31%); 28 barley lines are moderately resistant (MR) (19.29%); 65 barley lines are moderately susceptible (MS) (27.89%); 94 barley lines are susceptible (S) and (37.39%) 126 barley lines are highly susceptible (HS). The high degree of precision makes this an invaluable tool in the understanding of pathogen aggressiveness, host specialisation and parasitic fitness. Disease scale was strongly negative and had moderate correlation with germination (?0.309?? and ?0.649??) under normal and disease treatment. The correlation between yield and normal and disease treatment during two seasons was strong and negative (?0.834?? and ?0.847??, respectively were detected).     

PCR markers for selection of adult plant leaf rust resistance in barley (Hordeum vulgare L.)

Adult plant resistance (APR) is considered potentially more durable for controlling barley leaf rust than seedling Rph (Resistance to Puccinia hordei) genes. A major gene for adult plant resistance to barley leaf rust has been mapped to the telomere region of chromosome 5HS. PCR-based molecular markers were developed for saturation of this region based on previously mapped simple sequence repeat, restriction fragment length polymorphism and Diversity Arrays Technology markers. In addition, defence gene homologue (DGH) and wheat expressed sequence tags mapped in specific bins were used to develop new PCR markers. Seventeen PCR-based markers were mapped to the short arm of chromosome 5H in 292 doubled haploid lines from a cross of Pompadour × Stirling, in which seven markers were mapped within 5 cM of the APR gene. The closest linked marker was about 0.7 cM from the APR gene. The wheat deletion bin map together with defence gene homologues was demonstrated to be an efficient tool for development of new molecular markers associated with the disease resistance gene. Four DGH markers were associated with the APR gene. The new molecular markers are a useful tool for marker-assisted selection of the APR gene and provided a better understanding of the molecular mechanism for leaf rust resistance.