Development and characterization of Hordeum chilense chromosome-specific STS markers suitable for wheat introgression and marker-assisted selection

 RAPD markers were developed for octoploid×Tritordeum (amphiploid Hordeum chilense×Triticum aestivum) and its parents. Addition lines were used to identify specific RAPD markers for the Hordeum chilense chromosomes detectable in a wheat background. Twelve RAPD fragments have been cloned, sequenced and converted into STS markers. Eleven of these STSs have maintained both the chromosome specificity and the possibility of detection in a wheat background. The use of these markers in multiplexed PCRs facilitates both the efficient and reliable screening of new addition lines as well as the monitoring of introgression of H. chilense in bread and durum wheat. Received: 5 June 1998 / Accepted: 17 September 1998     

DNA markers and marker-assisted breeding for durable resistance to bacterial blight disease in rice

Bacterial leaf blight caused by the bacterial pathogen Xanthomonas oryzae pv oryzae (Xoo) limits rice yield in all major rice-growing regions of the world, especially in irrigated lowland and rainfed conditions where predisposition factors favor disease development to epidemic proportions. Since bacterial pathogens are difficult to manage, development of host plant resistance is the most effective means of disease management. As many as 24 major genes conferring resistance to various races of the pathogen have been identified and utilized in rice breeding programs. However, large-scale and long-term cultivation of varieties carrying a single gene for resistance resulted in a significant shift in pathogen race frequency with consequent breakdown of resistance in these cultivars. To combat the problem of resistance breakdown, pyramiding of resistance genes into different cultivars is being carried out. Pyramiding of resistance genes is now possible with molecular markers that are developed for individual genes. This review discusses the various bacterial blight resistance genes identified and their corresponding molecular markers developed for breeding durable resistance into modern rice cultivars.     

Transferability and polymorphism of barley EST-SSR markers used for phylogenetic analysis in Hordeum chilense

Background

Hordeum chilense, a native South American diploid wild barley, is a potential source of useful genes for cereal breeding. The use of this wild species to increase genetic variation in cereals will be greatly facilitated by marker-assisted selection. Different economically feasible approaches have been undertaken for this wild species with limited direct agricultural use in a search for suitable and cost-effective markers. The availability of Expressed Sequence Tags (EST) derived microsatellites or simple sequence repeat (SSR) markers, commonly called as EST-SSRs, for barley (Hordeum vulgare) represents a promising source to increase the number of genetic markers available for the H. chilense genome.

Results

All of the 82 barley EST-derived SSR primer pairs tested for transferability to H. chilense amplified products of correct size from this species. Of these 82 barley EST-SSRs, 21 (26%) showed polymorphism among H. chilense lines. Identified polymorphic markers were used to test the transferability and polymorphism in other Poaceae family species with the aim of establishing H. chilense phylogenetic relationships. Triticum aestivum-H. chilense addition lines allowed us to determine the chromosomal localizations of EST-SSR markers and confirm conservation of the linkage group.

Conclusion

From the present study a set of 21 polymorphic EST-SSR markers have been identified to be useful for diversity analysis of H. chilense, related wild barleys like H. murinum, and for wheat marker-assisted introgression breeding. Across-genera transferability of the barley EST-SSR markers has allowed phylogenetic inference within the Triticeae complex.     

Development of wild barley (Hordeum chilense)-derived DArT markers and their use into genetic and physical mapping

Diversity arrays technology (DArT) genomic libraries were developed from H. chilense accessions to support robust genotyping of this species and a novel crop comprising H. chilense genome (e.g., tritordeums). Over 11,000 DArT clones were obtained using two complexity reduction methods. A subset of 2,209 DArT markers was identified on the arrays containing these clones as polymorphic between parents and segregating in a population of 92 recombinant inbred lines (RIL) developed from the cross between H. chilense accessions H1 and H7. Using the segregation data a high-density map of 1,503 cM was constructed with average inter-bin density of 2.33 cM. A subset of DArT markers was also mapped physically using a set of wheat-H. chilense chromosome addition lines. It allowed the unambiguous assignment of linkage groups to chromosomes. Four segregation distortion regions (SDRs) were found on the chromosomes 2H(ch), 3H(ch) and 5H(ch) in agreement with previous findings in barley. The new map improves the genome coverage of previous H. chilense maps. H. chilense-derived DArT markers will enable further genetic studies in ongoing projects on hybrid wheat, seed carotenoid content improvement or tritordeum breeding program. Besides, the genetic map reported here will be very useful as the basis to develop comparative genomics studies with barley and model species.     

Development of molecular markers for breeding of double flowers in Japanese gentian

Double flowers are valuable floral traits in most floricultural plants. We recently revealed that a double-flowered mutant of Gentiana scabra was caused by an insertion of a retrotransposable element (Tgs1) into GsAG1, one of the C-class MADS-box genes in gentian. In this study, we developed a PCR-based molecular DNA marker to distinguish double- and single-flower phenotypes at the young seedling stage in Japanese gentian plants. To test the validity of the markers, 17 F₂ populations were produced by selfing F₁ plants crossed between the double-flower mutant and seven breeding lines. Multiplex PCR demonstrated that the Tgs1 insertion in GsAG1 cosegregated with the double-flower phenotype in two F₂ populations, indicating that the PCR-based DNA marker was useful to discriminate between double- and single-flower phenotypes in advance of flowering in Japanese gentian. Given that Japanese gentians lack variation in flower shape and require a long breeding period, the DNA marker developed here will be helpful for efficient breeding of double-flowered cultivars in the future.     

Cloning and molecular characterization of B-hordeins from Hordeum chilense (Roem. et Schult.)

One of the main limitations of cereal breeding is the lack of genetic variability within cultivated crops. Hordeum chilense is a wild relative of Hordeum vulgare, which has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. Among the agronomic traits of these new amphiploids, the allelic variation in the endosperm storage proteins and their influence on breadmaking and malting quality are of special interest. B-hordeins are sulfur rich prolamins, which account for 70–80% of the total hordein fraction in barley. In this work, rapid amplification of cDNA ends by PCR (RACE-PCR) has been used for the cloning of the full-length open reading frame (ORF) of six sequences of B3-hordeins from two lines of H. chilense. Two consensus sequences of 813 and 822 bp for the H1 and H7 lines, respectively, were determined by alignment of all the sequences generated. Between both lines, differences involving single base changes, which could correspond to single nucleotide polymorphisms (SNP), insertions and deletions were observed. Of these differences, only six out of the 13 within the ORF caused a change of amino acid. Two insertions/deletions of 9 and 12 bp were also observed between both lines. The derived amino acid sequences showed a similar structure to the B-hordeins from cultivated barley and other prolamins. The repetitive region is based on the repetition of the motif PQQPFPQQ. The copy number of the B3-hordeins was estimated as a minimum of nine and five copies for the H1 and H7 lines, respectively. The expression profile of the B-hordeins through the developing endosperm is also described in this work. This study of the storage proteins of H. chilense is a useful contribution to the knowledge of the genetic diversity available in wild relatives of cultivated barley. In addition, the origin of the different prolamins can be better understood with an in-depth knowledge of its wild equivalent.     

Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice

We prevent an ultra-simple DNA extraction method for microsatellite analysis of rice. Each extraction requires only one microtube, one disposable pipette tip, TE buffer and few pieces (about 5 mm) of rice leaf tissue. This is sufficient for 200 PCR reactions. The extract can be kept in the freezer for long-term storage. Also, DNA can be extracted from 200–300 individuals in a few hours. These features enabled us to perform rapid largescale seedling genotyping required for marker-assisted selection. We have also examined the applicability of this method for other PCR-based markers: RAPDs, nuclear STS, chloroplast STS and chloroplast microsatellites.     

A PCR-based molecular marker applicable for marker-assisted selection for anthracnose disease resistance in lupin breeding

Selection for anthracnose disease resistance is one of the major objectives in lupin breeding programs. The aim of this study was to develop a molecular marker linked to a gene conferring anthracnose resistance in narrow-leafed lupin (Lupinus angustifolius L.), which can be widely used for MAS in lupin breeding. A F(8)derived RIL population from a cross between cultivar Tanjil (resistant to anthracnose) and Unicrop (susceptible) was used for marker development. DNA fingerprinting was conducted on 12 representative plants by combining the AFLP method with primers designed based on conserved sequences of plant disease resistance genes. A co-dominant candidate marker was detected from a DNA fingerprint. The candidate marker was cloned, sequenced, and converted into a sequence-specific, simple PCR based marker. Linkage analysis based on a segregating population consisting of 184 RILs suggested that the marker, designated as AntjM2, is located 2.3 cM away from the R gene conferring anthracnose resistance in L. angustifolius. The marker has now being implemented for MAS in the Australian national lupin breeding program.     

Utility of barley and wheat simple sequence repeat (SSR) markers for genetic analysis of Hordeum chilense and tritordeum

A selection of 36 wheat and 35 barley simple sequence repeat markers (SSRs) were studied for their utility in Hordeum chilense. Nineteen wheat and nineteen barley primer pairs amplified consistent H. chilense products. Nine wheat and two barley SSRs were polymorphic in a H. chilense mapping population, producing codominant markers that mapped to the expected homoeologous linkage groups in all but one case. Thirteen wheat and 10 barley primer pairs were suitable for studying the introgression of H. chilense into wheat because they amplified H. chilense products of distinct size. Analysis of wheat/H. chilense addition lines showed that the H. chilense products derived from the expected homoeologous linkage groups. The results showed that wheat and barley SSRs provide a valuable resource for the genetic characterization of H. chilense, tritordeums and derived introgression lines. Received: 20 November 2000 / Accepted: 12 April 2001     

Evaluation of diagnostic molecular markers for DUS phenotypic assessment in the cereal crop, barley (Hordeum vulgare ssp. vulgare L.)

The deployment of genetic markers is of interest in crop assessment and breeding programmes, due to the potential savings in cost and time afforded. As part of the internationally recognised framework for the awarding of Plant Breeders’ Rights (PBR), new barley variety submissions are evaluated using a suite of morphological traits to ensure they are distinct, uniform and stable (DUS) in comparison to all previous submissions. Increasing knowledge of the genetic control of many of these traits provides the opportunity to assess the potential of deploying diagnostic/perfect genetic markers in place of phenotypic assessment. Here, we identify a suite of 25 genetic markers assaying for 14 DUS traits, and implement them using a single genotyping platform (KASPar). Using a panel of 169 UK barley varieties, we show that phenotypic state at three of these traits can be perfectly predicted by genotype. Predictive values for an additional nine traits ranged from 81 to 99?%. Finally, by comparison of varietal discrimination based on phenotype and genotype resulted in correlation of 0.72, indicating that deployment of molecular markers for varietal discrimination could be feasible in the near future. Due to the flexibility of the genotyping platform used, the genetic markers described here can be used in any number or combination, in-house or by outsourcing, allowing flexible deployment by users. These markers are likely to find application where tracking of specific alleles is required in breeding programmes, or for potential use within national assessment programmes for the awarding of PBRs.     

Prospects for exploitation of disease resistance from Hordeum chilense in cultivated cereals

Hordeum chilense is a South American wild barley with high potential for cereal breeding given its high crossability with other members of the Triticeae. In the present paper we consider the resistance of H. chilense to several fungal diseases and the prospects for its transference to cultivated cereals. All H. chilense accessions studied are resistant to the barley, wheat and rye brown rusts, the powdery mildews of wheat, barley, rye and oat, to Septoria leaf blotch, common bunt and to loose smuts, which suggests that H. chilense is a non-host of these diseases. There are also lines resistant to wheat and barley yellow rust, stem rust and to Agropyron leaf rust, as well as lines giving moderate levels of resistance to Septoria glume blotch, tan spot and Fusarium head blight. Some H. chilense lines display pre-appressorial avoidance to brown rust. Lines differ in the degree of haustorium formation by rust and mildew fungi they permit, and in the degree to which a hypersensitive response occurs after haustoria are formed. Unfortunately, resistance of H. chilense to rust fungi is not expressed in tritordeum hybrids, nor in chromosome addition lines in wheat. In tritordeum, H. chilense contributes quantitative resistance to wheat powdery mildew, tan spot and loose smut. The resistance to mildew, expressed as a reduced disease severity, is not associated with macroscopically visible necrosis. Hexaploid tritordeums are immune to Septoria leaf blotch and to common bunt although resistance to both is slightly diluted in octoploid tritordeums. Studies with addition lines in wheat indicate that the resistance of H. chilense to powdery mildew, Septoria leaf blotch and common bunt is of broad genetic basis, conferred by genes present on various chromosomes.     

Identification of SSR markers for a broad-spectrum blast resistance gene Pi20(t) for marker-assisted breeding

The Pi20(t) gene was determined to confer a broad-spectrum resistance against diverse blast pathotypes (races) in China based on inoculation experiments utilizing 160 Chinese Magnaporthe oryzae (formerly Magnaporthe grisea) isolates, among which isolate 98095 can specifically differentiate the Pi20(t) gene present in cv. IR24. Two flanking and three co-segregating simple sequence repeat (SSR) markers for Pi20(t), located near the centromere region of chromosome 12, were identified using 526 extremely susceptible F₂ plants derived from a cross of Asominori, an extremely susceptible cultivar, with resistant cultivar IR24. The SSR OSR32 was mapped at a distance of 0.2 cM from Pi20(t), and the SSR RM28050 was mapped to the other side of Pi20(t) at a distance of 0.4 cM. The other three SSR markers, RM1337, RM5364 and RM7102, co-segregated with Pi20(t). RM1337 and RM5364 were found to be reliable markers of resistance conditioned by Pi20(t) in a wide range of elite rice germplasm in China. As such, they are useful tags in marker-assisted rice breeding programs aimed at incorporating Pi20(t) into advanced rice breeding lines and, ultimately, at obtaining a durable and broad spectrum of resistance to M. oryaze. Wei Li and Cailin Lei contributed equally to this work.     

Strategies for efficient implementation of molecular markers in wheat breeding

Although molecular markers allow more accurate selection in early generations than conventional screens, large numbers can make selection impracticable while screening in later generations may provide little or no advantage over conventional selection techniques. Investigation of different crossing strategies and consideration of when to screen, what proportion to retain and the impacts of dominant vs. codominant marker expression revealed important choices in the design of marker-assisted selection programs that can produce large efficiency gains. Using F₂ enrichment increased the frequency of selected alleles allowing large reductions in minimum population size for recovery of target genotypes (commonly around 90%) and/or selection at a greater number of loci. Increasing homozygosity by inbreeding from F₂ to F_2:3 also reduced population size by around 90% in some crosses with smaller incremental reductions in subsequent generations. Backcrossing was found to be a useful strategy to reduce population size compared with a biparental population where one parent contributed more target alleles than the other and was complementary to F₂ enrichment and increasing homozygosity. Codominant markers removed the need for progeny testing reducing the number of individuals that had to be screened to identify a target genotype. However, although codominant markers allow target alleles to be fixed in early generations, minimum population sizes are often so large in F₂ that it is not efficient to do so at this stage. Formulae and tables for calculating genotypic frequencies and minimum population sizes are provided to allow extension to different breeding systems, numbers of target loci, and probabilities of failure. Principles outlined are applicable to implementation of markers for both quantitative trait loci (QTL) and major genes.     

Characterization of Hordeum chilense chromosomes by C-banding and in situ hybridization using highly repeated DNA probes.

C-banding patterns of Hordeum chilense and of Triticum aestivum 'Chinese Spring' - H. chilense disomic addition lines were analyzed and compared with in situ hybridization patterns using a biotin-labeled highly repetitive Triticum tauschii DNA sequence, pAs1, and a wheat 18S-26S rDNA probe. All seven H. chilense chromosomes pairs and the added H. chilense chromosomes present in the addition lines were identified by their characteristic C-banding pattern. Chromosome morphology and banding patterns were similar to those of the corresponding chromosomes present in the parent H. chilense accession. A C-banded karyotype of the added H. chilense chromosomes was constructed and chromosome lengths, arm ratios, and relative length, as compared with chromosome 3B, were determined. The probe pAs1 was found to hybridize to specific areas on telomeres and interstitial sites along the chromosomes, allowing the identification of all seven pairs of the H. chilense chromosomes. Comparison of the patterns of distribution of the hybridization sites of clone pAs1 in the T. tauschii and H. chilense chromosomes was carried out by in situ hybridization on somatic metaphase chromosomes of the HchHchDD amphiploid. In situ hybridization using the 18S-26S rDNA probe confirmed that the H. chilense chromosomes 5Hch and 6Hch were carrying nucleolus organizer regions. The results are discussed on the basis of phylogenetic relationships between D and Hch genomes.     

Preliminary research of the RAPD molecular marker-assisted breeding of the edible basidiomycete Stropharia rugoso-annulata

Summary The average RAPD molecular genetic distance was proposed as a criterion in selecting monokaryotic parents for cross breeding and predicting the performance of hybrids of the mushroom Stropharia rugoso-annulata. Three groups of cross pairs or hybrids were recognized based on the average RAPD genetic distance of the monokaryotic parental population. The RAPD-based molecular genetic distance significantly correlated with hybrid mycelial growth rate and mycelial growth heterosis, and their determination coefficients were 0.9237 and 0.8464 respectively. One of the hybrids in group I showed more vigorous mycelial growth in different pH conditions, incubation temperatures, carbon and nitrogen sources, and higher mushroom yield compared with its dikaryotic parent. These results suggested that RAPD-based molecular genetic distance of the monokaryotic parents might be a suitable criterion for selecting monokaryotic parents and predicting the performance of hybrids in mushroom cross breeding.     

Estimation of hymenopteran parasitism in cereal aphids by using molecular markers

Polymerase chain reaction (PCR) primers were designed and tested for identification of immature parasitoids in small grain cereal aphids and for estimation of parasitism rates. PCR technique was evaluated for 1) greenhouse-reared greenbugs, Schizaphis graminum (Rondani), parasitized by Lysiphlebus testaceipes Cresson and 2) aphids collected from winter wheat fields in Caddo County, Oklahoma. For greenhouse samples, parasitism frequencies for greenbugs examined by PCR at 0, 24, and 48 h after removal of L. testaceipes parasitoids were compared with parasitism frequencies as determined by greenbug dissection. PCR was unable to detect parasitism in greenbugs at 0 and 24 h postparasitism, but it was able to detect parasitoids 48 h after parasitoid removal at frequencies that were not significantly different from dissected samples. Field-collected samples were analyzed by rearing 25 aphids from each sample and by comparing parasitoid frequencies of mummies developed and PCR performed on another 50 aphids. Aphid samples included corn leaf aphids, Rhopalosiphum maidis (Fitch); bird cherry-oat aphids, Rhopalosiphum padi (L.); English grain aphids, Sitobion avenae (F.); and greenbugs. Mummies were isolated until adult emergence, whereupon each parasitoid was identified to species (L. testaceipes was the only parasitoid species found). Parasitism detection frequencies for PCR also were not statistically different from parasitism frequencies of reared aphids. These results indicate that PCR is a useful tool for providing accurate estimates of parasitism rates and especially for identification of immature parasitoids to species.     

Development of highly polymorphic SNP markers from the complexity reduced portion of maize [Zea mays L.] genome for use in marker-assisted breeding

The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.     

Methodology: a modified method for high-quality DNA extraction for molecular analysis in cereal plants

Cereal crops that have rigid non-cellulose components in the cell wall tissues of leaves and high starch and protein content in grains face limitations in DNA extraction. Advanced molecular genetic techniques such as mapping and marker-assisted selection programs require pure and quick DNA extraction. In this study, we developed methods for isolating high-quality genomic DNA from leaves and seeds of major cereal crops with minor modifications. DNA yields ranged from 300 to 1800 ng for 0.01 g seed or leaf tissue.     

Chromosomal location of genes coding for endosperm proteins of Hordeum chilense,determined by two-dimensional electrophoresis of wheat-H. chilense chromosome addition lines

The proteins of Hordeum chilense grain were resolved into 25 major components by two-dimensional electrophoresis. Their solubilities in aqueous alcohol solutions were determined to distinguish prolamin storage proteins from metabolic and structural proteins. The prolamins were divided into two groups, based on the presence or absence of intermolecular disulfide bonds determined by gel-filtration chromatography. Using an incomplete set of Chinese Spring wheat-H. chilense disomic addition lines, the structural genes of 21 of the 26 most dominant seed proteins were assigned to chromosomes. The great majority of the prolamin genes, including those coding for a high molecular weight (HMW) prolamin subunit, was present on chromosome 1H^ch. However, a small number of prolamin genes also occurred on chromosomes 5H^ch and 7H^ch. A minor protein, probably belonging to the nonstorage group of proteins, is coded by genes on 5H^ch. Various ditelosomic addition lines and ditelosomic and disomic substitution lines for chromosome 7H^ch were also analyzed by electrophoresis. This technique revealed that the genes for three major prolamins occur on the arm of chromosome 7H^ch and that a gene for a minor protein, also thought to be a prolamin, occurs on the arm. These results are discussed in relation to the evolution of prolamin genes in the Triticeae.     

Mind the gap: analysis of marker-assisted breeding strategies for inbred mouse strains

The development of congenic mouse strains is the principal approach for confirming and fine mapping quantitative trait loci, as well as for comparing the phenotypic effect of a transgene or gene-targeted disruption between different inbred mouse strains. The traditional breeding scheme calls for at least nine consecutive backcrosses before establishing a congenic mouse strain. Recent availability of genome sequence and high-throughput genotyping now permit the use of polymorphic DNA markers to reduce this number of backcrosses, and empirical data suggest that marker-assisted breeding may require as few as four backcrosses. We used simulation studies to investigate the efficiency of different marker-assisted breeding schemes by examining the trade-off between the number of backcrosses, the number of mice produced per generation, and the number of genotypes per mouse required to achieve a quality congenic mouse strain. An established model of crossover interference was also incorporated into these simulations. The quality of the strain produced was assessed by the probability of an undetected region of heterozygosity (i.e., “gaps”) in the recipient genetic background, while maintaining the desired donor-derived interval. Somewhat surprisingly, we found that there is a relatively high probability for undetected gaps in potential breeders for establishing a congenic mouse strain. Marker-assisted breeding may decrease the number of backcross generations required to generate a congenic strain, but only additional backcrossing will guarantee a reduction in the number and length of undetected gaps harboring contaminating donor alleles. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.