Potential and limits to unravel the genetic architecture and predict the variation of Fusarium head blight resistance in European winter wheat (Triticum aestivum L.).

Genome-wide mapping approaches in diverse populations are powerful tools to unravel the genetic architecture of complex traits. The main goals of our study were to investigate the potential and limits to unravel the genetic architecture and to identify the factors determining the accuracy of prediction of the genotypic variation of Fusarium head blight (FHB) resistance in wheat (Triticum aestivum L.) based on data collected with a diverse panel of 372 European varieties. The wheat lines were phenotyped in multi-location field trials for FHB resistance and genotyped with 782 simple sequence repeat (SSR) markers, and 9k and 90k single-nucleotide polymorphism (SNP) arrays. We applied genome-wide association mapping in combination with fivefold cross-validations and observed surprisingly high accuracies of prediction for marker-assisted selection based on the detected quantitative trait loci (QTLs). Using a random sample of markers not selected for marker-trait associations revealed only a slight decrease in prediction accuracy compared with marker-based selection exploiting the QTL information. The same picture was confirmed in a simulation study, suggesting that relatedness is a main driver of the accuracy of prediction in marker-assisted selection of FHB resistance. When the accuracy of prediction of three genomic selection models was contrasted for the three marker data sets, no significant differences in accuracies among marker platforms and genomic selection models were observed. Marker density impacted the accuracy of prediction only marginally. Consequently, genomic selection of FHB resistance can be implemented most cost-efficiently based on low- to medium-density SNP arrays.

[Comparative Characteristic of Triticum aestivum/Triticum durum and Triticum aestivum/Triticum dicoccum hybrid lines by genomic composition and resistance to fungal diseases under different environmental conditions].

The genetic diversity of common wheat hybrid lines Triticum aestivum/Triticum durum and Triticum aestivum/Triticum dicoccum (2n = 42, F(6-7)) using chromosome-specific microsatellite (SSR) markers and C-staining of chromosomes was studied. Cluster analysis of data obtained by 42 SSR markers indicated that the hybrid lines can be broken into three groups according to their origin. There were two cases of complete genetic similarity between lines 183(2)-2/184(1)-6 and-208-3/213-1, which were obtained using common wheat as the parental plants. In cross combinations, when the stabilization of the nuclear genome of hexaploid lines occurred against a background of the cytoplasmic genome of tetraploid wheats, there was a high level of divergence between sister lines, in some cases exceeding 50%. The evaluation of the degree of susceptibility of the lines to powdery mildew, leaf and stem rust, and septoria leafblotch was performed under different environmental conditions. It was shown that resistance to powdery mildew and leaf rust significantly depended on the region where assays were conducted. An evaluation of the field data showed that he lines 195-3, 196-1, and 221-1 with T. durum genetic material displayed complex resistance to fungal pathogens in Western Siberia and the Republic of Belarus. For lines 195-3 and 196-1, one shows a possible contribution of chromosomes 4B and 5B in the formation of complex resistance to diseases. Hybrid lines with complex resistance can be used to expand the genetic diversity of modern common wheat cultivars for genes of immunity.

Assessment of genetic diversity among Syrian durum (Triticum turgidum ssp. durum) and bread wheat (Triticum aestivum L.) using SSR markers.

Genetic diversity among 49 wheat varieties (37 durum and 12 bread wheat) was assayed using 32 microsatellites representing 34 loci covering almost the whole wheat genome. The polymorphic information content (PIC) across the tested loci ranged from 0 to 0.88 with average values of 0.57 and 0.65 for durum and bread wheat respectively. B genome had the highest mean number of alleles (10.91) followed by A genome (8.3) whereas D genome had the lowest number (4.73). The correlation between PIC and allele number was significant in all genome groups accounting for 0.87, 074 and 0.84 for A, B and D genomes respectively, and over all genomes, the correlation was higher in tetraploid (0.8) than in hexaploid wheat varieties (0.5). The cluster analysis discriminated all varieties and clearly divided the two ploidy levels into two separate clusters that reflect the differences in genetic diversity within each cluster. This study demonstrates that microsatellites markers have unique advantages compared to other molecular and biochemical fingerprinting techniques in revealing the genetic diversity in Syrian wheat varieties that is crucial for wheat improvement.

[Genetic analysis and location of loci controlling leaf rust resistance of introgressive Triticum aestivum x Triticum timopheevii].

Introgressive lines resulting from crossing common wheat Triticum aestivum with the tetraploid T. timopheevii are characterized by effective resistance to leaf rust caused by Puccinia triticina Eriks. Molecular analysis using 350 specific short sequence repeat (SSR) markers was used to locate the T. timopheevii genome to chromosomes 1A, 2A, 2B, 5A, 5B, and 6B. A population of F2 offspring of crossing hybrid line 842-2 with common wheat cultivar Skala was obtained for mapping the loci controlling leaf rust resistance. Analysis of association of phenotypic and genotypic data by means of simple interval mapping (SIM) and composite interval mapping (CIM) has shown that the resistance of adult plants is determined by two loci in chromosomes 5B and 2A. The major locus QLr.icg-5B transferred from T. timopheevii chromosome 5G mapped to the interval of microsatellite loci Xgwm408-Xgwm1257 controls 72% of the phenotypic diversity of the trait. The other, minor locus QLr.icg-2A located to chromosome 2A at a distance of 10 cM from Xgwm312 accounts for 7% of the trait expression. Microsatellite markers located near these loci may be used for controlling the transfer of commercially valuable loci when new lines and cultivars are created.

A high density barley microsatellite consensus map with 775 SSR loci.

A microsatellite or simple sequence repeat (SSR) consensus map of barley was constructed by joining six independent genetic maps based on the mapping populations 'Igri x Franka', 'Steptoe x Morex', 'OWB(Rec) x OWB(Dom)', 'Lina x Canada Park', 'L94 x Vada' and 'SusPtrit x Vada'. Segregation data for microsatellite markers from different research groups including SCRI (Bmac, Bmag, EBmac, EBmag, HVGeneName, scsssr), IPK (GBM, GBMS), WUR (GBM), Virginia Polytechnic Institute (HVM), and MPI for Plant Breeding (HVGeneName), generated in above mapping populations, were used in the computer program RECORD to order the markers of the individual linkage data sets. Subsequently, a framework map was constructed for each chromosome by integrating the 496 "bridge markers" common to two or more individual maps with the help of the computer programme JoinMap 3.0. The final map was calculated by following a "neighbours" map approach. The integrated map contained 775 unique microsatellite loci, from 688 primer pairs, ranging from 93 (6H) to 132 (2H) and with an average of 111 markers per linkage group. The genomic DNA-derived SSR marker loci had a higher polymorphism information content value (average 0.61) as compared to the EST/gene-derived SSR loci (average 0.48). The consensus map spans 1,068 cM providing an average density of one SSR marker every 1.38 cM. Such a high-density consensus SSR map provides barley molecular breeding programmes with a better choice regarding the quality of markers and a higher probability of polymorphic markers in an important chromosomal interval. This map also offers the possibilities of thorough alignment for the (future) physical map and implementation in haplotype diversity studies of barley.

Comparative mapping of genes for glume colouration and pubescence in hexaploid wheat (Triticum aestivum L.).

Microsatellite markers were used to map the major genes Bg (determining black glume colour), Rg1 and Rg3 (red glume), and a locus determining smokey-grey coloured glume to the distal ends of the short arms of the homoeologous group 1 chromosomes, proximally (or closely linked) to Xgwm1223 and distal to Xgwm0033. On this basis, we propose that these genes represent a set of homoeoloci, designated Rg-A1, Rg-B1, and Rg-D1. Rg3 and Bg appear to be variant alleles of Rg-A1. Both Rg3 and Bg are closely linked with the major glume pubescence gene Hg. Similarly, the hexaploid wheat smokey-grey glume gene and Rg2 represent alleles at Rg-D1. The microsatellite markers linked to the Rg genes were used to analyse a phenotypically and genotypically characterized set of Siberian spring wheats. A coincidence between the presence of the 264-bp allele of Xgwm0136 and Rg-A1b (Rg3) was observed; so Xgwm0136 can probably be used as a diagnostic marker for this gene.

[Production of wheat-rye substitution lines and identification of chromosome composition of karyotypes using C-banding, GISH, and SSR markers].

Based on the cross (Triticum aestivum L. x Secale cereale L.) x T. aestivum L., wheat-rye substitution lines (2n = 42) were produced with karyotypes containing, instead of a pair of homologous wheat chromosomes, a homeologous pair of rye chromosomes. The chromosome composition of these lines was described by GISH and C-banding methods, and SSR analysis. The results of genomic in situ hybridization demonstrated that karyotype of these lines included one pair of rye chromosomes each and lacked wheat--rye translocations. C-banding and SSR markers were used to identify rye chromosomes and determine the wheat chromosomes at which the substitution occurred. The lines were designated 1R(1D), 2R(2D)2, 2R(2D)3, 3R(3B), 6R(6A)2. The chromosome composition of lines IR(1A), 2R(W)1, 5R(W), 5R(5A), and 6R(W)1, which were earlier obtained according to the same scheme for crossing, was characterized using methods of telocentric analysis, GISH, C-banding, and SSR analysis. These lines were identified as 1R(1A), 2R(2D)1, 5R(5D), 5R(5A), and 6R(6A)1, C-banding of chromosomes belonging to line 1R(1A) revealed the presence of two translocated chromosomes (3DS.3DL-del. and 4AL.W) during simultaneous amplification of SSR markers located on 3DL and 4AS arms. The "combined" long arm of the newly derived chromosome 4A is assumed to be formed from the long arm of chromosome 4AS itself and a deleted segment 3DL. All examined lines are cytologically stable, except for 3R(3B), which does not affect the stability of rye 3R chromosome transfer. Chromosome identification and classification of the lines will permit them to be models for genetic studies that can be used thereafter as promising "secondary gene pools" for the purpose of plant breeding.

Analysis of QTLs for yield components, agronomic traits, and disease resistance in an advanced backcross population of spring barley.

Hordeum vulgare subsp. spontaneum, the wild progenitor of barley, is a potential source of useful genetic variation for barley breeding programs. The objective of this study was to map quantitative trait loci (QTLs) in an advanced backcross population of barley. A total of 207 BC3 lines were developed using the 2-rowed German spring cultivar Hordeum vulgare subsp. vulgare 'Brenda' as a recurrent parent and the H. vulgare subsp. spontaneum accession HS584 as a donor parent. The lines were genotyped by 108 simple-sequence repeat (SSR) markers and evaluated in field tests for the measurement of grain yield and its components, such as ear length, spikelet number per spike, grain number per spike, spike number, and 1000-grain mass, as well as heading date and plant height. A total of 100 QTLs were detected. Ten QTLs with increasing effects were found for ear length, spikelet number, and grain number per spike. Three QTLs contributed by HS584 were found to significantly decrease days to heading across all years at 2 locations. In addition, 2 QTLs from HS584 on chromosomes 2H and 3H were associated with resistance to leaf rust. Based on genotypic data obtained from this population, 55 introgression lines carrying 1 or 2 donor segments were selected to develop a set of doubled-haploid lines, which will be used to reconfirm and investigate the effects of 100 QTLs for future genetic studies.

Expression genetics and haplotype analysis reveal cis regulation of serine carboxypeptidase I (Cxp1), a candidate gene for malting quality in barley (Hordeum vulgare L.).

Using a cDNA array-based functional genomics approach in barley, several candidate genes for malting quality including serine carboxypeptidase I (Cxp1) were previously identified (Potokina et al. in Mol Breed 14:153, 2004). The gene was mapped as a single nucleotide polymorphism (SNP) marker on chromosome 3H using the Steptoe (feeding grade)xMorex (malting grade) mapping population. Subsequently, the relative level of Cxp1 expression was determined by real-time RT-PCR for each of the 134 progeny lines and mapped as a quantitative trait. Only one quantitative trait locus (QTL) could be identified that significantly influenced the level of the Cxp1 expression. The expressed QTL maps to the same region on chromosome 3H as does the structural gene and corresponds to a QTL for "diastatic power," one among several traits measured to assess malting quality. An analysis of 90 barley cultivars sampled from a worldwide collection revealed six SNPs at the Cxp1 locus, three of which display complete linkage disequilibrium and define two haplotypes. The Cxp1 expression level in a set of barley accessions showing haplotype I was significantly higher than that of accessions displaying haplotype II. The data provide evidence that (1) the expression of Cxp1 is regulated in cis and that (2) the level of diastatic power in the barley seed is influenced by the level of Cxp1 expression.

Rationalising germplasm collections: a case study for wheat.

In total 70 genebank accessions comprising 50 hexaploid, 12 tetraploid and 8 diploid wheats of the Gatersleben collection were selected based on the screening of the passport data for identical cultivar names or accession numbers of the donor genebanks. Twelve potential duplicate groups consisting of three to nine accessions with identical names/numbers were selected and analysed with DNA markers (microsatellites). A bootstrap approach based on re-sampling of both microsatellite markers and alleles within marker loci was used to test for homogeneity. Although several homogeneous groups were identified it became clear that cultivar name identity alone did not allow the determination of duplicates. A combination of SSR-analysis followed by the bootstrap method and database survey considering the botanical classification and other data (origin, growth habit and donor) available is recommended in order to determine duplicates. A procedure for the identification of duplicates and their further handling in ex situ genebanks is discussed.

Transferability of wheat microsatellites to diploid Aegilops species and determination of chromosomal localizations of microsatellites in the S genome.

Overall, 253 genomic wheat (Triticum aestivum) microsatellite markers were studied for their transferability to the diploid species Aegilops speltoides, Aegilops longissima, and Aegilops searsii, representing the S genome. In total, 88% of all the analyzed primer pairs of markers derived from the B genome of hexaploid wheat amplified DNA fragments in the genomes of the studied species. The transferability of simple sequence repeat (SSR) markers of the T. aestivum A and D genomes totaled 74%. Triticum aestivum-Ae. speltoides, T. aestivum-Ae. longissima, and T. aestivum-Ae. searsii chromosome addition lines allowed us to determine the chromosomal localizations of 103 microsatellite markers in the Aegilops genomes. The majority of them were localized to homoeologous chromosomes in the genome of Aegilops. Several instances of nonhomoeologous localization of T. aestivum SSR markers in the Aegilops genome were considered to be either amplification of other loci or putative translocations. The results of microsatellite analysis were used to study phylogenetic relationships among the 3 species of the Sitopsis section (Ae. speltoides, Ae. longissima, and Ae. searsii) and T. aestivum. The dendrogram obtained generally reflects the current views on phylogenetic relationships among these species.

Analysis of QTLs for yield, yield components, and malting quality in a BC3-DH population of spring barley.

Advanced backcross (AB)-quantitative trait locus (QTL) analysis has been successfully applied for detecting and transferring QTLs from unadapted germplasm into elite breeding lines in various plant species. Here, we describe the application of a modified AB breeding scheme to spring barley. A BC3-doubled haploid (DH) population consisting of 181 lines derived from the German spring barley cultivar 'Brenda' (Hordeum vulgare subsp. vulgare) as the recurrent parent and the wild species line 'HS213' (H. vulgare subsp. spontaneum) as the donor line was evaluated for yield and its components as well as malting quality traits. A set of 60 microsatellite markers was used to genotype the population, and phenotypic data were collected at two locations in Germany in continuous years. Altogether, 25 significant QTLs were detected by single-marker regression analysis and interval mapping. Most positive QTLs originated from the recurrent parent 'Brenda'. A QTL, Qhd2.1, on chromosome 2HS from 'Brenda' explained 18.3% and 20.7% of the phenotypic variation for yield and heading date, respectively. Due to the small percentage of donor-parent genome of 6.25%, the BC3-DH lines could be directly used for the extraction of near-isogenic lines (NILs) for Qhd2.1. Consequently, it was possible to determine the precise location of the locus hd2.1 within a region of 6.5 cM, using an F2 population consisting of 234 individuals developed from a cross between an NIL containing a defined donor segment at this locus and 'Brenda'. The location of this QTL was consistent with the presence of a major photoperiod response gene, Ppd-H1, previously reported in this region, which is associated with pleiotropic effects on yield components. In summary, the analysis of a BC3-DH population in barley provides a compromise between the analysis of QTLs by means of an AB scheme and the generation of defined substitution lines. Several lines carrying defined different donor segments for only one single chromosome or trait in the genetic background of 'Brenda' could be selected for further genetic studies.

Advanced backcross QTL analysis in progenies derived from a cross between a German elite winter wheat variety and a synthetic wheat (Triticum aestivum L.).

We report here the second advanced backcross quantitative trait locus (AB-QTL) analysis carried out in winter wheat. Seven agronomic traits were studied in a BC2F1 population derived from a cross between the German winter wheat variety Flair and the synthetic wheat line XX86 developed in Japan. We selected 111 BC2F1 lines and genotyped these with 197 microsatellite markers. Field data for seven agronomic traits were collected from corresponding BC2F3 families that were grown at up to six locations in Germany. QTL analyses for yield and yield components were performed using single-marker regression and interval mapping. A total of 57 putative QTLs derived from XX86 were detected, of which 24 (42.1%) were found to have a positive effect from the synthetic wheat XX86. These favourable QQTLs were mainly associated with thousand-grain weight and grain weight per ear. Many QTLs for correlated traits were mapped in similar chromosomal regions. The AB-QTL data obtained in the present study are discussed and compared with results from previous QTL analyses.

Genetic diversity in cultivated plants-loss or stability?

Human activities like urbanisation, the replacement of traditional agriculture systems by modern industrial methods or the introduction of modern high-yielding varieties may pose a danger to the biological diversity. Using microsatellite markers, we analysed samples of cultivated wheat ( Triticum aestivum L.) collected over an interval of 40-50 years in four comparable geographical regions of Europe and Asia. No significant differences in both the total number of alleles per locus and in the PIC values were detected when the material collected in the repeated collection missions in all four regions were compared. About two-thirds of the alleles were common to both collection periods, while one-third represented collection mission-specific alleles. These findings demonstrate that an allele flow took place during the adaptation of traditional agriculture to modern systems, whereas the level of genetic diversity was not significantly influenced.

Molecular studies on genetic integrity of open-pollinating species rye (Secale cereale L.) after long-term genebank maintenance.

The genetic integrity of six accessions represented by 14 sub-populations of the open-pollinating species rye ( Secale cereale L.) was investigated. Seeds available from a herbarium collection (first regeneration) and from the cold store (most recent regeneration) were multiplied two to fourteen times and fingerprinted using microsatellite markers. Four accessions had significantly different allele frequencies. These were multiplied seven to thirteen times. Nearly 50% of the alleles discovered in the original samples were not found in the material present in the cold store. However alleles were detected in the most recently propagated sub-populations, that were not observed in the investigated plants of the original one. The change in allele frequencies is a continuous process. Reasons for the occurrence of genetic changes and consequences for managing open pollinating species maintained in ex situ genebanks are discussed.

Development and genetic mapping of 127 new microsatellite markers in barley.

To enhance the marker density of existing genetic maps of barley ( Hordeum vulgare L.), a new set of microsatellite markers containing dinucleotide motifs was developed from genomic clones. Out of 254 primer pairs tested, a total of 167 primer pairs were classifed as functional in a panel of six barley cultivars and three H. spontaneum accessions, and of those, 127 primer pairs resulting in 133 loci were either mapped or located onto chromosomes. The polymorphism information content (PIC) ranged from 0.05 to 0.94 with an average of 0.60. The number of alleles per locus varied from 1 to 9. On average, 3.9 alleles per primer pair were observed. The RFLP frameworks of two previously published linkage maps were used to locate a total of 115 new microsatellite loci on at least one mapping population. The chromosomal assignment of 48 mapped loci was corroborated on a set of wheat-barley chromosome addition lines; 18 additional loci which were not polymorphic in the mapping populations were assigned to chromosomes by this method. The microsatellites were located on all seven linkage groups with four significant clusters in the centromeric regions of 2H, 3H, 6H and 7H. These newly developed microsatellites improve the density of existing barley microsatellite maps and can be used in genetic studies and breeding research.

Advanced backcross QTL analysis for the identification of quantitative trait loci alleles from wild relatives of wheat ( Triticum aestivum L.).

Advanced backcross QTL (AB-QTL) analysis was used to identify quantitative trait loci (QTLs) for yield and yield components in a BC(2)F(2) population derived from a cross between the German winter wheat variety 'Prinz' and the synthetic wheat line W-7984 developed by CIMMYT. Two hundred and ten microsatellite markers were employed to genotype 72 pre-selected BC(2)F(2) plants and phenotypic data were collected for five agronomic traits from corresponding BC(2)F(3) families that were grown at four locations in Germany. Using single-marker regression and interval mapping, a total of 40 putative QTLs derived from W-7984 were detected, of which 11 were for yield, 16 for yield components, eight for ear emergence time and five for plant height. For 24 (60.0%) of them, alleles from the synthetic wheat W-7984 were associated with a positive effect on agronomic traits, despite the fact that synthetic wheat was overall inferior with respect to agronomic appearance and performance. The present study indicated that favorable QTL alleles could be transferred from wild relatives of wheat into an elite wheat variety for improvement of quantitative trait loci like yield by the advanced backcross QTL strategy and molecular breeding. To our knowledge, the results presented here were the first report on AB-QTL analysis in wheat.

Microsatellite mapping of the powdery mildew resistance gene Pm5e in common wheat (Triticum aestivum L.).

Powdery mildew, caused by Erysiphe graminis DM f. sp. tritici (Em. Marchal), is one of the most important diseases of common wheat world-wide. Chinese wheat variety 'Fuzhuang 30' carries the powdery mildew resistance gene Pm5e and has proven to be a valuable resistance source of powdery mildew for wheat breeding. Microsatellite markers were employed to identify the gene Pm5e in a F(2) progeny from the cross 'Nongda 15' (susceptible) x 'Fuzhuang 30' (resistant). The gene Pm5e was mapped in the distal region of chromosome 7BL. Seven microsatellite markers were found to be linked to the gene Pm5e, of which two codominant markers Xgwm783 and Xgwm1267 were relatively close to Pm5e with a linkage distance of 11.0 cM and 6.6 cM, respectively. It is possible to use the 136-bp allele of Xgwm1267 in 'Fuzhuang 30' for marker-assisted selection during the wheat resistance breeding process for facilitation of gene pyramiding. The mapping information in the present study provides a starting point for fine mapping of the Pm5 locus and map-based cloning to clarify the molecular structure and function of the different alleles at the Pm5 locus. A microsatellite linkage map of chromosome 7B was constructed with 20 microsatellite loci, nine on the short arm and 11 on the long arm. This information will be very useful for further mapping of agronomically important genes of interest on chromosome 7B.

Genetic effects on microsatellite diversity in wild emmer wheat (Triticum dicoccoides) at the Yehudiyya microsite, Israel.

This study investigated allele size constraints and clustering, and genetic effects on microsatellite (simple sequence repeat, SSR) diversity at 28 loci comprising seven types of tandem repeated dinucleotide motifs in a natural population of wild emmer wheat, Triticum dicoccoides, from a shade vs sun microsite in Yehudiyya, northeast of the Sea of Galilee, Israel. It was found that allele distribution at SSR loci is clustered and constrained with lower or higher boundary. This may imply that SSR have functional significance and natural constraints. Genetic factors, involving genome, chromosome, motif, and locus significantly affected SSR diversity. Genome B appeared to have a larger average repeat number (ARN), but lower variance in repeat number (sigma(ARN)(2)), and smaller number of alleles per locus than genome A. SSRs with compound motifs showed larger ARN than those with perfect motifs. The effects of replication slippage and recombinational effects (eg, unequal crossing over) on SSR diversity varied with SSR motifs. Ecological stresses (sun vs shade) may affect mutational mechanisms, influencing the level of SSR diversity by both processes.

Inheritance of microsatellite alleles in pedigrees of Latvian barley varieties and related European ancestors.

Genetic diversity and inheritance of 65 microsatellite (SSR) loci were studied in a set of 37 barley varieties involved in the pedigrees of seven Latvian barley varieties: Abava, Agra, Balga, Imula, Linga, Priekulu 1 and Stendes. Cluster analysis divided all the varieties into two large groups according to their geographic distribution. Moravian, Swedish and Danish varieties clustered separately from varieties from Norway and Finland. The pattern of subgroups of both European and Latvian varieties was in accordance with their pedigree information. Graphical genotypes of microsatellite alleles of all seven barley chromosomes were determined for all the 37 varieties studied. Parental inheritance and transmission of microsatellite alleles through the generations of the pedigrees were analysed. The results confirmed the importance and informative value of microsatellite markers for genetic studies in barley and their utility for barley breeding and other applications in fundamental and applied barley genetics.