ToxA Is Present in the U.S. Bipolaris sorokiniana Population and Is a Significant Virulence Factor on Wheat Harboring Tsn1.

ToxA, a necrotrophic effector originally identified from the tan spot fungus Pyrenophora tritici-repentis in 1987, was subsequently identified from Parastagonospora nodorum in 2006. More recently, the ToxA gene was identified in the spot blotch fungus Bipolaris sorokiniana in Australia. Here we show that the ToxA gene is also present in the B. sorokiniana population in the winter wheat region of southcentral Texas. Leaves from 'Duster' wheat showing strong necrotic lesions were collected in Castroville, TX. Fifteen single-spore isolates were collected from separate lesions, and 13 of them harbored the BsToxA gene and secreted ToxA in culture based on sensitivity of BG261, the differential line containing the dominant ToxA sensitivity gene, Tsn1. Four isolates harboring BsToxA and one deficient in BsToxA were used to infiltrate two wheat lines harboring Tsn1 as well as their corresponding tsn1 mutant lines. Culture filtrates of the isolate lacking BsToxA did not induce necrosis on any of the lines. Culture filtrates of the four BsToxA-containing isolates induced necrosis on the wild type (Tsn1) lines but not on the corresponding tsn1 mutant lines. Sensitivity to these culture filtrates also mapped to the previously identified location for Tsn1 in the winter wheat mapping population Arina × Forno. Inoculation of one of these ToxA-producing isolates on the same population showed that the Tsn1 locus accounted for 24.4% of the disease variation. All 13 isolates harbored the same BsToxA nucleotide sequence, which was identical to one of the two haplotypes previously identified in Australia. Sensitivity to ToxA is prevalent in popular hard winter wheat cultivars in the central and southcentral winter wheat regions of the United States, showing the potential of a selective advantage for B. sorokiniana isolates that harbor the ToxA gene.

Genetic analysis of net form net blotch resistance in barley lines CIho 5791 and Tifang against a global collection of P. teres f. teres isolates.

KEY MESSAGE: A CIho 5791 × Tifang recombinant inbred mapping population was developed and used to identify major dominant resistance genes on barley chromosomes 6H and 3H in CI5791 and on 3H in Tifang. The barley line CIho 5791 confers high levels of resistance to Pyrenophora teres f. teres, causal agent of net form net blotch (NFNB), with few documented isolates overcoming this resistance. Tifang barley also harbors resistance to P. teres f. teres which was previously shown to localize to barley chromosome 3H. A CIho 5791 × Tifang F6 recombinant inbred line (RIL) population was developed using single seed descent. The Illumina iSelect SNP platform was used to identify 2562 single nucleotide polymorphism (SNP) markers across the barley genome, resulting in seven linkage maps, one for each barley chromosome. The CIho 5791 × Tifang RIL population was evaluated for NFNB resistance using nine P. teres f. teres isolates collected globally. Tifang was resistant to four of the isolates tested whereas CIho 5791 was highly resistant to all nine isolates. QTL analysis indicated that the CIho 5791 resistance mapped to chromosome 6H whereas the Tifang resistance mapped to chromosome 3H. Additionally, CIho 5791 also harbored resistance to two Japanese isolates that mapped to a 3H region similar to that of Tifang. SNP markers and RILs harboring both 3H and 6H resistance will be useful in resistance breeding against NFNB.

Identification and Characterization of the SnTox6-Snn6 Interaction in the Parastagonospora nodorum-Wheat Pathosystem.

Parastagonospora nodorum is a necrotrophic fungal pathogen that causes Septoria nodorum blotch (SNB) (formerly Stagonospora nodorum blotch) on wheat. P. nodorum produces necrotrophic effectors (NE) that are recognized by dominant host sensitivity gene products resulting in disease development. The NE-host interaction is critical to inducing NE-triggered susceptibility (NETS). To date, seven NE-host sensitivity gene interactions, following an inverse gene-for-gene model, have been identified in the P. nodorum-wheat pathosystem. Here, we used a wheat mapping population that segregated for sensitivity to two previously characterized interactions (SnTox1-Snn1 and SnTox3-Snn3-B1) to identify and characterize a new interaction involving the NE designated SnTox6 and the host sensitivity gene designated Snn6. SnTox6 is a small secreted protein that induces necrosis on wheat lines harboring Snn6. Sensitivity to SnTox6, conferred by Snn6, was light-dependent and was shown to underlie a major disease susceptibility quantitative trait locus (QTL). No other QTL were identified, even though the P. nodorum isolate used in this study harbored both the SnTox1 and SnTox3 genes. Reverse transcription-polymerase chain reaction showed that the expression of SnTox1 was not detectable, whereas SnTox3 was expressed and, yet, did not play a significant role in disease development. This work expands our knowledge of the wheat-P. nodorum interaction and further establishes this system as a model for necrotrophic specialist pathosystems.

Targeted mapping of Cdu1, a major locus regulating grain cadmium concentration in durum wheat (Triticum turgidum L. var durum).

Some durum wheat (Triticum turgidum L. var durum) cultivars have the genetic propensity to accumulate cadmium (Cd) in the grain. A major gene controlling grain Cd concentration designated as Cdu1 has been reported on 5B, but the genetic factor(s) conferring the low Cd phenotype are currently unknown. The objectives of this study were to saturate the chromosomal region harboring Cdu1 with newly developed PCR-based markers and to investigate the colinearity of this wheat chromosomal region with rice (Oryza sativa L.) and Brachypodium distachyon genomes. Genetic mapping of markers linked to Cdu1 in a population of recombinant inbred substitution lines revealed that the gene(s) associated with variation in Cd concentration resides in wheat bin 5BL9 between fraction breakpoints 0.76 and 0.79. Genetic mapping and quantitative trait locus (QTL) analysis of grain Cd concentration was performed in 155 doubled haploid lines from the cross W9262-260D3 (low Cd) by Kofa (high Cd) revealed two expressed sequence tag markers (ESMs) and one sequence tagged site (STS) marker that co-segregated with Cdu1 and explained >80% of the phenotypic variation in grain Cd concentration. A second, minor QTL for grain Cd concentration was also identified on 5B, 67 cM proximal to Cdu1. The Cdu1 interval spans 286 kbp of rice chromosome 3 and 282 kbp of Brachypodium chromosome 1. The markers and rice and Brachypodium colinearity described here represent tools that will assist in the positional cloning of Cdu1 and can be used to select for low Cd accumulation in durum wheat breeding programs targeting this trait. The isolation of Cdu1 will further our knowledge of Cd accumulation in cereals as well as metal accumulation in general.

Marker-assisted characterization of durum wheat Langdon-Golden Ball disomic substitution lines.

The durum wheat cultivar 'Golden Ball' (GB) is a source of resistance to wheat sawfly due to its superior solid stem. In the late 1980s, Dr. Leonard Joppa developed a complete set of 14 'Langdon' (LDN)-GB disomic substitution (DS) lines by using GB as the chromosome donor and LDN as the recipient. However, these substitution lines have not been previously characterized and reported in the literature. The objectives of this study were to confirm the authenticity of the substituted chromosomes and to analyze the genetic background of the 14 LDN-GB DS lines with the aid of molecular markers, and to further use the substitution lines for chromosomal localization of DNA markers and genes conferring the superior stem solidness in GB. Results from simple sequence repeat marker analysis validated the authenticity of the substituted chromosomes in 14 LDN-GB DS lines. Genome-wide scans using the target region amplification polymorphism (TRAP) marker system produced a total of 359 polymorphic fragments that were used to compare the genetic background of substitution lines with that of LDN. Among the polymorphic TRAP markers, 134 (37.3%) and 185 (51.5%) were present in LDN and GB, respectively, with only 10 (2.8%) derived from Chinese Spring. Therefore, marker analysis demonstrated that each LDN-GB DS line had a pair of chromosomes from GB with a genetic background similar to that of LDN. Of the TRAP markers generated in this study, 200 were successfully assigned to specific chromosomes based on their presence or absence in the corresponding LDN-GB DS lines. Also, evaluation of stem solidness in the substitution lines verified the presence of a major gene for stem solidness in chromosome 3B. Results from this research provides useful information for the utilization of GB and LDN-GB DS lines for genetic and genomic studies in tetraploid wheat and for the improvement of stem solidness in both durum and bread wheat.

Genetic analysis of disease susceptibility contributed by the compatible Tsn1-SnToxA and Snn1-SnTox1 interactions in the wheat-Stagonospora nodorum pathosystem.

Stagonospora nodorum is a foliar pathogen of wheat that produces several host-selective toxins (HSTs) and causes the disease Stagonospora nodorum blotch (SNB). The wheat genes Snn1 and Tsn1 confer sensitivity to the HSTs SnTox1 and SnToxA, respectively. The objectives of this study were to dissect, quantify, and compare the effects of compatible Snn1-SnTox1 and Tsn1-SnToxA interactions on susceptibility in the wheat-S. nodorum pathosystem. Inoculation of a wheat doubled haploid population that segregates for both Snn1 and Tsn1 with an S. nodorum isolate that produces both SnTox1 and SnToxA indicated that both interactions were strongly associated with SNB susceptibility. The Snn1-SnTox1 and Tsn1-SnToxA interactions explained 22 and 28% of the variation in disease, respectively, and together they explained 48% indicating that their effects are largely additive. The Snn1-SnTox1 interaction accounted for 50% of the variation when the population was inoculated with an S. nodorum strain where the SnToxA gene had been mutated, eliminating the Tsn1-SnToxA interaction. These results support the theory that the wheat-S. nodorum pathosystem is largely based on multiple host-toxin interactions that follow an inverse gene-for-gene scenario at the host-toxin interface, but disease exhibits quantitative variation due to the mainly additive nature of compatible interactions. The elimination of either Snn1 or Tsn1 toxin sensitivity alleles resulted in decreased susceptibility, but the elimination of both interactions was required to obtain high levels of resistance. We propose the use of molecular markers to select against Snn1, Tsn1, and other toxin sensitivity alleles to develop wheat varieties with high levels of SNB resistance.

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) (xAegilotriticum 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.

Host-selective toxins produced by Stagonospora nodorum confer disease susceptibility in adult wheat plants under field conditions.

Stagonospora nodorum, causal agent of Stagonospora nodorum blotch (SNB), is a destructive pathogen of wheat worldwide. As is true for many necrotrophic host-pathogen systems, the wheat-S. nodorum system is complex and resistance to SNB is usually quantitatively inherited. We recently showed that S. nodorum produces at least four proteinaceous host-selective toxins that interact with dominant host sensitivity/susceptibility gene products to induce SNB in seedlings. Here, we evaluated a population of wheat recombinant inbred lines that segregates for Tsn1, Snn2, and Snn3, which confer sensitivity to the toxins SnToxA, SnTox2, and SnTox3, respectively, to determine if compatible host-toxin interactions are associated with adult plant susceptibility to SNB foliar disease under field conditions. Artificial inoculation of the population in 2 years and two locations with a fungal isolate known to produce SnToxA and SnTox2 indicated that compatible SnToxA-Tsn1 and SnTox2-Snn2 interactions accounted for as much as 18 and 15% of the variation in disease severity on the flag leaf, respectively. As previously reported for seedlings, the effects of these two interactions in conferring adult plant susceptibility were largely additive. Additional adult plant resistance QTLs were identified on chromosomes 1B, 4B, and 5A, of which, the 1B and 5A QTLs were previously reported to be associated with seedling resistance to SNB. Therefore, in this population, some of the same QTLs are responsible for seedling and adult plant resistance/susceptibility. This is the first report showing that host-selective toxins confer susceptibility of adult plants to SNB, further substantiating the importance of compatible toxin-host interactions in the wheat-S. nodorum pathosystem.

Development and characterization of expressed sequence tag-derived microsatellite markers for the wheat stem rust fungus Puccinia graminis f. sp. tritici.

Puccinia graminis f. sp. tritici is the causal agent of stem rust disease in wheat. The rust fungus has caused devastating disease epidemics throughout history and is still posing a potential threat to wheat production in some regions of the world due to the appearance of new races. To develop microsatellite or simple sequence repeat (SSR) markers for use in population genetics studies, a total of 60,579 expressed sequence tag (EST) sequences (reads) generated from P. graminis f. sp. tritici were screened for tandemly repeated di- and tri-nucleotide units using a bioinformatics approach and 708 unisequences containing putative SSR loci with six or more repeat units were identified. Flanking primers were designed for 384 unique SSR loci, which mapped to different locations of the draft genome sequence of the fungus. Of the 384 primer pairs tested, 72 EST-SSR markers were eventually developed, which showed polymorphism among 19 isolates of P. graminis f. sp. tritici and 4 isolates of P. graminis f. sp. secalis evaluated. Thirty-two of the SSR loci were also evaluated in three other rust fungi (P. triticina, P. hordei, and P. coronata f. sp. hordei) for cross-species transferability. These SSR markers derived from ESTs will be useful for characterization of population structures and for gene mapping in P. graminis.

Emergence of tan spot disease caused by toxigenic Pyrenophora tritici-repentis in Australia is not associated with increased deployment of toxin-sensitive cultivars.

The wheat disease tan (or yellow leaf) spot, caused by Pyrenophora tritici-repentis, was first described in the period 1934 to 1941 in Canada, India, and the United States. It was first noted in Australia in 1953 and only became a serious disease in the 1970s. The emergence of this disease has recently been linked to the acquisition by P. tritici-repentis of the ToxA gene from the wheat leaf and glume blotch pathogen, Stagonospora nodorum. ToxA encodes a host-specific toxin that interacts with the product of the wheat gene Tsn1. Interaction of ToxA with the dominant allele of Tsn1 causes host necrosis. P. tritici-repentis races lacking ToxA give minor indistinct lesions on wheat lines, whereas wheat lines expressing the recessive tsn1 are significantly less susceptible to the disease. Although the emergence and spread of tan spot had been attributed to the adoption of minimum tillage practices, we wished to test the alternative idea that the planting of Tsn1 wheat lines may have contributed to the establishment of the pathogen in Australia. To do this, wheat cultivars released in Australia from 1911 to 1986 were tested for their sensitivity to ToxA. Prior to 1941, 16% of wheat cultivars were ToxA-insensitive and hence, all other factors being equal, would be more resistant to the disease. Surprisingly, only one of the cultivars released since 1940 was ToxA insensitive, and the area planted to ToxA-insensitive cultivars varied from 0 to a maximum of only 14% in New South Wales. Thus, the majority of the cultivars were ToxA-sensitive both before and during the period of emergence and spread of the disease. We therefore conclude that the spread of P. tritici-repentis in Australia cannot be causally linked to the deployment of ToxA-sensitive cultivars.

A region of barley chromosome 6H harbors multiple major genes associated with net type net blotch resistance.

Net type net blotch (NTNB), caused by Pyrenophora teres f. teres Drechs., is prevalent in barley growing regions worldwide. A population of 118 doubled haploid (DH) lines developed from a cross between barley cultivars 'Rika' and 'Kombar' were used to evaluate resistance to NTNB due to their differential reaction to various isolates of P. teres f. teres. Rika was resistant to P. teres f. teres isolate 15A and susceptible to isolate 6A. Conversely, Kombar was resistant to 6A, but susceptible to 15A. A progeny isolate of a 15A x 6A cross identified as 15A x 6A#4 was virulent on both parental lines. The Rika/Kombar (RK) DH population was evaluated for disease reactions to the three isolates. Isolate 15A induced a resistant:susceptible ratio of 78:40 (R:S) whereas isolate 6A induced a resistant:susceptible ratio of 40:78. All but two lines had opposite disease reactions indicating two major resistance genes linked in repulsion. Progeny isolate 15A x 6A#4 showed a resistant:susceptible ratio of 1:117 with the one resistant line also being the single line that was resistant to both 15A and 6A. An RK F(2) population segregated in a 1:3 (R:S) ratio for both 15A and 6A indicating that resistance is recessive. Molecular markers were used to identify a region on chromosome 6H that harbors the two NTNB resistance genes. This work shows that multiple NTNB resistance genes exist at the locus on chromosome 6H, and the recombinant DH line harboring the resistance alleles from both parents will be useful for the development of NTNB-resistant barley germplasm.

Acc homoeoloci and the evolution of wheat genomes.

The DNA sequences of wheat Acc-1 and Acc-2 loci, encoding the plastid and cytosolic forms of the enzyme acetyl-CoA carboxylase, were analyzed with a view to understanding the evolution of these genes and the origin of the three genomes in modern hexaploid wheat. Acc-1 and Acc-2 loci from each of the wheats Triticum urartu (A genome), Aegilops tauschii (D genome), Triticum turgidum (AB genome), and Triticum aestivum (ABD genome), as well as two Acc-2-related pseudogenes from T. urartu were sequenced. The 2.3-2.4 Mya divergence time calculated here for the three homoeologous chromosomes, on the basis of coding and intron sequences of the Acc-1 genes, is at the low end of other estimates. Our clock was calibrated by using 60 Mya for the divergence between wheat and maize. On the same time scale, wheat and barley diverged 11.6 Mya, based on sequences of Acc and other genes. The regions flanking the Acc genes are not conserved among the A, B, and D genomes. They are conserved when comparing homoeologous genomes of diploid, tetraploid, and hexaploid wheats. Substitution rates in intergenic regions consisting primarily of repetitive sequences vary substantially along the loci and on average are 3.5-fold higher than the Acc intron substitution rates. The composition of the Acc homoeoloci suggests haplotype divergence exceeding in some cases 0.5 Mya. Such variation might result in a significant overestimate of the time since tetraploid wheat formation, which occurred no more than 0.5 Mya.

Identification of novel tan spot resistance loci beyond the known host-selective toxin insensitivity genes in wheat.

Tan spot, caused by Pyrenophora tritici-repentis, is a destructive foliar disease of wheat causing significant yield reduction in major wheat growing areas throughout the world. The objective of this study was to identify quantitative trait loci (QTL) conferring resistance to tan spot in the synthetic hexaploid wheat (SHW) line TA4152-60. A doubled haploid (DH) mapping population derived from TA4152-60 x ND495 was inoculated with conidia produced by isolates of each of four virulent races of P. tritici-repentis found in North America. QTL analysis revealed a total of five genomic regions significantly associated with tan spot resistance, all of which were contributed by the SHW line. Among them, two novel QTLs located on chromosome arms 2AS and 5BL conferred resistance to all isolates tested. Another novel QTL on chromosome arm 5AL conferred resistance to isolates of races 1, 2 and 5, and a QTL specific to a race 3 isolate was detected on chromosome arm 4AL. None of these QTLs corresponded to known host selective toxin (HST) insensitivity loci, but a second QTL on chromosome arm 5BL conferred resistance to the Ptr ToxA producing isolates of races 1 and 2 and corresponded to the Tsn1 (Ptr ToxA sensitivity) locus. This indicates that the wheat-P. tritici-repentis pathosystem is much more complex than previously thought and that selecting for toxin insensitivity alone will not necessarily lead to tan spot resistance. The markers associated with the QTLs identified in this work will be useful for deploying the SHW line as a tan spot resistance source in wheat breeding.

Seedling Resistance to Tan Spot and Stagonospora nodorum Leaf Blotch in Wild Emmer Wheat (Triticum dicoccoides).

Tan spot and Stagonospora nodorum blotch (SNB), caused by Pyrenophora tritici-repentis and Stagonospora nodorum, respectively, are two destructive foliar diseases of wheat, causing significant yield reduction worldwide. The objective of this study was to evaluate 172 accessions of wild emmer wheat (Triticum dicoccoides) for seedling resistance to tan spot and SNB. All accessions were inoculated with P. tritici-repentis race 1 and a mixture of three diverse isolates of S. nodorum, respectively. The accessions were also evaluated for sensitivity to host-selective toxins (HSTs), including ToxA produced by both S. nodorum and P. tritici-repentis and culture filtrate produced by S. nodorum. A total of 34 accessions were resistant to tan spot, and 136 accessions were resistant to SNB. Among these accessions, 31 were resistant to both diseases. Significant correlations between HST insensitivity and disease resistance were observed. Our results showed that T. dicoccoides is a good genetic source of resistance to tan spot and SNB in wheat.

Identification of a Novel Fusarium Head Blight Resistance Quantitative Trait Locus on Chromosome 7A in Tetraploid Wheat.

ABSTRACT Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most destructive diseases of durum (Triticum turgidum sp. durum) and common wheat (T. aestivum). Promising sources of FHB resistance have been identified among common (hexaploid) wheats, but the same is not true for durum (tetraploid) wheats. A previous study indicated that chromosome 7A from T. turgidum sp. dicoccoides accession PI478742 contributed significant levels of resistance to FHB. The objectives of this research were to develop a genetic linkage map of chromosome 7A in a population of 118 recombinant inbred lines derived from a cross between the durum cv. Langdon (LDN) and a disomic LDN-T. turgidum sp. dicoccoides PI478742 chromosome 7A substitution line [LDN-DIC 7A(742)], and identify a putative FHB resistance quantitative trait locus (QTL) on chromosome 7A derived from LDN-DIC 7A(742). The population was evaluated for type II FHB resistance in three greenhouse environments. Interval regression analysis indicated that a single QTL designated Qfhs.fcu-7AL explained 19% of the phenotypic variation and spanned an interval of 39.6 cM. Comparisons between the genetic map and a previously constructed physical map of chromosome 7A indicated that Qfhs.fcu-7AL is located in the proximal region of the long arm. This is only the second FHB QTL to be identified in a tetraploid source, and it may be useful to combine it with the QTL Qfhs.ndsu-3AS in order to develop durum wheat germ plasm and cultivars with higher levels of FHB resistance.

Identification and chromosomal location of major genes for resistance to Pyrenophora teres in a doubled-haploid barley population.

Net blotch, caused by Pyrenophora teres, is one of the most economically important diseases of barley worldwide. Here, we used a barley doubled-haploid population derived from the lines SM89010 and Q21861 to identify major quantitative trait loci (QTLs) associated with seedling resistance to P. teres f. teres (net-type net blotch (NTNB)) and P. teres f. maculata (spot-type net blotch (STNB)). A map consisting of simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers was used to identify chromosome locations of resistance loci. Major QTLs for NTNB and STNB resistance were located on chromosomes 6H and 4H, respectively. The 6H locus (NTNB) accounted for as much as 89% of the disease variation, whereas the 4H locus (STNB resistance) accounted for 64%. The markers closely linked to the resistance gene loci will be useful for marker-assisted selection.

Molecular mapping of hybrid necrosis genes Ne1 and Ne2 in hexaploid wheat using microsatellite markers.

Hybrid necrosis is the gradual premature death of leaves or plants in certain F1 hybrids of wheat (Triticum aestivum L.), and it is caused by the interaction of two dominant complementary genes Ne1 and Ne2 located on chromosome arms 5BL and 2BS, respectively. To date, molecular markers linked to these genes have not been identified and linkage relationships of the two genes with other important genes in wheat have not been established. We observed that the F1 hybrids from the crosses between the bread wheat variety 'Alsen' and four synthetic hexaploid wheat (SHW) lines (TA4152-19, TA4152-37, TA4152-44, and TA4152-60) developed at the International Maize and Wheat Improvement Center (CIMMYT) exhibited hybrid necrosis. This study was conducted to determine the genotypes of TA4152-60 and Alsen at the Ne1 and Ne2 loci, and to map the genes using microsatellite markers in backcross populations. Genetic analysis indicated that Alsen has the genotype ne1ne1Ne2Ne2 whereas the SHW lines have Ne1Ne1ne2ne2. The microsatellite marker Xbarc74 was linked to Ne1 at a genetic distance of 2.0 cM on chromosome arm 5BL, and Xbarc55 was 3.2 cM from Ne2 on 2BS. Comparison of the genetic maps with the chromosome deletion-based physical maps indicated that Ne1 lies in the proximal half of 5BL, whereas Ne2 is in the distal half of 2BS. Genetic linkage analysis showed that Ne1 was about 35 cM proximal to Tsn1, a locus conferring sensitivity to the host selective toxin Ptr ToxA produced by the tan spot fungus. The closely linked microsatellite markers identified in this study can be used to genotype parental lines for Ne1 and Ne2 or to eliminate the two hybrid necrosis genes using marker-assisted selection.

Genomic analysis and marker development for the Tsn1 locus in wheat using bin-mapped ESTs and flanking BAC contigs.

The wheat Tsn1 gene confers sensitivity to the host-selective toxin Ptr ToxA produced by the tan spot fungus (Pyrenophora tritici-repentis). The long-term goal of this research is to isolate Tsn1 using a positional cloning approach. Here, we evaluated 54 ESTs (expressed sequence tags) physically mapped to deletion bin 5BL 0.75-0.76, which is a gene-rich region containing Tsn1. Twenty-three EST loci were mapped as either PCR-based single-stranded conformational polymorphism or RFLP markers in a low-resolution wheat population. The genetic map corresponding to the 5BL 0.75-0.76 deletion bin spans 18.5 cM and contains 37 markers for a density of 2 markers/cM. The EST-based genetic map will be useful for tagging other genes, establishing colinearity with rice, and anchoring sequence ready BAC contigs of the 5BL 0.75-0.76 deletion bin. High-resolution mapping showed that EST-derived markers together with previously developed AFLP-derived markers delineated Tsn1 to a 0.8 cM interval. Flanking markers were used to screen the Langdon durum BAC library and contigs of 205 and 228 kb flanking Tsn1 were assembled, sequenced, and anchored to the genetic map. Recombination frequency averaged 760 kb/cM across the 228 kb contig, but no recombination was observed across the 205 kb contig resulting in an expected recombination frequency of more than 10 Mb/cM. Therefore, chromosome walking within the Tsn1 region may be difficult. However, the sequenced BACs allowed the identification of one microsatellite in each contig for which markers were developed and shown to be highly suitable for marker-assisted selection of Tsn1.

Molecular cytogenetic characterization and seed storage protein analysis of 1A/1D translocation lines of durum wheat.

Two durum wheat [Triticum turgidum L. ssp. durum (Desf.) Husn.] lines carrying the high-molecular-weight (HMW) glutenin subunits (GS) 1 D x 5 + 1Dy10 encoded by Glu-D1d, L252 and S99B34, were characterized using fluorescent genomic in-situ hybridization (FGISH) and microsatellite markers. These two durum lines were derived from the crosses in which the common wheat (T. aestivum L.) 'Len' and durum wheat 'Langdon' (LDN) and 'Renville' were involved. FGISH patterns of the mitotic chromosomes indicated that these two durum lines have one pair of 1AS.1AL-1DL translocated chromosomes in which the terminal region of 1AL was replaced by a homoeologous segment of 1DL. The 1DL segment spans approximately 31% of the long arm of the translocated chromosome. Microsatellite marker analysis confirmed the 1AS.1AL-1DL translocation and determined the translocation breakpoint to be distal to Xgwm357 on 1AL. Seed storage proteins (GS and gliadins) were analysed in these two 1AS.1AL-1DL translocation lines and three sib lines (L092, S99B19 and S99B33) using SDS-PAGE and A-PAGE. The SDS-PAGE and A-PAGE profiles demonstrated that the two low yielding lines (L252 and S99B19) had the low-molecular-weight (LMW) -1 GS encoded by Glu-A3k and Glu-B3s and 1B-encoded gliadins from LDN, and the other three lines (L092, S99B33 and S99B34) with higher yield had LMW-2 GS and 1B-encoded gliadins from Renville, suggesting that undesirable genetic components from LDN might limit substantial improvement of yield. Thus, the translocation lines with 1 D x 5 + 1Dy10 and LMW-2, which are associated with good bread-making and pasta qualities, respectively, in a good genetic background will be useful for developing durum cultivars with dual-purpose end-use. Results from this study demonstrate that the D-genome could play an important role in the genetic improvement of durum wheat and evolution of the A- and B-genomes in tetraploid wheat.

A wheat intervarietal genetic linkage map based on microsatellite and target region amplified polymorphism markers and its utility for detecting quantitative trait loci.

Efficient user-friendly methods for mapping plant genomes are highly desirable for the identification of quantitative trait loci (QTLs), genotypic profiling, genomic studies, and marker-assisted selection. SSR (microsatellite) markers are user-friendly and efficient in detecting polymorphism, but they detect few loci. Target region amplification polymorphism (TRAP) is a relatively new PCR-based technique that detects a large number of loci from a single reaction without extensive pre-PCR processing of samples. In the investigation reported here, we used both SSRs and TRAPs to generate over 700 markers for the construction of a genetic linkage map in a hard red spring wheat intervarietal recombinant inbred population. A framework map consisting of 352 markers accounted for 3,045 cM with an average density of one marker per 8.7 cM. On average, SSRs detected 1.9 polymorphic loci per reaction, while TRAPs detected 24. Both marker systems were suitable for assigning linkage groups to chromosomes using wheat aneuploid stocks. We demonstrated the utility of the maps by identifying major QTLs for days to heading and reduced plant height on chromosomes 5A and 4B, respectively. Our results indicate that TRAPs are highly efficient for genetic mapping in wheat. The maps developed will be useful for the identification of quality and disease resistance QTLs that segregate in this population.