A Novel Retrotransposon Inserted in the Dominant Vrn-B1 Allele Confers Spring Growth Habit in Tetraploid Wheat (Triticum turgidum L.).

Vernalization genes determine winter/spring growth habit in temperate cereals and play important roles in plant development and environmental adaptation. In wheat (Triticum L. sp.), it was previously shown that allelic variation in the vernalization gene VRN1 was due to deletions or insertions either in the promoter or in the first intron. Here, we report a novel Vrn-B1 allele that has a retrotransposon in its promoter conferring spring growth habit. The VRN-B1 gene was mapped in a doubled haploid population that segregated for winter-spring growth habit but was derived from two spring tetraploid wheat genotypes, the durum wheat (T. turgidum subsp. durum) variety 'Lebsock' and T. turgidum subsp. carthlicum accession PI 94749. Genetic analysis revealed that Lebsock carried the dominant Vrn-A1 and recessive vrn-B1 alleles, whereas PI 94749 had the recessive vrn-A1 and dominant Vrn-B1 alleles. The Vrn-A1 allele in Lebsock was the same as the Vrn-A1c allele previously reported in hexaploid wheat. No differences existed between the vrn-B1 and Vrn-B1 alleles, except that a 5463-bp insertion was detected in the 5'-UTR region of the Vrn-B1 allele. This insertion was a novel retrotransposon (designated as retrotrans_VRN), which was flanked by a 5-bp target site duplication and contained primer binding site and polypurine tract motifs, a 325-bp long terminal repeat, and an open reading frame encoding 1231 amino acids. The insertion of retrotrans_VRN resulted in expression of Vrn-B1 without vernalization. Retrotrans_VRN is prevalent among T. turgidum subsp. carthlicum accessions, less prevalent among T. turgidum subsp. dicoccum accessions, and rarely found in other tetraploid wheat subspecies.

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.

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.

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.

The effect of talisay (Terminalia catappa) extract on Staphylococcus aureus growth in vitro and Staphylococcus aureus-infected wounds in guinea pig (Cavia porcellus)

Seventeen Philippine plants were subjected to antimicrobial screening against Staphylococcus aureus and Escherichia coli using antimicrobial disc assay. The result showed that Terminalia catappa, locally known as "talisay", exhibited the highest antimicrobial activity against S. aureus and none against E. coli. The growth of Staphylococcus aureus inoculated in nutrient broth with different concentrations of the T. catappa extract was determined by measuring cell density at 0 hours and 24 hours of inoculation. The results showed that bacterial cell density decreased significantly after 24 hours of inoculation in the plant extract. Talisay was further tested for its wound healing properties on 2 groups (group 1: standard drug vs. negative control; group 2: herbal extract vs. negative control) of guinea pigs using the abrasion method. A swab of inoculum of S. aureus was applied for infecting the wound. Differences in degrees of wound healing determined by free radical scavenging activity, colony forming units (CFU) counting and histopathologic analysis were noted. Samples from wound abscesses remaining after 48 hours of application of extract were swabbed in petriplates containing 20 ml nutrient agar and were verified using the catalase test. The CFUs were counted 24 hours after incubation. Crude extract was further subjected to High Pressure Liquid Chromatography (HPLC) yielding a polar substance suspected to be of the aromatic tannin family. ANOVA revealed significant difference in the positive control and negative control results against the T. catappa extract treatment in the in vivo antimicrobial activity model. Among these setups, the wounds treated with the extract exhibited advanced healing as supported by significantly lower absorbance levels in the antioxidant assay, lower CFU count, and significantly higher grade in wound healing parameters for histopathologic analysis. The T. catappa extract under study showed significant inhibition of growth of S. aureus and effective healing of infected wounds