Tillering Characteristic And Genetic Analysis Of Scald Resistance In Cultivated Barley Vlamingh

Barley（Hordeum vulgare L.） is the fourth most important cereal crop in the world, which has wide adaptability and high nutritional value. Due to the scarce germplasm resources and limited genetic information, it is difficult to develop barley cultivars with high yield and durable disease resistance. Exploitation of elite germplasm is regarded as an efficient strategy for barley breeding. The cultivated barley variety, Vlamingh, originally introduced from Australia to China, processes wide genetic variations and multiple disease-resistance genes. The mining of the important traits associated with yield and disease resistance from Vlamingh is of critical importance in barley breeding.In the present study, a mutant deprived from Vlamingh was used to investigate the tillering ability, and the mechanism of tillering was systematically resolved by molecular biological, metabonomic, histological and anatomical methods. Meanwhile, the QTL analysis of scald resistance was carried out using a DH population deprived from a cross of Vlamingh and WABAR2147. The main results are as follows:1. High-tillering mutant, hnil, was isolated from Barley cultivar Vlamingh treated with gramma rays. Morphologically, the mutant exhibited dwarfism. excessive tillers, and narrow leaf compared to that of the wild-type. Anatomical analysis suggests that the increased tiller number of hut1was a consequence of accelerating tiller buds outgrowth rather than more tiller buds initiation. Among the DH population, we demonstrated that hntl was controlled genetically by a single locus based on the segregation ratio that148out of269DH lines exhibited the mutant phenotype. Linkage analysis with360SSR markers showed that the locus was primarily mapped between the markers Ebmatc0039and GBM1385on the long arm of chromosome2H.2. Based on the sequence difference between barley cultivar Morex and Barke, we developed additional PCR-based markers and then narrowed down the locus between the INDELs markers M39589and M54556. Within this region, there were25high confidential genes which were defined the function in comparison with sequenced model plant genomes. Four genes were related to branch regulation.3. Sequence comparison of four genes between mutant hntl and the wild-type revealed that a2-bp deletion existed in MLOC₆7307.2. No mutation was found in the other three genes. Two molecular markers, which were developed from the2-bp deletion, could co-segregate with the mutated phenotype in the DH population. Therefore. MLOC67307.2was considered as the candidate gene of the hntl locus. MLOC₆7307.2encoded a trypsin-like cysteine protease with648amino acids. The2-bp deletion at the fourth exon caused a frame shift and generated a premature translation termination product in the hntl mutant. Real-time PCR provided the evidence that the MLOC₆7307.2was mainly expressed in shoot bases and leaves. Phylogenetic tree analysis indicated that the MLOC67307.2had various homologies in many plant species. Notablely, rice Nall gene shared85%sequence similarity to the MLOC₆7307.2. Loss-of-function mutation in Nall gene showed a similar characteristic phenotype with that of the hntl mutant.4. In order to reveal mechanisms of hormonal signaling in tillering, the mutant hntl and Vlamingh were used to detect the contents of trans-Zeatin and indole-3-acetic acid in leaves and basal stem by HPLC-MS. For mutant hntl and Vlamingh, there were obvious differences in the contents of trans-Zeatin and indole-3-acetic acid between leaves and basal stem, respectively. Compared with that of Vlamingh, the basal stem of mutant hntl had higher level of trans-Zeatin. Meanwhile, we selected3homologies of At-PINl gene from barley genome and investigated the expression of them in basal stem between Vlamingh and mutant hntl. Realtime PCR assays indicated that the expression levels of AK357068gene were significantly reduced in mutant hntl.5. A population of220double haploid （DH） lines was developed from a cross of Vlamingh and WABAR2147. in which Vlamingh showed adult plant resistance and WABAR2147showed seedling resistance to a group of isolates. The population was tested for adult plant resistance to scald under natural infection in two consecutive seasons in addition to seedling screen with three isolates. One single gene was mapped to chromosome6H based to the seedling test and two QTLs（QSc.VlWa.4H and QSc.VlWa.6H） were mapped to chromosomes4H and6H based on the adult plant resistance. Epistatic interaction was observed between the two QTLs and environment/QTL interaction was only observed for QSc.VlWa.6H which co-segregated with the seedling resistance gene and contributed to the basal resistance against scald during the whole growth stages. OSc. VlWa.4H explained42.5%and57.8%of the phenotypic variations in the two independent trials when the effect of QSc. VlWa.6H was excluded for analysis. In order to compare with the known genes and QTLs for scald resistance, we developed a high density consensus genetic map with7876molecular makers.27QTLs and7genes for scald resistance from different mapping populations. No known QTL or gene was reported in the similar position of OSc. VlWa.4H and it was the first major QTL for adult plant resistance of scald on chromosome4HS. Integration of the two QTL achieved better and stable scald resistance across4different environments.