Allelic variation analysis and development of gene-specific molecular markers confering acid soil tolerance in barley (Hordeum vulgare L.)

Acid soil is a prevalent problem over the world. The high concentration of Al in the acid soil is one of the major production limiting factors to many plants. Decades of studies have resulted in a significant progress in revealing the mechanism of Al tolerance in plants. Several key genes have been identified and the Al tolerance was validated to be related with gene sequence variations in some plants. Barley (Hordeum vulgare L.) is one of most sensitive cereals to aluminum toxicity. This thesis was aimed at revealing the mechanisms of Al tolerance in barley using the marker development, QTL, association mapping and sequencing techniques. The major findings of the thesis are listed below: 1. The genetics of barley Al tolerance was studied in two double haploid populations, Hamelin/Svanhals and Br2/Hamelin, through QTL mapping. The phenotypic vitiation was investigated in both hydroponic and acid soil experiments. The phenotypic result suggested that a single gene is responsible for Al tolerance in barley. Al tolerance is controlled by single QTL on chromosome 4H and flanked by commonly used SSR makers. 2. Gene-specific markers were developed covering the whole sequence of HvAACT1 gene (also named as HvMATE). The polymorphic gene-specific markers were incorporated with the other commonly used SSR markers to conduct the QTL mapping. The result showed that the QTL interval for acid soil tolerance was narrowed and the phenotypic variations explained by the QTLs were increased. The gene-specific markers could also explain more phenotype variation than these commonly used SSR markers. These new gene-specific markers provide effective and simple molecular tools for marker assisted selection in acid soil tolerant barley breeding. 3. The genetic diversity analysis and candidate gene association mapping based on HvAACT1 gene were conducted using accessions with different Al tolerance. Twenty eight gene-specific markers were polymorphic among different accessions. The sequencing analysis showed that these polymorphisms among accessions varied from over 1Kb to one SNP. These markers clearly identified the genetic relationship of different accessions using cluster analysis. Several gene specific markers were found to be associated with the Al tolerance in accessions. These significant polymorphisms detected by these markers could be considered candidate variation sites related to Al tolerance. 4. The allele variations of HvALMT were also studied in different accessions. Ten pairs of primers (6 in the coding region, 4 in the upstream region) showed polymorphisms. Sequencing analysis showed that these polymorphisms varied from one SNP to over 400bp insertion/deletion. Stepwise regression analysis revealed one gene-specific marker, UA21, was significantly correlated with the phenotypic variation of root length in acid soil and the relative root length in acid soil, as well as acid soil treated with lime. In conclusion, the findings from this thesis suggest that: 1) While citrate exudation was validated to be responsible for Al tolerance in barley, it cannot fully explain the genetic variability in Al tolerance in barley; 2) Malate exudation may play an important role in detoxifying Al in some barley accessions; 3) The gene specific markers developed from HvAACT1 and HvALMT can be used in molecular marker assisted selection in breeding; and 4) The significant polymorphisms detected by the association mapping can be used for further gene expression study.