Genetic analysis of Barley (Hordeum vulgare L.) grain (1,3;1,4)-β-glucan concentration and fine structure.
Date
2015-03-19
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Degree Level
Doctoral
Abstract
(1,3;1,4)-β-glucan accumulated in barley (Hordeum vulgare L.) cell walls is an important determinant for grain end-use as food, malt, feed or fuel. As a trait affected by multiple genes and the environment, grain (1,3;1,4)-β-glucan concentration qualifies as a quantitative trait. A major QTL on chromosome 7H contains a cellulose synthase like gene HvCslF6, coding for an enzyme associated with (1,3;1,4)-β-glucan biosynthesis. To develop gene based perfect markers, HvCslF6 was analyzed to determine allelic variation between CDC Bold, a low (1,3;1,4)-β-glucan (~ 3.3 %) cultivar and TR251, a high (1,3;1,4)-β-glucan (~ 5.2 %) genotype. Comparison of the CDC Bold and TR251 nucleotide sequences downstream of the ATG start codon in HvCslF6 alleles revealed 16 single nucleotide polymorphisms (SNPs) and two indels. The two indels added 16 nucleotides to the first intron of HvCslF6 of CDC Bold and a single SNP in the third exon changed alanine 590 codon in the CDC Bold sequence to a threonine codon in TR251 allele. Five polymorphic sites were converted into genetic markers and confirmed to select low and high (1,3;1,4)-β-glucan lines in a previously characterized CDC Bold / TR251 doubled haploid genetic mapping population and a novel F5 recombinant inbred line (RIL) population derived from a Merit / H93174006 (4.8 and 5.3 % (1,3;1,4)-β-glucan) cross. An analysis of parental lines of six populations segregating for (1,3;1,4)-β-glucan concentration validated the association between the TR251 HvCslF6 haplotype and high (1,3;1,4)-β-glucan concentration in populations showing a (1,3;1,4)-β-glucan quantitative trait locus (QTL) on chromosome 7H.
To further investigate the role of HvCslF6 alleles, 91 lines of the Merit / H93174006 RIL grown in two environments were phenotyped for (1,3;1,4)-β-glucan grain concentration, cellotriose content (DP3), cellotetraose content (DP4) and cellotriose:cellotetraose (DP3:DP4) ratio. DP3, DP4, (1,3;1,4)-β-glucan and total DP3+DP4 were strongly positively correlated (r>0.9) to each other, suggesting no preference for DP3 or DP4 subunit production in high or low (1,3;1,4)-β-glucan lines. DP3:DP4 ratio showed no strong correlation with any other measured trait. Significant effects arising from genotype and environment were associated with grain (1,3;1,4)-β-glucan concentration, DP3, DP4 and DP3:DP4 ratio. Only DP3:DP4 ratio showed a significant GxE (genotype by environment) interaction. Hereditability of grain (1,3;1,4)-β-glucan concentration was moderate (~ 30 %), DP3 and DP4 had low heritability (> 21 %) and DP3:DP4 ratio had moderate heritability (~ 43 %). Single marker analysis showed an association between marker CSLF6_4105 and (1,3;1,4)-β-glucan fine structure in Vegreville but not in Castor, supporting significant GxE interaction in (1,3;1,4)-β-glucan fine structure. Association mapping of candidate markers in 119 barley genotypes of diverse origin grown in greenhouses showed that on chromosome 7H, marker CSLF6_4105 was associated only with (1,3;1,4)-β-glucan concentration, while Bmac273e was associated with both (1,3;1,4)-β-glucan concentration and DP3:DP4 ratio. In addition on chromosome 1H, markers Bmac504 and Bmac211 were associated only with DP3:DP4 ratio. This study suggests that DP3:DP4 ratio is strongly affected by genotype and environment.
To identify new markers with (1,3;1,4)-β-glucan concentration, ninety-four two-row spring varieties were genotyped using double digestion Restriction-site Associated DNA (ddRAD) sequencing on an Illumina sequencer. Two bioinformatics pipelines were used to discover and call SNPs for association linkage analysis. SAMtools bioinformatics pipeline identified 9,062 markers and UNEAK identified 3,060 markers, 2,311 of which were identical between both bioinformatics pipelines. Both sets of markers showed excellent coverage of the genome and distinguished the ninety-four varieties into the same subgroups based on geographical region of origin. Association mapping was performed using TASSEL 3.0 and grain (1,3;1,4)-β-glucan concentration was associated with a region on the 5HS telomere by markers generated using both UNEAK and SAMtools. Some putative candidate genes were identified, including a UDP-glucosyltransferase, two phosphorylation signaling proteins and two transcription factors. The markers developed and tested in this study can be used in marker assisted selection to develop barley genotypes with desired (1,3;1,4)-β-glucan concentration.
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Keywords
1,3, 1,4)-β-glucan, CslF6, Barley, Association mapping
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Plant Sciences
Program
Plant Science