| Lignin content of stem in common wheat(Triticum aestivum L.) exerts an important effect on lodging resistance, and the caffeic acid O-methyltransferase(COMT) is a key enzyme in the pathway of lignin biosynthesis. Characterization of COMT genes and development of functional markers are of great importance for marker-assisted selection in wheat breeding targeting for lignin content improvement. In the present study, stem lignin contents of 166 cultivars and advanced lines grown in four environments were assayed; TaCOMT genes were cloned using the homologous cloning approach in combination with PCR amplification, and functional markers were developed and validated. In addition, a genome-wide association study(GWAS) for stem lignin content was conducted using the wheat 90 K iSelect SNP chip. The main results are summarized as follows,1. Stem lignin content was significantly influenced by genotypes, environments and their interaction. The heritability of stem lignin content across four environments was 0.90, indicating that stem lignin content is mainly affected by genetic factors and selection for it at the early generation can be effectively applied in wheat breeding program.2. The full-length genomic DNA(gDNA) sequences of wheat COMT genes located on chromosomes 3A, 3B and 3D were cloned, and designated as TaCOMT-3A, TaCOMT-3B and TaCOMT-3D, respectively. Each of the three genes comprised two exons and one intron, and a 1,071-bp coding sequence(CDS) which encoding a COMT precursor peptide of 356 amino acids. Sequence similarity for the coding regions and the deduced peptide sequences were 97.9% and 99.1%, respectively, while the similarity of introns was 81.7% among three homoeologous genes, indicating that the exons of TaCOMT genes have been under higher selective pressure than the introns. Homology analysis showed that there was a closer relationship between TaCOMT genes and COMT genes from monocotyledoneaes compared with those from dicotyledoneaes, indicating there has been a divergence of COMT genes from monocotyledoneaes and dicotyledoneaes in the evolution process.3. Two allelic variants, TaCOMT-3Aa and TaCOMT-3Ab, were detected at TaCOMT-3A locus. A single base deletion was observed at the second exon of TaCOMT-3Ab, leading to a premature stop for the translation. Based on this characterization, a cleaved amplified polymorphic sequence(CAPS) marker, designated as COMT3A-CAPS, was developed to discriminate the two alleles. Unfortunately, there was no signinficant difference of stem lignin contents between the two genotypes.4. For TaCOMT-3B locus, two allelic variants TaCOMT-3Ba and TaCOMT-3Bb were also detected. A 222-bp insertion/deletion(InDel) was found in the 3’-untranslated region(3’-UTR). Based on this InDel, a co-dominant functional marker, COMT-S3 B, was developed, yielding 638- and 416-bp fragements from genotypes TaCOMT-3Ba and TaCOMT-3Bb, respectively. The association between the polymorphic band patterns and stem lignin contents was validated using the 166 cultivars, and the results showed the functional marker COMT-S3 B was significantly associated with stem lignin content, and it can be used in wheat breeding targeting for the improvement of stem lignin content.5. A total of 18,207 high quality SNP markers were derived from the 90 K iSelect array and were used for GWAS of stem lignin content. Fifteen loci significantly related with stem lignin content were detected, explaining 6.78–10.35% of the phenotypic variation. |
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