Mapping QTLs For Lipoxygenase Activity And Development Of Functional Markers For LOX Gene In Common Wheat

Flour color is an important trait in the assessment of flour quality and exerts significant influence on qualities of noodles and other related products. Lipoxygenase (LOX) activity in grain influences the processing quality of wheat-based products. Characterization of LOX genes and development of functional markers are of importance for marker-assisted selection in wheat breeding. In the present study,71 doubled haploid (DH) lines derived from Zhongyou 9507/CA9632 and 198 Chinese winter wheat cultivars and advanced lines were used and their grain LOX activity was evaluated. LOX gene associated with grain LOX activity, was cloned by the method of in silico cloning in combination with PCR amplification, and functional markers were developed for the genes according to their allelic variants in different wheat cultivars, and their associations with phenotypes were then analyzed. The main results obtained in this study are summarized below.1. LOX activity varies among wheat genotypes and it is also significantly affected by environments. However, the LOX activities of the DH lines across four environments have a high heritability (hl= 0.94), indicating that it was mainly affected by genetypes. Significant differences in grain LOX activity were observed in the Chinese winter wheat cultivars. Grain LOX activity among Chinese winter wheats ranged from 48.4 to 89.6 A234.min-1.g-1. Therefore, selection for LOX activity can be effectively applied at the earlier generation of breeding program. It is possible to breed new wheat cultivars with high LOX activity by choosing parents and making selection in early generations.2. Quantitative trait loci (QTLs) for LOX activity in common wheat were mapped using 71 doubled haploid (DH) lines derived from a Zhongyou 9507/CA9632 and SSR markers. Two QTLs QLpx.caas.lAL and QLpx.caas-4B were identified on chromosomes 1AL and 4BS, closely related to LOX activity. The SSR loci Xwmc312 and Xgwm251 were proven to be diagnostic and explained 13.4-25.2% of phenotypic variance the 1AL locus and 14.3-27.0% for the 4B locus across four environments. The SSR markers Xwmc312 and Xgwm251 were validated across 198 Chinese wheat cultivars and advanced lines and showed highly significant (P<0.01) association with LOX activity. We further established a multiplexed PCR with SSR marker combination Xwmc312/Xgwm251 to test these wheat cultivars and advanced lines. The results suggested that the marker combination Xwmc312/Xgwm251 is efficient and reliable for evaluating LOX activity and can be used in marker-assisted selection (MAS) targeting for flour color attributes to noodle and other wheat-based products.3. The full-length genomic DNA sequence of TaLox-B1 and TaLox-B2 on chromosome 4BS and 5B, respectively, were characterized by in silico cloning and experimental validation. The cloned TaLox-B1 comprises seven exons and six introns, with 4,263 bp in total and an open reading frame (ORF) of 2,586 bp, and it encodes a LOX precursor peptide of 861 amino acids with a predicted molecular weight of-96.0 kDa. The genomic DNA sequence of TaLox-B2 comprised 3,325 base pairs, including six exons and five introns, and partial 3’UTR. The gene harbored an ORF of 2,595 bp, encoding a polypeptide of 864 amino acids with a predicted molecular weight of-96.4 kDa. TaLox-B1 and TaLOX1 were mapped on the homologous group 4, and TaLox-B2 and TaLOX2 were mapped on the homologous group 5 in wheat. Cluster analysis of TaLox-B1-TaLox-B2、TaLOXl、TaLOX2 and ZmLOX3 indicated that these LOX genes from the same homologous group showed the highest sequence identities in gDNA, cDNA and AA levels, respectively.4. Functional markers for the LOX gene on chromosomes 4B based on single nucleotide polymorphism were developed and validated in common wheat. Two complementary dominant STS markers, LOX 16 and LOX 18, were developed based on the single nucleotide polymorphism (SNP) and amplifying 489-bp and 791-bp fragments in cultivars with higher and lower LOX activitives, respectively. The two markers were mapped on chromosome 4BS using a doubled haploid (DH) population derived from Zhongyou 9507/CA9632, and a set of Chinese Spring nullisomic-tetrasomic lines and ditelosomic line 4B. QTL analysis indicated that TaLox-B1 co-segregated with two functional markers and was closely linked to SSR marker Xgwm251 on chromosome 4BS with a genetic distance of 5.4 cM, explaining 15.9-26.2% of the phenotypic variance for LOX activity across four environments. Two complementary dominant functional markers LOX 16 and LOX 18, were validated on 198 Chinese wheat cultivars and advanced lines and showed highly significant (P<0.01) association with LOX activity. The results suggested that these two STS markers are closely related to LOX activity and could be used for the improvement of flour color attributes for noodle and other wheat-based products.5. TaLox-B1 b in Northern China Plain Winter Wheat Rgion and the Yellow and the Huai River Valley Winter Wheat Region was dominant. TaLox-B1a was a superior allele for flour color and dough rheology. TaLox-Bla associated with higher LOX activitive had low frequency (34.3%) in these two regions. The frequency of TaLox-Bla in Yellow and Huai River Valley Winter Wheat Region (36.0%) were higher than those in Northern China Plain Winter Wheat Region (25.0%) (P0.01). These results indicated the preferred TaLox-B1a has not yet widely used in Chinese winter wheat regions. Therefore, it has a large potential to select new cultivars with high LOX activity.