Development And Application Of Functional Gene Markers For Wheat Grain Lipoxygenase Gene In Common Wheat

The activity of lipoxygenase(LOX) is closely linked to the storage stability of wheat, and reducing the LOX content of wheat seed is a effected way to delay the time of storage. Breeding wheat cultivars with low LOX activity is the best way to improve their quality in appearance. Molecular marker-assisted selection (MAS) is more effective and faster than conventional selection in breeding program. Studying on the variation of LOX activity and the relationship between genotype and phenotype in203wheat varieties (lines) could take good advantage of germplasm and effectively delay the storage time. The main results obtained in this study are summarized below.1. Based on a wheat grain LOX gene sequence GU167920.1(GenBank Accession Number), some pairs of primers were designed. Two group of wheat cultivars with high and low LOX activity,respectively, were amplified to screen the STS marker. One pair of primer, designated as LOX1-wp01, amplified three fragments (a, b, c) in the cultivars with high LOX activity, while two fragments (a, c) were detected in the cultivars with low LOX activity. In a test of203cultivars using LOX1-wp01, three fragments (a, b, c) were detected in164genotypes, accounting for80.79%of the total cultivars (lines), but not in the remaining39genotypes, occupying19.21%, and the mean value of LOX activity of the former cultivars (10.46) was significantly (p<0.01) higher than that of the latter (7.36). Thus LOX1-wp01is an efficient and reliable molecular marker for LOX activity, and can be applied in wheat breeding programs aimed for low LOX activity.2. To meet the requirements of quality breeding, LOX1-wp01was used to screene10cultivars, Hemai026, Neibai, W1032, Zhengmai9405, Lumai22, Annongnuo1, ZWY-10, Zhoumai16, Wanxie497,Yannong21, with high LOX activity between14.65nkat· g-1and19.43nkat·g-1,and10low cultivars, Zhoumai11, Hanyou9409, Huaimai19, Neixiang188, P206, Shan228, Yangfumai5242, Wan0606, Annong95081-8, Wan0202, with LOX activities between4.56and5.93nkat·g-1. These material resources were of practical significance to the research on wheat LOX activity and the improvement of storage quality.3. The sequence of the band ’a’ showed100%identity with the Lpx-B1.2(HM126467.1) gene. The DNA sequence of the band ’b’ showed polymorphisms between cultivars with low LOX activity and cultivars with high activity and was99%identical to the sequence from the Lpx-B1.1(HM126468.1) gene. The sequence of the band’c’showed96%identical with Ipx-B1.1(HM126468.1) gene. These results indicate that there are three LOX1loci in common wheat. We propose to designate the locus corresponding to GenBank JQ715609as LOX1.1and the locus corresponding to GenBank JQ715610as LOX1.2and the locus corresponding to GenBank JQ715611as LOX1.3, and the deletion of LOX1.1was associated with a reduction in lipoxygenase activity. Further analysis revealed that one intron sequence,which have the typical characteristics of exon intron boundary ’GT-AG’, are present in the amplified fragments.The major reason for the diversity of the bands(a,b,c) is that the intron sequences are different in length.4. LOX sequences of poaceae crop were searched/blasted in NCBI and aligned using MEGA5.0software. The results showed that wheat LOX genes could be divided into4clades(G1, G2, G3, and G4). G1, contained L37359.1, HQ913602.1, and DQ474244.1, encoded lipoxygenase3; G2consisted of7members including NM001112503.1, X64396.1, L37358, and GQ166691.1, which encoded lipoxygenase2; G3included14members, such as HM126467.1, DQ474241.1, JQ715610, L35931.1,and GQ369443.1,and their encoding product was lipoxygenase1; G4with EU725461.1and AB099850.1encoded lipoxygenase1. Further analysis of the phylogenetic tree revealed LOX1gene from poaceae crops was divided into two groups(G3ang G4); LOX1gene of oryza sativa was individually assigned to a group(G4); the genetic relationship between Triticum and Hordeum was closest, while the genetic relationship between Triticum and Hordeum, Sorghum and Oryza became farther gradually.5. A cDNA sequence of LOX1gene was cloned by adopting RT-PCR from Jimai21, which was submitted to GenBank (Accession No:JX126806). Results of the sequence analysis showed that this cDNA was384bp in length encoding127amino acids with a molecular weight of11.15kD and a theoretical pI of4.93. The multiple comparison sequence analysis indicated that the homologies of nucleotide of this gene with Triticum durum,Hordeum vulgare, Sorghum bicolor and Oryza sativa Indica Group were99.7%,94.8%,73.7%, and56.2%. The phylogenetic tree showed that the genetic relationship between Triticum aestivum and Triticum durum was closest,while the genetic relationship between Triticum aestivum and Hordeum vulgare, Sorghum bicolor and Oryza sativa Indica Group became farther gradually. cDNA sequence of LOX1gene was cloned, and the result of the phylogenetic evolution analysis by LOX1gene of different species is similar to the relative species relationship,and therefore the LOX1gene could be used as an ideal marker to study the species relationships or hereditary distance.