Molecular Characterization Of Lipoxygenase And Puroindoline B-2Variants In Wheat And Its Relatives

1. The yellow colour of pasta products is one of the main criteria used byconsumers to assess pasta quality. This character is due to the presence of carotenoidpigments in semolina. During pasta processing,oxidative degradation of carotenoidpigments occurs mainly due to lipoxygenase (LOX). Lipoxygenase (LOX) activity isan important factor determining the color of flour and end-use products of wheat.1.1A total of218durum wheat germplasms in this study were provided by fivecountries and regions as materials. The collected materials were used to identifyLpx-B1genes and to analysis of LOX activity, respectively, by use of PCRamplification with specific primers, site-specific cleavage with the restriction enzyme,DNA clone and sequencing, Bio-Rad smartspec plus and ELIASA.1.2This result suggested Lpx-B1genes family can divide into three locusincluding Lpx-B1.1、Lpx-B1.2and Lpx-B1.3in durum wheat germplasm, Lpx-B1.1a、Lpx-B1.1b and Lpx-B1.1c alleles belong to Lpx-B1.1locus, while Lpx-B1.2andLpx-B1.3is of a pair of alleles. In218durum wheat,118varieties belong toLpx-B1.1a genotype, account for54.1%;21varieties belong to Lpx-B1.1b genotype,account for9.6%;79varieties belong to Lpx-B1.1c genotype, account for36.2%,indicated that Lpx-B1.1a and Lpx-B1.1c alleles played an important role in Lpx-B1.1locus. On the other hand,193varieties belong to Lpx-B1.2genotype, account for88.5%;25varieties belong to Lpx-B1.3genotype, account for11.5%amongLpx-B1.2and Lpx-B1.3locus, demonstrated that Lpx-B1.2was major genotype indurum wheat germplasm.1.3This test by means of detected LOX activity of durum wheat gerplasm,indicated that LOX activity of different genotype was different. The result of Varianceanalysis demonstrated LOX activity of Lpx-B1.1b allele was highest, the second wasLpx-B1.1a allele, that of Lpx-B1.1c allele was lowest, and divergence was significant(P<0.01). Meanwhile, the deletion of Lpx-B1.1c maybe associate with a strong reduction in LOX activity in semolina; and variance analysis suggested LOX activityof Lpx-B1.3allele was higher than that of Lpx-B1.2allele (P<0.01).1.4Based on Lpx-B1genes distribution in durum wheat, four differenthaplotypes were distinguished: haplotype I, carrying Lpx-B1.3and the Lpx-B1.1ballele; haplotype II carrying Lpx-B1.2and the Lpx-B1.1a allele; and haplotype IIIcarrying Lpx-B1.2and the Lpx-B1.1c allele; haplotypeⅣ, carrying Lpx-B1.3and theLpx-B1.1a allele. Determination of Lpx-B1total LOX activity in mature grainsrevealed differences among these four haplotypes: haplotypes I, Ⅳ,II and III showedfrom high to low levels enzymatic activity.1.5In the present study, we characterized the relationship between thedistribution of Lpx-B1gene family and activity of LOX in durum wheat germplasm,developed novel molecular marker:Lpx-B1-23, provided useful information forestablishing the accurate breeding objective to meet all kinds of consumer group.2. Cloning and phylogenetic analysis of puroindoline b-2variants in commonwheat (Triticum aestivum L.) and its relatives would advance the understanding of thegenetic diversity and evolution of puroindoline b-2gene in common wheat and itsrelated species.2.1In the present study, common wheat (AABBDD) and four related species,including T. urartu (Au Au), Aegilops speltoides (SS), Ae. Tauschii (DD), and T.turgidum (AABB) were sampled for the presence of novel alleles at Pinb2v-A1,Pinb2v-B1/Pinb2v-S1and Pinb2v-D1loci corresponding to common wheatpuroindoline b-2variants.2.2Nine new alleles were identified at these loci, designated Pinb2v-A1a throughPinb2v-A1c, Pinb2v-S1a through Pinb2v-S1e, and Pinb2v-D1a. Alignment ofpuroindoline variants or alleles from common wheat and its relatives indicated that allalleles in diploid wheats are attributed to single nucleotide substitution whencompared with puroindoline b-2variants in polyploids.2.3Deduced amino acid sequences showed that all three alleles at Pinb2v-A1locus and four alleles (Pinb2v-S1a, Pinb2v-S1b, Pinb2v-S1c and Pinb2v-S1e)at thePinb2v-S1locus could not be normally translated due to the presence of prematurestop codons, whereas Pinb2v-D1a at the Pinb2v-D1locus and Pinb2v-S1d at thePinb2v-S1locus could be normally translated, possibly suggesting that thepuroindoline b-2variant in Ae. tauschii was more highly conserved than those in T.urartu and Ae. speltoides. Meanwhile, puroindoline b-2variant could be normally translated in all of the durum and common wheat cultivars surveyed.2.4None of the puroindoline b-2alleles previously identified in durum andcommon wheat were found in the diploid genome donors examined here, even thougha greater diversity of alleles were found in diploid wheat compared to polyploid wheat.These results likely reflect the evolutionary history of tetraploid and hexaploid wheats,although it may be that puroindoline b-2variant alleles have been selected for stabilityand functionality in common and durum wheat.2.5This study provides a survey of puroindoline b-2variants in common wheatand its relatives, and provides useful information for understanding the geneticdiversity of puroindoline-like genes and their duplication events in wheat.