Molecular Identification Of Hardness-related Genes And Analysis Of Molecular Mechanism Of PINA Null In Bread Wheat

Kernel hardness is one of the important traits of wheat quality and possesses significant influence on the amount of water addition for tempering, milling yield, the amount of damaged starch and flour granule size. It serves as evaluation for wheat classification and marketing cultivars because of its big impact on milling quality and end-use processing quality,.A total of 278 Wheat germplasms in this study are provided by Wheat Genetics and Breeding Laboratory of Henan Agricultural University of Huang-Huai region including landraces cultivars, the current cultivars and new developed lines for wheat regional test. The collected materials were used to identify traits relating to yield, phenotype of kernel hardness, genotype of puroindoline alleles and puroindoline b-2 variants, respectively, by use of Single Kernel Characterization System (SKCS), PCR amplification with specific primers, site-specific cleavage with the restriction enzyme Pf1MI, DNA sequencing and real-time fluorescence quantitative polymerase chain reaction (PCR). In addition, 12 bread wheat varieties from China and CIMMYT were used to exam the molecular mechanism of PINA protein null with the method of primer walking and corresponding Pina-N marker were developed for rapid identifying Pina-D1b alleles. The main results obtained in this study are summarized below.1. Hard wheat was dominant in Huang-Huai Winter Wheat Area of China and its hardness variations with wider range. Hard wheat with percentage of 57.4% was predominant while percentages of the mixed and soft wheats were only 8.9% and 33.7%, respectively. Based on SKCS results, grain hardness index of wheat surveyed ranged widely from -2 to 78.6. Four puroindoline alleles of Pinb-D1b, Pinb-D1p, Pina-D1b and Pina-D1l were detected in hard wheat surveyed. Of them, Pinb-D1b is the most prevalent with percentage of 79.4%, Secondly Pinb-D1p were 12.4%, while Pina-D1b and Pina-D1l were only 4.1% and 1.0%, respectively. A total of 109 wheat advanced lines from recently for Regional Trial Observation of Huang-huai wheat region surveyed hard wheat with higher percentage of 61.5% was predominant while percentages of the mixed and soft wheats were only 15.6% and 22.9%, respectively. Three puroindoline alleles of Pinb-D1b, Pinb-D1p and Pina-D1b were detected in hard wheat surveyed. Of them, Pinb-D1b is the most prevalent with percentage of 86.5%, while Pinb-D1p and Pina-D1b were only 7.5% and 6.0%, respectively. Therefore, the Huang-Huai wheat region should further expand the using scope of wheat germplasms with relatively superior genotypes, such as cultivars with Pinb-D1d, Pinb-D1e or Pinb-D1g, which can speed breeding process of the bread and noodle wheat varieties.2. Molecular mechanism of Pina-D1b type, the predominant genotype resulting from PINA protein null, were illustrated by primer walking strategy. Restults indicated that Pina-D1b alleles have a 15, 380-bp fragment deletion from the beginning of 23rd bases (relative to the Pina gene ATG) in Ha locus compared with wild-type, and the primers were designed from spanning deletion fragment and successfully molecular marker Pina-N were developed for directly detecting the Pina-D1b allele at the DNA level。The functional molecular mark Pina-N can substitute SDS-PAGE technology widely used for detection of Pina-D1b for many years, which will greatly improve the detecting efficiency and reduce testing costs.3. Landraces varieties Jiangdongmen from Jiangsu, Heshangtou and Hongquanmang from Henan have been discovered to have a new type of the PINA null, designated Pina-D1r. Its molecular mechanism was examed by the above-mentioned primer walking strategy. Results indicated that compared with the wild type, Pina-D1r type Ha locus has a 10, 415 bp of the large deletions, the deletions from -5117 bp to +5298 bp (Pina gene relative to ATG). Functional molecular marker Pina-N2 were developed based on its molecular characteristics to design the primers spanning the 10, 415-bp deletion on purpose of identifying the Pina-D1r at the DNA level.4. Identifying puroindo1ine b-2 variants in all the 246 materials surveyed (not including mixed wheat) indicated that they all contained Pinb-D2v1 and Pinb-A2v4 two variants. Of them, however, 58 varieties possessed Pinb-B2v2 variant with the percentage of 23.6% and the remaining 188 species had Pinb-B2v3 variant with the percentage of 76.4%. Therefore, the combination of Pinb-A2v4/Pinb-B2v3/Pinb-D2v1 at Puroindoline b-2 locus is the most common types in Huang-huai wheat region. In the soft wheat cultivars (Pina-D1a/Pinb-D1a), SKCS average hardness index (27.2) of Pinb-B2v3 cultivars was significantly higher than that of Pinb-B2v2 cultivar (22.2). However, in hard wheat cultivar with Pinb-D1b or Pinb-D1p gene types of sub-group, respectively, the SKCS hardness of Pinb-B2v2 and Pinb-B2v3 group has no any significant difference. Therefore, Puroindoline b-B2 loci possibly performed more big impact on soft wheat than hard wheat. Interestingly, further analyzing yield-related traits between Pinb-B2v2 and Pinb-B2v3 variants indicated that the averages of grain number, single grain weight, flag leaf length and flag leaf area of genotypes with Pinb-B2v3 variant were significantly higher than those of genotypes with Pinb-B2v2 variant. It suggested that the agronomic traits of the cultivars with Pinb-B2v3 were more preferable than those of the cultivars with Pinb-B2v2 variants.5. Real-time PCR results showed that the relative mRNA expressing amount of Pinb-2v3 variant was significantly higher than that of Pinb-2v2 variant. Among three different types of Pinb-2v3 alleles, Pinb-2v3b presented the highest relative expressing amount. Based on above-mentioned results that Pinb-2v3 wheat varieties possessed obviously superior yield-related traits than those of Pinb-2v2 variant, it possibly suggested that Puroindo1ine b-B2 gene expression amount may be positively related to the superiority of wheat yield. Amongest the relative expression amount of Pinb-B2v gene in the different tissues, the expression amount of Puroindo1ine b-B2 variant in leaves was significantly higher than that in roots.