Analysis Of Quality Parameters Associated With Grain Hardness Genes And Molecular Mechanism Of PINA Null In Bread Wheat

Grain texture is one of the most important characteristics as it largely determinesmilling and processing qualities, and possesses significant influence on the amount ofwater addition for tempering, milling yield, damaged starch and flour granule size.In this study, a total of536bread wheat cultivars and advanced lines, including43Chinese winter wheat cultivars and advanced lines,204Chinese landrace wheatcultivars,104CIMMYT cultivars,88Australian cultivars,53Chilean cultivars and44cultivars from Netherlands are exchanged or introduced from different countries orregions to Seed Bank of Henan Agricultural University, possessed certain or multiplesuperior agronomic traits and are being popularly used as parents in wheat breedingprograms in the Yellow and Huai Valleys of China. The collected materials were usedto identify phenotype of kernel hardness, thousand-grain weight, flour proteincontent, Alveograph parameters, Mixolab parameters,grain diameter and genotype ofpuroindoline alleles respectively, by use of Single Kernel Characterization System(SKCS), PCR amplification with specific primers, site-specific cleavage with therestriction enzyme Pf1MI and DNA sequencing. Four landrace cultivars Yumai,Changyaomai, Yangqingke and Bailaolaibian were used to examine the molecularmechanism of PINA-null protein using primer walking and corresponding Pina-Nmarker were developed for rapid identification of PINA-null alleles. In addition, thebread wheat cultivar Yunong202that developed in the wheat breeding program atHenan Agricultural University was used to identify the Puroindoline b-2variants. Themain results obtained in this study are summarized below.1. Of the surveyed493bread wheat cultivars and advanced lines (including204Chinese landrace wheat cultivars,104CIMMYT cultivars,88Australian cultivars,53 Chilean cultivars and44Netherlands cultivars),139and354were classified as softand hard genotypes, respectively. This suggests that hard wheat is predominant insurveyed Chinese landraces and cultivars of Mexico, Australia, Chile and Netherlands.Pina-D1b、Pina-D1l、Pina-D1n、Pina-D1r、Pina-D1s、Pina-D1u、Pinb-D1、Pinb-D1c、Pinb-D1d、Pinb-D1p、Pinb-D1q、Pinb-D1t、Pina-D1r/Pinb-D1p、Pina-null/Pina-nullalleles have been found amongst hard cultivars from different countries. Among fivedifferent countries, Chinese landraces showed the highest diversity onPuroindoline-D1genes and possessed11types of Puroindoline-D1alleles in hardwheat landraces. CIMMYT hard wheat, only composed of two kinds ofPuroindoline-D1alleles, Pina-D1b was predominant with the high percentage of94.6%. In wheat cultivars from Australia and Netherlands, Pinb-D1b was predominantwith the high percentage of73.6%and56.7%, respectively. In Chile, Pina-D1b andPinb-D1b are almost equally distributed in hard wheat cultivars. Based on fivekernels’ results, one Chinese landrace cultivar, Bailaolaibian, possesses a doublemutation genotype Pina-D1r/Pinb-D1p and its SKCS hardness index is73.2. This canprovide useful information for the improvement of wheat quality.2. Chinese landraces varieties have been discovered to have a new type of thePINA null, designated Pina-Ds. Results indicated that Pina-D1s alleles have a4422-bp fragment deletion from+371to+4792bp (relative to the Pina gene ATG) in Halocus compared with wild-type by a primer walking strategy. The primers weredesigned from spanning deletion fragment and successfully molecular markerPina-N3was developed for directly detection of the Pina-D1s allele at the DNA level.The functional molecular mark Pina-N3will contribute to significant improvement ofthe detecting efficiency and reducing test cost. The new marker Pina-N3was used toidentify the Pina-D1s allele and the results revealed that28Chinese landrace cultivarspossess the Pina-D1s allele, suggesting that the Pina-D1s is prevalent in hard wheatof the Chinese landrace cultivars surveyed.3. Chinese landraces variety Zuantoubaike has been discovered to have a newtype of the PINA null, designated Pina-D1u. Its molecular mechanism was examinedby the above-mentioned primer walking strategy. Results indicated that comparedwith the wild type, Pina-D1u type Ha locus has a large6460-bp deletions, from-4435bp to+2024bp (reference to the ATG of Pina gene). Functional molecular markerPina-N4was developed based on its molecular characteristics to design the primers spanning the6460bp deletion on purpose of identifying the Pina-D1u at the DNAlevel.4. Further analysis of molecular characterization of Puroindoline-D1alleles insome cultivars indicated that three landrace cultivars Yumai, Changyaomai andYangqingke with an absence of Pina and Pinb genes. Results from the primer walkingstrategy showed a valid marker spanning the big deletion for delimiting the location ofthis new allele was not obtained due to this mutation with a single≈33-kb deletioncontaining Pina and Pinb coding regions. This mutation was temporarily designatedas Pina-null/Pinb-null because of the large deletion simultaneously related to Pinaand Pinb genes.5. In this study, we used dCAPS (derived Cleaved Amplified PolymorphicSequences) technique to design two sets of primers BalI-Pina-D1l, BsrDI-Pina-D1nfor amplifying fragments containing SNPs in cultivars with Pina-D1l or Pina-D1nallele with software dCAPS Finder2.0, then restriction enzymes BalI and BsrDI wereused to directly digest the PCR products amplified with primer sets of BalI_Pina-D1land BsrDI_Pina-D1n, respectively, for the detection of Pina-D1l and Pina-D1n alleles.The cultivars with176-bp and124-bp digested fragments belong to Pina-D1n andPina-D1l alleles, respectively.6. Hard wheat was dominant in43Chinese winter wheat cultivars and advancedlines and its hardness index variations have wide range. Hard wheat with percentageof57.4%was predominant while percentages of the mixed and soft wheat were only8.9%and33.7%, respectively. Based on SKCS results, grain hardness index of wheatsurveyed ranged widely from-2to78.6. This investigation could suggest that weshould expand the scope of the use of the genetic resources in Henan. Analysis ofassociation of SKCS hardness with Alveograph and Mixolab parameters, flour proteincontent, thousand-grain weight and grain diameter indicated that SKCS hardnessshowed significant positive correlations with flour protein content, Alveograph P andP/L and Mixolab C2and water absorption and showed significant negativecorrelations with Mixolab C3, C4, C5, C3C2and C5C4. Flour protein contentshowed significant positive correlations with Alveograph P, L, G, W and Ie andMixolab C1, C2and water absorption and showed significant negative correlationswith Mixolab C4, C5, C3C2and C5C4. Between Alveograph and Mixolabparameters, Alveograph P was significantly positively correlated with Mixolab stability, water absorption, C2and C2C1, while Alveograph W was significantlypositively correlated with Mixolab stability, C2and C2C1.In addition, graindiameter was significantly correlated with thousand-grain weight but not with SKCShardness.7. Identificatin of association of Puroindoline allelic variations with SKCShardness, flour protein content, Alveograph and Mixolab parameters under the tripleenvironments the allelic variations at the Puroindoline-D1locus contributedsignificantly to variation in SKCS hardness, flour protein content and Alveograph P,L, G and W, Mixolab C2, C3, C4, C5, water absorption and stability. Among thedifferent Mixolab parameters in the three Puroindoline-D1genotypes, t hePina-D1a/Pinb-D1a genotype possessed the highest C3, C4and C5values, but thelowest Mixolab C2, water absorption and stability at all three locations.PINA-null/Pinb-D1a genotype possessed the highest SKCS hardness, flour proteincontent, Alveograph P, W and P/L values, water absorption and stability, but thelowest C3, C4and C5values at all three locations.8. In addition, a new Puroindoline-2variant was discovered in Chinese currentpopular wheat cultivar Yunong202and it was designated as Pinb-2v6. Full alignmentshowed that Pinb-2v6possessed74.0%,95.4%,94.7%,92.3%,98.7%and98.0%identity at the DNA level with Pinb-D1a, Pinb-2v1, Pinb-2v2, Pinb-2v3a, Pinb-2v4andPinb-2v5alleles, respectively. Full alignment of the deduced amino acid sequences ofPinb-D1a and six Puroindoline b-2variants indicated that Pinb-2v6possesses96.0%,92.0%,88.0%,97.3%and95.3%identify at the amino acid level with Pinb-D1a,Pinb-2v1, Pinb-2v2, Pinb-2v3a, Pinb-2v4and Pinb-2v5, respectively.