Study On The Relationship Between Puroindoline Genes And Wheat Grain Hardness

Kernel hardness is a key trait that greatly affects the milling and baking qualities of common wheat.Kernel hardness is largely controlled by Puroindoline genes(Pina and Pinb).If there is any mutations in either Pina or Pinb gene,the wheat is hard kernel.Only both wild-type genes impart a soft kernel phenotype.Grain hardness is determined not only by Pin,but also by environment,kernel weight and some slightly effective quantitative trait loci(QTL)of hardness.According to some previous researches,the Puroindoline b-like(Pinb-2v)genes may be one of the QTLs.How is the relationship between Pin genes and hardness phenotypes? and whether Pinb-like,as genes very similar to the sequence of Pinb,has some relationship with hardness phenotypes? These questions remain to be further explored.In this study,the phenotype of grain hardness was determined by Single Kernel Characterization System(SKCS)and the genotype of Pin-D1 was identified by PCR to obtain the composition for the 107 wheat accessions.Western blotting was used to further determine the effect of Pin mutation or deletion on PIN protein expression.Based on the known Pin genotypes and hardness phenotypes,the primers of Pinb-like genes were designed for the different chromosomes.Accordance to the latest genome database of triticeae crops,the genotypes composition of Pinb-like was analyzed by PCR,nested PCR and derived c1 eaved amplified polymorphic sequences(dCAPS),and then the relationship between Pinb-like genotypes and hardness phenotypes can be speculated.In addition,in order to confirm the relationship between Pina gene and milling quality,the RNA interference vector of Pina and its linear gene cassette were transformed into wheat variety Emai 12 by particle bombardment.The progeny of transgenic plants were analyzed after screening.The main results are as follows:(1)The results of Pin genotypes and hardness phenotypes of Chinese wheat accessions showed that the most frequent genotypes in these accessions were Pina-D1a/Pinb-D1a(39.3%)and Pina-D1a/Pinb-D1b(34.6%)and Chinese landraces have higher allelic diversity in the Pina gene compared to elite cultivars.We identified a novel Pina-null allele(designated Pina-D1z)and its specific molecular marker was designed.We also identified a Pina/Pinb double null allele.(2)A set of gene identification process and primers were designed for Pinb-like genes,and then the genotype identification was carried out.Seven new Pinb-like genes were identified.They are highly expressed in wheat endosperm.The syntenic relationships within the A,B and D genomes of the Pinb-like homologous fragments in hexaploid wheat,are not as good as that between the genome and its donor genome.The differentiation time of B genome is earlier than that of A and D genomes,and the evolution speed is also faster.(3)The analysis of hardness index and PINA protein expression in Pina-RNAi transgenic plants showed that the hardness index of some positive transgenic lines increased,and the expression of PINB protein probably increased by the PINA protein decreased.Based on the genome database of triticeae crops,primers designed to identified the composition of Pinb-like and Pin genes,hardness measured by SKCS,supplemented by the synteny of gene loci,and all these proved that Pinb-like genes are only similar to Pinb in sequence,but not related to hardness phenotypes.The systematic analysis of Pin genotypes and hardness phenotypes demonstrated that 21 Pin alleles may be correlated with significant changes in hardness.The investigation on the relationship between Pins,Pinb-like genes and grain hardness could help us to further understanding the kernel hardness characteristics.These will be useful to wheat quality improvement.