Grain Hardness And Allelic Variation And Hardness Related Molecular Marker Development Of Vin-1/3 Genes In Avena Nuda

Oat is an annual herbaceous plant of the genus Avena,which is a worldwide cultivated crop.Large-grain naked oat（Avena nuda L.）is an allohexaploid（2n=6x=42）,which originated in China and is an important food and feed crop.Oat grain hardness is closely related to its damage during harvesting,washing,threshing and hulling,and has an important impact on the processing quality and eating quality of oat.In order to explore the grain hardness formation mechanism of naked oat,260 core germplasm resources of naked oats were collected in this study.The aspects about methods of grain hardness detection,the classification and distribution of grain hardness,the differences in environment,grain structure,grain composition,and sequence and expression differences of Vromindoline-1 and Vromindoline-3 genes among different hardness materials were studied.The main results of this study were as follows:（1）Detection of grain hardness of naked oat:The grain hardness of naked oats was detected by single grain characteristic analyzer and texture analyzer,and it was determined that the texture analyzer was more suitable for testing the hardness of naked oats.It was determined that the best value of the hardness value of the texture analyzer was the position of 0.3 mm displacement.260 naked oats have been planted and tested for 3 years and 2 points,and the kernel hardness was from 17.72 N to 37.26 N.The hardness value of soft naked oats was less than 24.4 N,the medium types were from 24.4to 29.3 N,and hard types were more than 29.3 N.Planting environment,year and variety had significant effects on grain hardness.The proportion of materials with different hardness was that the intermediate type was larger than the soft type,and the hard type had the lowest proportion.The proportion of medium types in the materials tested in the North China and Northwest production areas was the highest,while the materials from the Southwest production area in the southwest production area were mainly soft types.（2）Kernel hardness and structure characteristics of naked oat:The aleurone cells and endosperm cells of soft oat grains were more rigid and tidy,with loose endosperm structure and large gaps,while the secondary aleurone layer of hard oats was thicker than that of soft ones,with compact endosperm structure and small gaps.Oat grains were mainly composed of small（particle size<6μm）and medium（6μm<particle size<40μm）starch granules.There were many attachments on the surface of starch granules.The number,surface area and volume of starch granules showed an unimodal distribution.The number and volume percentages of small starch granules in soft oat were higher than those in hard,and the number and volume percentage of medium and large starch granules was lower than those in hard.The percentages of surface area of small and medium starch granules in soft oat were higher than that of hard ones,while that of large starch granules was lower than that of hard ones.（3）Kernel hardness and main components of naked oat:Amylopectin content was significantly positively correlated with hardness.A quantitative lipidomic study of soft and hard oat mature grains identified a total of 797 lipids.The total lipid content identified in the hard and soft groups was 54765.38 nmol/g and 63492.09 nmol/g,respectively.Differential lipids were screened according to VIP and FC value,99differential lipids were shared by soft and hard group,72 were down-regulated and 27were up-regulated.Correlation analysis of 21 core differential lipids revealed significant negative correlations between 19 triglycerides and 2 glycerophospholipids.The above results indicated that glycerolipid content was decreased and glycerophospholipid content was increased in hard oat,while soft oats were observed oppositely,and 21 core differential lipids could be used as biomarkers related to kernel hardness of naked oat.An extensive targeted metabolomic study of soft and hard naked oat identified a total of754 metabolites.142 differential metabolites were screened according to VIP and FC value,and 67 were up-regulated and 75 were down-regulated in them.Flavonoids were the main metabolites responding to differences in hardness.There were 11 core differential metabolites,which|log₂FC|were large than 8,and the correlation coefficient of them was strongly correlated.All 11 core differential metabolites could be used as biomarkers related to kernel hardness of naked oat.Membrane proteins were extracted from the whole flour and the surface of starch granules of soft and hard naked oat mature grains.The results showed that the protein content at 14 k D was not significantly different in the whole flour,but the content on the surface of starch granules was higher in soft than hard naked oats.A total of 41 proteins were identified from the 14 k D peptide mixture by HPLC/MS,including Vromindoline protein and oatα-amylase trypsin inhibitor,etc.The above results showed that the grains with low amylopectin content,high glycerolipid content,low glycerophospholipid content,and high protein content such as Vromindoline were soft.（4）Kernel hardness and Vromindoline:A total of 18 AnVin-1 and 9 AnVin-3 gene sequences were identified in this study,all of which contained only one complete ORF,with lengths of 444 bp and 429 bp,respectively.Based on the sequence differences between the A,C and D genomes of oat,a genomic marker of the AnVin-1 gene was developed.AnVin-1A,AnVin-1C,AnVin-1D and AnVin-3C were expressed to different degrees in roots,stems,leaves,and other organization.The expression levels of them in different developmental grains increased first and then decreased,and the highest expression was found in grains with 14 DAP.In the 14 DAP grains of oat with different hardness,the expression levels of AnVin-1C and AnVin-3C were higher in soft naked oat than in hard.Based on the SNPs between 27 copies of sequences,gene markers of 15copies of sequences were developed,and allelic variation identification and analysis among different hardness materials were carried out.The results showed that AnVin-1C6and AnVin-3C2 genes have two genotypes in different hardness oats,and a CAPS marker had been developed according to the two genotypes of AnVin-1C6,which could explain1.44%of the hardness variation rate.The subcellular localization of AnVin-1C6 and AnVin-3C2 genes was found to be localized in the nucleus and cytoplasm,respectively.The above results indicate that AnVin-1 and AnVin-3 genes were involved in grain development and may affect grain hardness,and the degree of variation in natural populations was low.In summary,a hardness testing method was established,and revealed its relationship of grain hardness with grain hardness from the aspects of growth environment,grain structure,grain composition and Vromindoline gene,and predicted its action mode.This study provides a theoretical basis for a comprehensive analysis of the formation mechanism of naked oat grain hardness,and lays a foundation for the breeding of special varieties for oat processing.