Studies On Genetic Regulation Of β-Glucan Content In Barley Grains

Barley ranks the fourth in terms of planting area among the cereal crops in the world.It is mainly used for feed and beer production,and in small quantities used for human food.Improvement of barley quality for meeting the different end uses is the most efficient pathway to promote barley production in China.β-glucan is an important trait that affects the use of barley and the quality of various barley products.Barley varieties with low β-glucan content are suitable for livestock feed and beer production.While barley varieties with high β-glucan content are suitable for edible and functional food production.It is quite imperative to reveal the genetic mechanism of β-glucan accumulation and explore the related genes in barley for developing the elite barley varieties.In this study,GWAS(genome-wide association study),transcriptome,CRISPR/Cas9 gene editing were used to identify the genes related to β-glucan content in barley grains,and reveal their genetic regulation mechanism.In addition,the practicability of barley CslF6 gene used in breeding to increase β-glucan content in rice grains was also investigated.The main research results are as follows:1.Identifying seven candidate genes related to β-glucan content by GWAS100 barley genotypes of a international core collection were used as materials and cultivated in three different environments(Changxing-2018,Changxing-2019 and Cixi-2019),to determine grain β-glucan content.It was found that there were significant differences in grain β-glucan content among 100 genotypes,with range of 1.40%6.07%,1.16%-6.53%and 1.35%-6.51%,and average of 3.75%,3.45%and 3.75%for the three environments,respectively.Barley genotypes with high/low β-glucan content and little change in three environments were screened from the population.GWAS was performed on β-glucan content of the 100 barley genotypes using high-quality SNP markers.Totally,14 significant marker-trait associations for grain β-glucan content were identified,and 7 putative candidate genes were found to be associated with βglucan content.This study provides gene resources for further research on genetic regulation and the functions of β-glucan content related genes.2.Revealing the difference in transcriptome of developing grains among barley genotypes differing in grain β-glucan contentIn this study,the transcriptome profiles of developing grains of two barley genotypes(BCS192 and BCS297)differing in grain β-glucan content were compared.Totally,1,160 co-modulated DEGs were identified.Functional enrichment analysis showed that the DEGs involved in carbohydrate metabolic process and starch and sucrose metabolism played the significant roles in β-glucan synthesis.These DEGs were involved in hydrolase activity and glucan metabolic process.Moreover,there were 1,131 DEGs expressed commonly in the two genotypes during different grain developmental stages.A functional analysis of the up-regulated DEGs showed that βglucan accumulation in barley grains was positively regulated by photosynthesis and carbon metabolism.The identified DEGs and their functions reported in this study may provide theoretical basis for analyzing the molecular mechanisms of β-glucan synthesis in barley grains and explaining the genotypic differences of β-glucan content.3.HvBGlu3 and HvGH43 negatively regulate β-glucan content in barley grainsTo further analyze the effects and regulatory mechanism of candidate genes identified by the previous GWAS on barley β-glucan content,CRISPR/Cas9 gene editing technology was used to knock out the two GH family genes HvBGlu3 and HvGH43 to reveal their biological functions.The results showed the knock-out mutant lines had significantly higher β-glucan content than wild type in mature grains,accompanied by the distinct difference in other grain composition(including total dietary fiber,and total starch)between the mutant lines and WT.Therefore,it can be concluded that HvBGlu3 and HvGH43 negatively regulate the β-glucan content in barley grains.Transcriptomic analysis showed that knockout of HvBGlu3 and HvGH43 promoted the starch and sucrose metabolism pathway in barley developing grains,and significantly affected the sugar metabolism,and finally increased the β-glucan content in mature grains.4.Heterologous overexpression of barley CslF6 gene increased β-glucan content in rice grainsBarley CslF6 gene was overexpressed in japonica rice Zhonghua 11 and indica rice Huazhan under the control of 35S promoter and rice endosperm-specific promoter GluB-1 using transgenic technology.Barley CslF3 gene was overexpressed under the control of rice endosperm-specific promoter GluB-1.The results showed that overexpression of CslF6 under the control of 35S promoter and overexpression of CslF3 under the control of GluB-1 promoter did not affect β-glucan content in rice grains.Overexpression of CslF6 gene under the control of GluB-1 promoter could significantly increase grain β-glucan content of Zhonghua 11 and Huazhan,but accompanied by a reduction of grain weight.The results showed the feasibility of improving β-glucan content in rice grains by genetic manipulation of CslF6 gene,and important issues to be solved in the coming research.