| β-Glucosidase(β-Glucosidase,EC3.2.1.21)is an important aromatic synthetase,which can effectively catalyze the conversion of glycoside aroma precursors,thereby producing aromatic substances,such as linalool,geraniol and other volatile organic compounds;These aroma substances not only affect the aroma quality of tea,but also effectively resist the damage caused by microorganisms such as herbivorous insects and bacteria to tea plants,and can also regulate commensal bacteria to improve tea plants to resist the stress of various external environments.Therefore,mining candidate genes associated with β-glucosidase activity is of great theoretical and practical significance for cultivating tea varieties with high β-glucosidase activity,promoting the formation of tea quality,and enhancing the resistance and adaptability of tea plants to environmental changes.In this study,Specific-Locus Amplified Fragment Sequencing(SLAF-seq)technology was used to sequence the simplified genomes of 95 single plants of the collected Camellia sinensis cv.Guiding-niaowangzhong,and high-quality single nucleotide polymorphisms(SNPs)were obtained and analyzed.At the same time,the activities of three endogenous enzymes,β-glucosidase,peroxidase and polyphenol oxidase,were determined,and the obtained SNPs were analyzed by genome-wide association study(GWAS),and candidate genes associated with β-glucosidase activity,peroxidase activity and polyphenol oxidase activity were obtained.RT-q PCR was used to predict the candidate genes associated with β-glucosidase activity in a single strain represented by low,medium and high enzyme activity.The following results are obtained:(1)SLAF-seq technology was used to perform simplified genome sequencing analysis on 95 single plants of Camellia sinensis cv.Guiding-niaowangzhong,and a total of 555.17 Mb Reads were obtained.Through bioinformatics analysis,1,801,337 SLAF tags were obtained,of which 64,530 were polymorphic SLAF tags,and a total of 7,572,089 population SNPs were obtained.The information annotation of the obtained SNPs showed that the most SNP variant types were A/G and C/T,and there were more conversion variants than reversal variants,indicating that base conversion was the main SNP variant type in the single plant of the Camellia sinensis cv.Guidingniaowangzhong.Based on SNP,the genetic evolution analysis of 95 single plants of Camellia sinensis cv.Guiding-niaowangzhong showed that the genetic differences of 95 single plants of Camellia sinensis cv.Guiding-niaowangzhong were relatively small,derived from the same ancestor,and did not form obvious population differentiation,and the results of genetic structure analysis were highly consistent with the results of phylogenetic analysis and principal component analysis.(2)The activities of three endogenous enzymes,namely β-glucosidase,peroxidase and polyphenol oxidase,were measured and analyzed in 95 single plants of the Camellia sinensis cv.Guiding-niaowangzhong of Guiding,indicating that the same population showed rich variation in the same environment,but the materials with higher enzyme activity accounted for a minority.Enzyme activity is not only affected by environmental factors,but also by genotype.The amplitude variability,mean,standard deviation and coefficient of variation of β-glucosidase activity in leaflets were higher than those in middle leaves and large leaves,while peroxidase activity and polyphenol oxidase activity variations,standard deviations and coefficients of variation were higher in middle leaves than those in leaflets and large leaves.At the same time,some single plant materials with high enzyme activity were screened,which could be used for the selection and breeding of highquality varieties in the later stage.Based on the EMMAX model,FASTLMM model,LM model and LMM model,the GWAS analysis of the three enzyme activities was carried out,and the number of sites detected by different models was different,and most of the significant sites were detected in each model.The gene annotation and function prediction of the upstream and downstream of the association site were analyzed by the genome analysis of Camellia＿sinensis:CSS＿Chr Lev plant,and the prediction of related candidate genes was carried out by combining the linkage imbalance analysis of the associated segment,and 7 genes associated with β-glucosidase activity,5 genes associated with peroxidase activity,and 6 genes associated with polyphenol oxidase activity were predicted.(3)Using 12 single plants of the representative Camellia sinensis cv.Guiding-niaowangzhong at the low,medium and high levels of enzyme activity as materials,the 7 candidate genes predicted to be associated with β-glucosidase activity were preliminarily verified by RT-q PCR.The candidate genes were predicted to be CSS0028616.1,CSS0024936.1,CSS0030286.1,CSS0012859.1,CSS0022910.1,CSS0005683.1,CSS0026950.1,and the association between gene expression and enzyme activity was analyzed.The results showed that there was no obvious trend between the expression of the seven candidate genes associated with the change of β-glucosidase activity,and the expression of each single plant was different,and there was a lack of obvious change trend,among which the gene expression levels of CSS0012859.1,CSS0022910.1,CSS0005683.1 and CSS0026950.1 may be one of the key reasons affecting the high activity of NW62 monoenzyme numbered C4.There is a very complex process from gene to enzyme activity,including gene translation,protein modification and enzyme activity expression,although the expression of these associated genes is not significantly related to the high and low changes of β-glucosidase activity,but the expression of these genes is one of the reasons affecting β-glucosidase activity. |
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