| Sichuan is not only one of the birthplaces of tea plants,but also the second largest province of tea planting area in China.Tea germplasm resources are very rich,which has laid a solid foundation for the cultivation of high-quality new varieties of tea.Chongzhou loquat tea（CZLT）,also known as Chongqing loquat tea,is a local tea cultivars formed through long-term natural evolution and semi-artificial domestication,has the characteristics of strong branching capacity,strong adaptability,strong cold tolerance and large leaves similar to loquat leaves.However,due to natural disasters,man-made logging,overexploitation and other factors,the number of wild CZLT has decreased sharply.In order to protect,develop and utilize the resources of CZLT,the research team has selected 26 new CZLT strains from its excellent individual plants based on systematic breeding method for decades.In order to further develop this excellent local tea resource,in this paper,the above-mentioned new strains were multiply evaluated through whose morphological characteristics,physiological characteristics,photosynthetic performance,cold resistance ability,high throughput transcriptome sequencing and metabonomics were used to evaluate the molecular regulation and metabolic response mechanism of cold resistance to provide a scientific theoretical basis for the cultivation of new varieties of Chongzhou loquat tea with excellent cold resistance and quality.The results are as follows:1.The 26 new CZLT strains mainly showed the characteristics of evolution from the wild type to the cultivated type,such as elliptical leaf shape and gradually pointed leaf tip,but there were still some wild-type tea characters,such as big leaves and big flowers,sparse and blunt leaf teeth,raised leaf surface and no hair in ovary.The mean variation coefficient of the morphological traits of the new strains was 34.17%,and its diversity index was 0.70.The genetic diversity level based on SSR marker was PIC=0.636,indicating that the new strains of CZLT had a high level of phenotype and genetic diversity,and its morphological and phenotypic stability was Bud>leaf>tree>flower.The one hundred bud weight of one bud and three leaves,leaf length,leaf width and the other yield-related factors had a higher genetic differentiation level,the new strains were potential tea resource for breeding for high-yield tea varieties.CZLT 24,26,23,20 and 16 showed strong growth potential,and one hundred buds weight of one bud and three leaves was relatively high,which had the potential to breed high-yield tea varieties.The buds and leaves of CZLT 3,5,11,and 20 were purple,which had the potential to breed purple tea varieties and high-anthocyanin tea varieties.CZLT 3,5,11,17 and 20 belonged to special tea plant germplasm resources.The germinating dates of CZLT 13 and 26 were 12 and 10 days earlier than CK,respectively,which indicated that such strains belonged to the early-germination tea strains.2.The polyphenols（TP）content of new shoots of CZLT strains ranged from 16.75%to28.28%,the content of TP in CZLT 3,11 and 23 in spring,summer and autumn was all>25%,indicating that which were specific tea strains with high TP.The amino acid content of new shoots of CZLT strains ranged from 1.74%to 5.69%,of which,the total amino acid content of new shoots of CZLT 25 and 26 was 5.69%and 5.18%,respectively,indicating they were high amino acid tea shoots.The caffeine content of new shoots was 3.24%-5.29%,and the content of CZLT 2 and 10 was 5.03%and 5.29%,respectively,which met the standard of high caffeine tea germplasm resources.The average values of catechins in CZLT11,12,22 and 23 at spring,summer and autumn were 23.04%,21.69%,23.48%and23.90%,respectively,they were belonging to the tea strains with high catechins.Additionally,the average values of EGCG content in CZLT 11,12,22,and 23 at different seasons was16.25%,15.31%,15.92%,and 15.95%,respectively,which indicated that such new strains belonged to the high EGCG tea germplasm resources.3.The results of photosynthetic performance research showed that Pn,Gs,Ci,Tr,WUE and CE of all the new CZLT strains were significantly different（P<0.01）,and the C.V.was Gs>CE>Pn>WUE>Tr>Ci.CZLT 14 had the highest net photosynthetic rate（Pn=11.57μmol/m2·s）.Twenty-six new CZLT strains were clustered into three different Pn groups,and the differences reached a significant level.The three strains with the highest light energy utilization were CZLT 8,22,25,and 26,which had the potential to be selected as high-yield tea varieties,the highest water utilization rate was CZTL 4,18,and 15,which had the potential to be a drought resistant tea variety.4.The cold resistance of the new CZLT strains was determined in the field and the laboratory.The results showed that the cold resistance of the 26 strains could be divided into4 grades,of which CZLT 10,12,20,21,and 26 had strong cold resistance,while CZLT 2,3,5,18,and 23 had weak cold resistance.The other strains showed relatively strong or medium cold resistance.In the low temperature treatment,the relative conductivity of CZLT 3,18,and 23 changed the most,and 12,20,and 26 changed the least.Under the conditions of natural growth and low temperature treatment,the average content of malondialdealdehyde of CZLT12 and 20 were the lowest(13.00μmol/g10-3 and 13.53μmol/g10-3),the highest content of proline existed in CZLT 21（62.21μg/g W）and the lowest content of which was discovered in CZLT 18（36.03g/g W）.The content of soluble sugars was the highest in CZLT26（89.23 mg/g）,and the lowest in CZLT 2 and 3（46.54 mg/g and 53.89 mg/g）.The activities of SOD,POD and CAT were the highest in CZLT26,and the lowest in CZLT 18 and 23.After comprehensive evaluation by the membership function method,CZLT12,20,21,and26 could be used as cold-tolerant tea strains.5.Metabonomics analysis showed that after treatment at-10℃for 2h,4h,8h,16h,and24h,the metabolic profile of CZLT 26（the strong cold resistance strain）and 23（the weak cold resistance strain）changed significantly,and the total number of significantly different metabolites is 953,of which 372 were up-regulated and 581 were down-regulated.Among them,8 metabolites such as leucine methionine,3-glucosyl-patchoulin,7,2’-dimethoxy-4’,5’-methylenedioxy isoflavone,1-salicylic acid glucoside acid,2,5-dihydroxybenzaldehyde,epicatechin,shikimic acid and thymine were significantly different at 5 time points.Different metabolites were concentrated in amino acids,sugars and sugar derivatives.The pathways of different metabolites enrichment were mainly concentrated in galactose metabolism,starch and sucrose metabolism,interaction between pentose and glucuronic acid,glycolysis/gluconeogenesis,arginine and ornithine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,β-alanine metabolism and so on.6.Transcriptomics analysis showed that after treatment at-10℃for 2h,4h,8h,16h and24h the gene expression of CZLT 26 and 23 changed significantly,and a total of 13369 genes differentially expressed,of which 7211 genes up-regulated and 6158 genes down-regulated,among them,1871 genes were significantly different at 5 time points.Genes with significant differences were mainly enriched in plant pathogen interaction,RNA translocation,glyoxylic acid and dicarboxylic acid metabolism,alanine,aspartic acid and glutamic acid metabolism,citric acid cycle,nitrogen metabolism and other pathways,and involved in cell physiological process,metabolic process,membrane,membrane part,binding,catalytic activity and other pathways.Under low temperature stress,the genes involved in carbohydrate metabolism including RS2,BAM,AMY,α-galactosidase,β-galactosidase,MFS,ERD6,SWEET,HXK,and Ca2+signaling pathway genes CDPK,CBL,CIPK,Ca M,CNGC,TPC,and transcription factors WRKY,MYB,b ZIP,b HLH,AP2/ERRBP,NAC had significant differences in expression,which may play an important role in the response of CZLT to low temperature stress.In summary,based on the results of morphological characteristics,physiological characteristics,photosynthetic performance,cold resistance characteristics,transcriptomics and metabolomics research of the new CZLT,CZLT 3,11,12,22,23 and 26 were the best among the 26 Chongzhou loquat tea lines.Chongpi No.3 can be further bred as purple,early-growing and high tea polyphenol tea variety,chongpi No.11 can be further bred as purple,high tea polyphenol,high catechin,high EGCG and strong cold resistance tea variety,chongpi No.12 can be further bred as high catechin,high EGCG and strong cold resistance tea variety,chongpi No.22 can be further bred as high catechin,high EGCG and high light efficiency tea variety Chongpi 23 can be further selected as Zaosheng tea variety with high tea polyphenols,catechins and EGCG,while chongpi 26 can be further selected as Zaosheng tea variety with high amino acids,high light efficiency and strong cold resistance. |
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