| ‘Ziyan’（Camellia Sinensis）is a new tea cultivar with high anthocyanin content.‘Ziyan’was authorized by the Ministry of Agriculture for the“New Plant Cultivar Protection”in 2017,and registrated as a“Non-major Crop Cultivar”in 2018.Because of it special characteristics,‘Ziyan’is widely concerned and welcomed by researchers and consumers.This research mainly focused on 75 naturally-pollinated progeny of‘Ziyan’,to perform paternity and selfing analysis based on the SSR（Sample Sequence Repeat）marker,and to explore the genetic characteristics of important traits,such as anthocyanins content.In addition,six individuals with known parents and breeding preferred traits were selected for DUS（Distinctness,Uniformity and Stability）traits characterization and DNA fingerprints development.The main results are as follows:（1）According to the phenotypes such as shoots color,plant size and timing of bud flush in the spring,75 individuals were preliminarily screened from over 200naturally-pollinated progeny of‘Ziyan’.Genotyping analyses of the 75 individuals,mother‘Ziyan’and 40 candidate fathers were performed using 15 SSR primers with fluorescence and capillary electrophoresis.A total of 91 alleles were detected among the tested samples,with an average polymorphism information content（PIC）of 0.509.The superimposed Probability of Exclusion（PE）of these markers was calculated to be>0.9999,and the superimposed PI（Probability of Identy）and PIsibs（Probability of Identy in sibling）were6.19×10-5 and 2.30×10-10,respectively,indicating that these 15 markers were highly polymorphic and reliable for parental analyses.（2）Paternity analysis was performed using the exclusion method and the likelihood method.Under the condition of Trio（offspring-‘Ziyan’-father）mismatching loci≤1 and LOD score>5.80（corresponding confidence of 95%）,21 individuals matched their optimal fathers.Among them,the most-likely father（MLF）of 6 individuals（201,1102,1112,1114,1312 and 1604）is‘Mingshan Baihao 131’,MLF of 3,23,24,202 and 1302 is‘Chuanmu 217’,MLF of 1004 and 1309 is‘Huangjinya’,MLF of 601 is‘Pingyang Tezao’,MLF of 801 is‘Zijuan’and so on.（3）Genetic analysis found that the genetic distance among 29 individuals and‘Ziyan’was very close（0.0195～0.1367）,and they clustered tightly with‘Ziyan’in the cluster analysis.We suspected that they were the self-pollination of‘Ziyan’.To confirm this,‘Ziyan’was added in the candidate father list in the paternal analysis.The results showed that the Trio（offspring-‘Ziyan’-‘Ziyan’）mismatching loci with≤1 for the 29individuals.Ten additional SSR markers were genotyped to obtain higher confidence,and the results showed that the Trio mismatching loci of 29 individuals were still≤1,and LOD score≥-0.35（corresponding confidence>95%）.In contrast,the other tested individuals had 10～18 Trio mismatching loci and a LOD score varied from-31.28 to-58.99.Therefore,we have a high confidence to consider the 29 individuals as the selfing progeny of‘Ziyan’.In addition,the proportion of homozygous loci in these selfing progeny（0.52～0.92）was significantly higher than that of other progeny（0.16～0.44）and the mother ZY（0.44）.Population structure analysis also classified the selfing progeny and other half-sibling into two distinct groups,further supporting the selfing results.（4）In order to explore the genetic characteristics of anthocyanin content and other traits in slef progeny（OP）and outcrossing progeny（OP）of‘Ziyan’,we measured and compared 12 traits of‘Ziyan’,SP（n=29）and OP（n=38）.The results showed that the average anthocyanin content of the SP was 3.67 times that of OP,indicating that the SP was more likely to inherit the high-anthocyanin trait of‘Ziyan’.The total catechin content of SP was slightly lower than that of OP,and their caffeine contents were similar.However,the adaptive traits of SP were significantly lower than those of OP.These adaptive traits included the root-collar diameter,crown circumference,matured leaf area,sprouting index of over-winter buds（observed on March 30）and fruit number per individual.Among them,the average fruit number per individual of SP was only 3.86,which was 9.0%of that of‘Ziyan’（42.67）and 7.3%of that of OP（52.55）.Therefore,there is a strong inbreeding depression in the SP of‘Ziyan’.（5）According to the parental analysis and the phenotypes observed in the field,6offspring individuals of‘Ziyan’（702,1402,1601,601,801 and 1102）were further selected for DUS related traits characterization.Compared with their mother‘Ziyan’,there were 8～13 traits were significant different.Among them,the selfing individuals 702,1402 and1601 grows relatively vigorously with dark-purple young leaves.The individual 601,which derived from‘Ziyan’בPingyang Tezao’bud breaks significantly earlier than‘Ziyan’.The individual 801 grows vigorously with dark-puple leaves,which anthocyanin content is 1.76 and 4.00 times that of its mother‘Ziyan’and father‘Zizuan’,respectively.No anthocyanin was detected in the young shoots of individual 1102,but its growth was the strongest among all observed offspring.We futher screened 10 core SSR markers to construct DNA fingerprints for the six selected individuals and their parents.These results laid a foundation for the breeding of new tea cultivar and the protection of breeders’rights. |
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