| Tea, made from the young shoots of tea plant (Camellia sinensis), is one of the most popular non-alcoholic beverages. Evidence is mounting that daily consumption of tea may reduce the risks of various types of cancer, obesity, neurological and cardiovascular diseases. The worldwide annual tea production reached 5.35 million tons in 2013 after a steady increase during the last 50 years. In addition to the traditional products that mainly consist of black, green and oolong tea, various extracts from tea leaves are also increasing used as natural additives in food and pharmaceutical products. The increased popularity of tea products has also increased the demand for breeding new tea cultivars that can satisfy the diversified market. However, tea plant breeding is very difficult due to a long breeding cycle and the large and highly heterozygous genome of C. sinensis. Furthermore, most economically important traits in tea are controlled by multiple genes, making it extremely difficult to create a new and desirable cultivar through traditional breeding methods.Genetic linkage maps constructed with DNA-based molecular markers are particularly useful for genetic studies of quantitative traits. To uncover the genetic controls of agronomic traits and facilitate marker-assisted selection (MAS) breeding in tea plant, we constructed a saturated SSR-based genetic linkage map using an F1 population derived from the crossing of'Longjin43'('L43') x'Baihaozao'('BHZ').The phenotype data were collected for several important traits from this segregation population and OTL mapping were conducted using the genetic map. Futhermore, the mapped SSR markers were further screen for DNA fingerprinting 128 clonal tea cultivars in China. The main results are as follows:1. To obtain enough markers for genetic mapping, we screened 711 candidate SSR markers using the genomic DNA of'L43','BHZ' and six F1 individuals as PCR templates. Among them,460 were novel SSRs derived from the tea floral transcriptome sequences and the other 251 were transferred from two other genetic maps of tea plant. In total 342 (48.1%) SSR markers including 206 from the novel group turned out to be informative for mapping with the'L43'x'BHZ'population. These informative markers were genotyped in the whole mapping population containing 170 Fi individuals.2. Genotyping data of the 342 new and 155 previous SSR markers were combined and then used for genetic map construction. A total of 483 SSR markers were successfully integrated into a new consensus map. The map has 15 linkage groups, covering 1226.2 cM of thee tea plant genome with an average marker distance of 2.5 cM. We also constructed two parental genetic maps based on the "pseudo-test cross" mapping strategy. For the maternal map ('L43'),323 segregating markers were assembled into a map, spanning 1036.2 cM in total; for the paternal map ('BHZ'),303 segregating markers were assembled into a map spanning 1322.5 cM. Both maps contain 15 linkage groups, which correspond to the number of chromosomes in the C. sinensis haploid genome. A higher percentage of distorted markers were found on the maternal map.3. Using 126 commonly mapped SSR markers, we successfully aligned the genetic map constructed in this study with two other maps of tea plant derived from two different mapping populations. Overall, all the 15 LGs from the three maps have one-to-one correspondences. The alignments between those maps are important for comparative mapping in tea plant.4. In 2014 and 2015, we collected the phenotype data of several important traits from the'L43' × 'BHZ' mapping population:timing of spring bud flush (TBF), young shoot color (YSC), mature leaf length (MLL), mature leaf width (MLW), leaf shape index (LSI, i.e. MLL/MLW). We also measured the contents of caffeine and seven types of catechins in young shoots using high performance liquid chromatography (HPLC) metood. QTL analyses were performed for these phenotype traits using the 'L43' × 'BHZ' consensus map. In total, we identified 43 QTLs, including two for TBF, five for YSC, five for MLL, three for LSI, two for caffeine content,20 for the seven types of catechins and 6 for the ratio of gallated catechins to non-galloylated catechins. A total of 14 QTLs were significant at genome-wide level (P<0.01) and were detected in several multiple phenotype datasets.5. We fingerprinted 128 elite clonal tea cultivars in China with thirty well-chosen SSR markers from the 'L43' × 'BHZ' map. The results showed that these markers are highly polymorphic among the tested cultivars with an average allele number of 10.4 per locus and average polymorphic information content (PIC) of 0.704. Robust fingerprinting power was demonstrated:it was possible to fully discriminate all 128 cultivars by a combination of four SSR markers. Eight SSRs were further screened and recommended as a core marker set for fingerprinting clonal tea cultivars.6. Based on the DNA fingerprint data, we performed parentage analyses among the 128 clonal tea cultivars, revealing 47 pairs of putative parent-offspring relationships, among which 33 were in agreement with the known pedigree information, whereas the other 14 were newly identified in this study.The novel SSR markers, genetic map and numerous QTLs reported here are valuable resources for future QTL mining, causal genes identification and MAS breeding in tea plant. The eight core SSR markers and DNA fingerprint database for clonal tea cultivars are also useful tools for many applications. |
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