Construction Of High-density Genetic Map And Its Application For QTL Analysis In Tea Plant

The tea plant is an economically important beverage crop cultivated in tropical and subtropicalregions. Recently, as the innovation of industrial technology and the rise of living standards, qualitytraits are becoming increasingly important in current tea plant breeding. However, improving these traitsgenetically is extremely difficult because most of them are controlled by quantitative traits loci (QTL).Moreover conventional breeding in tea plant is laborious, time-consuming due to its self-incompatibilityand long generation cycles. Genetic map construction and QTL mapping studies provide an abundanceof DNA marker-trait associations, and these trait linked markers have enormous potential to improve theefficiency and precision of conventional plant breeding via marker-assisted selection (MAS). In thisstudy, three genetic maps were constructed based on a F1population of inter-varietal hybrids of tea plant,using SSR markers and specific length amplified fragment sequencing (SLAF-seq), and QTLscontrolling caffeine (CAF) and theobromine (TBR) contents, leaf length (LL), leaf width (LW), leafindex (LL/LW) and length of ‘Two and a bud’(BL) were detected in the parental integrated map byrestricted multiple-QTL model (MQM) method. The results of this dissertation are as follows:1. A total of2982unigenes were predicted from8008tea plant ESTs, representing approximately4238kb of putative functional tea plant transcriptome. SSR searching detected561microsatellite lociamong462(15.5%) unigenes. The mined microsatellites were classified by repeat motif and size, andthe result showed that di-and tri-nucleotide repeats were the most common repeat types, accounting for45.5%and18.9%of the total. According to the flanking sequence of SSR,213primers weresuccessfully designed, of which104amplified unambiguous target bands with expected sizes.2. A set of1465SSR primers developed from different Camellia species were screened in the twoparents and six F1individuals. Of them,421EST-derived SSR and three genomic SSR yieldedpolymorphic amplicons. Paired comparison of allelic patterns obtained in parents showed that70(16.5%) loci were heterozygous in maternal parent while homozygous in paternal parent,168(39.6%)loci were heterozygous in paternal parent while homozygous in maternal parent, and186(43.9%) lociwere heterozygous in both parents. A chi-square test was performed on marker genotyping data for F1population, resulting in that130(30.7%) markers were distorted from Mendelian segregation ratios.3. Solexa sequencing of reduced-representation libraries generated a total of~130.35million readsfrom parents and150F1progenies. Reads were aggregated into101091SLAF tags, of which25014(24.7%) were polymorphic. After SNP genotype calling and removal of tags with incomplete data orunsuitable for CP population (outbreeder full-sib family), a total of6042SNP markers were finallyidentified. Of them,800markers were selected and analyses in F1population, and the result showed that289(36.1%) markers were available to for maternal map construction,276(34.5%) markers for paternalmap, and235(29.4%) markers for both parental maps. Chi-square test showed that164(20.5%)markers were distorted from Mendelian segregation ratios.4. Parental genetic maps were constructed separately based on pseudo-testcross theory. Thematernal map consisted of577markers (SSR226, SNP351), spaning a total length of1427.4cM with an average distance of2.5cM. The maternal map consisted of739markers (SSR313, SNP426),spaning a total length of1427.4cM with an average distance of2.5cM. Both maps contained15linkage groups, which corresponds to the basic chromosome number of tea plant. Based on homologousloci in parental maps, the consensus map was constructed, in which1101markers (SSR373, SNP728)were mapped on15linkage groups covering a total map length of1632.8cM with an average distanceof1.5cM.5. Significant differences were observed between the parents for all traits. The F1populationdisplayed continuous distribution of phenotypes and transgressive segregation. A total of18QTL with10.7-23.0%of the total phenotypic variance were identified. Of them, two common QTL, one for CAFcontent on LG05linkage group and one for LL/LW on LG07, were detected in different environments.