| Brassica rapa (syn. Brasscia campestris) are important vegetables and oil crops, it is belongs to the Brassica genus in Brassicaceae (Cruciferae) family. Flowering time and leaf traits are the most important agronomic traits for crop production. Flowering early or late and leaf traits will produce important impacts on the yield and quality. Flowering time and leaf traits have widely genetic variation in B. rapa. The gene expression are the basis for these variations, and regulated by endogenous cell growth status and environmental factors (mainly including photoperiod and temperature)The main objective of this thesis is to unravel the genetic variations of the Brassica rapa flowering time and leaf traits. To achieve this goal, we searched all flowering time candidate genes/paralogues in B. rapa (Chiifu), which has sequenced full-genome, based on the A.thaliana. To develop and map gene/paralog specific markers (PCR; CAPS; SSR) for flowering time genetic map and analysis the QTL and eQTL associated with flwering time. Furthermore, we validated the reference genes for real-time quantitative PCR normalization in B. rapa. To quantify the candidate flowering time genes expression patterns using qRT-PCR and Microarrays. Additionally, association studies were performed in order to take advantage of the genetic diversity and flowering time variation evaluated through candidate gene strategy in nature population. At last, leaf traits genes were searched and mapped based on A.thaliana leaf traits candidate genes, which were analyzed through the dynamic QTL mapping. The main conclusions are as follows:1. This study aims at the identification of genes that regulate flowering time in a QTL approach using a Doubled Haploid population that has been phenotyped in multiple seasons and years. The DH population was from a cross between a Yellow sarson (annual oil) and a pak choi (leafy vegetable); the genetic map was complemented with125markers that were designed based on sequences of flowering time genes. In one season, RNA from six weeks old DH lines was isolated and gene expression was profiled using the105K Cogenics array assembled using mainly EST sequences from three species:B. napus, B. rapa and B. oleracea. Colocation of flowering time QTL (fQTL) and expression QTL, and network visualization of flowering time and expression of flowering time genes with eQTL, resulted in the identification of candidate genes for flowering time QTL and the visualization of coexpression gene networks. A major fQTL on A02at the BrFLC2locus collocated with a cis eQTL for BrFLC2and trans eQTL for the floral integrator genes SOCl and FT, and the photoperiod genes CO, SSR1and TCP11. Combination of phenotyping and genetic analysis of transcriptional profiles is a promising approach for identifying biological pathways and dissecting the genetics of complex traits.2. The expression of13candidate reference genes were assessed. Their expression stabilities were analyzed using two programs, geNorm and NormFinder, in20different samples that represent four strategic groups. Results showed no gene was uniformly expressed in all tested samples. ACTIN and CyP are proposed as good reference genes when studying developmental stages. CyP, Tuba and UBC30are good reference genes when studying different tissues (from flowering to seed set). CyP and Tub a are the most stable reference genes under biotic stress treatments using the fungi Peronospora parasitica and Alternaria brassicicola. UBC30, EF1-α and ACTIN were recommended for normalization in abiotic stress treatments, including hormone, salt, drought, cold and heath treatments. Moreover, at least five reference genesCACTIN, CyP, UBC30, EF1-α and UBQ) are required for accurate qRT-PCR data normalization when studying gene expression across all tested samples.3. The variations in photoperiod responses were obtained in the11morphotypes of B. rapa. The results showed that this photoperiod-sensitivity varies are different among11accessions. Statistical comparisons demonstrated that Komatsuna-130, Mizuna-128, Yellow sarson-143, Pak choi-175and Broccoletto-029show stronger responses to different day-lengths than others accessions. We selected three key genes (GI-CO-FT) components of circadian clock in the photoperiod pathway (every four hour). The results show that CO, GI mRNA are present under both LDs and SDs, and is regulated by the circadian clock. They raise in abundance between9to13h,1to9h after dawn, respectively. FT mRNA is present and raises in abundance between13to17h after dawn under LDs, whereas under SDs, it does not express except in CX and CC types. Three hundred and twelve fowering time genes in Chinese cabbage were found under short (8h light) or long (16h light) daylengths at9and at21h after dawn by Microarray, suggesting that the complexity of the photoperiodic for controling the flowering time mechanism. 4. We developed90new SSR markers whose physically position closely linked to promising flowering time candidate genes. A diverse collection of250B. rapa accessions based on two core collections (Wageningen/WUR and Vavilov/VIR) representing different morphotypes from different geographical origins were used by55simple sequence repeats (SSR) markers closely to the FT candidate gene for association mapping in B. rapa. A total of426polymorphic alleles(average7.7Were revealed and varied from2.1～5.8for per SSR. The association was done by the program Tassel using the General Linear Model CGLM) and the Mixed Linear Model (MLM). The GLM method detecded in total of67significant hits associated with FT at a statistical probability (P-value) of0.05. The second method MLM detected in total of39significant hits associated with FT at a P-value of0.05.5. The dynamic quantitative trait loci (QTL) for lamina length, lamina width, blade length, petiole length and leaf index at condition and un-condition under day-length, and QTL for leaf number and leaf chlorophy under different conditions, were mapped by92DH68lines. A total of252QTLs were detected with LOD>2. We found out186leaf traits genes/paralouges in B. rapa based on the90leaf function genes from Arabidopsis. We will select some candidate genes for leaf traits to be map on genetic map. One hundred sixteen leaf traits genes were ordered the specific gene primers. By now, eight controlling leaf traits genes have been mapped on the DH68population by Lightscanner. |
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