Localization Of QTLs For Yield-related Traits And Heterosis Dissection Using Functional Markers In Brassica Napus L.

Bracissa napus is one of the most important oilseed crops in China. Now, the major object for breeders is to increase the oil production by increasing the yield and the oil content of rapeseed, and heterosis utilization, which is the most effective way to increase the yield and improve the quality in crops, have been widely used in B. napus. However, the molecular basis of heterosis is unclear until now. Some researches have shown that the differentially expressed genes between parents and hybrid are related to the heterosis. Nevertheless, it is unclear whether all the differentially expressed genes in hybrid are contributors to the heterosis or QTLs for traits, or some of them merely the results of hybridization. Therefore, such genes were developed to molecular functional markers, and were tested for their association with interested traits when they are integrated into a functional map of the genome and the QTLs of important agronomical traits have been located. This work will be helpful to the studies of the molecular basis of heterosis and facilitate the cloning and utilization of QTLs for important traits in B. napus. In addition, the comparative mapping between B. napus and model plants (Arabidopsis and flee) will accelerate above studies in rapeseed.In this study, an F2 population of 184 individuals resulting from crossing "SI-1300×Eagle" was used for linkage analysis, and a molecular functional map was constructed using differentially expressed genes, where QTLs mapping were conducted for 12 yield-related traits. Furthermore, the genetic dissection of heterosis in B. napus was studied. In addition, the comparative mapping was done between B. napus and model plants, including Arabidopsis and rice. The major results are as follows:1. A total of 261 PCR primer pairs were designed according to the differentially expressed genes between parents and hybrid. Of these, each of the majority (247) amplified a clear, strong and single band from the total genomic DNA of two parents when detected by 1.5% agarose gels. Using SSCP analysis, a total of 111 B. napus ESTs or genes showed polymorphisms between parents of the mapping population, providing 177 marker loci.2. Based on the information from Gene Ontology website, these 111 ESTs or genes were grouped into 17 functional categories, and the largest group was allocated to the general metabolism.3. One hundred and fifty-two sequence-related amplified polymorphism (SRAP) markers, 177 functional markers, 180 simple sequence repeat (SSR) markers, and 191 amplified fragment length polymorphism (AFLP) markers were used to construct a genetic linkage map of B. napus. Under the condition of LOD≥3.0, a total of 582 markers were assigned to 20 linkage groups (LGs). This map covered 2054.51 eM with average marker interval of 3.53 cM. The public information of microsatellites (SSR markers), derived from the Piquemal et.al (2005), Lowe et.al (2004), and BBSRC, contributed to establish the links between the map of Parkin et.al (1995) and ours. The results showed that most of LGs were aligned with the standard map (N1-NI9) except one LG.4. All the 12 yield-related traits demonstrated the normal distribution. The highly significant variation among the 184 genotypes was observed for all the traits, and the heritability ranged from 0.41 for number of siliques on main infloresence (SMI) to 0.81 for height of primary effective branch (HPB). High correlation coefficients between the two locations were observed for most traits with the exception of number of seeds per silique (SS), And the coefficients between different traits in the same location are also high.5. QTLs were detected for 12 yield-related traits in Wuhan and Jingmen, respectively. A total of 133 QTLs were identified, including 14 consistent ones across two locations. Seven to fifteen QTLs were found per trait and individual QTLs explained 3.02% 36..68% of the variance with an average of 11.62%. A total of 45 functional markers involved in 39 expressed sequence tags were linked with the QTLs of 12 traits, and the metabolic genes were the largest group.6. The results of mapping QTLs showed that most of the QTLs were clustered, especially on LGs N2 and N7. Some QTLs related to different traits located in the similar region, which are tightly linked or pleiotropy. Both parents had QTLs with positive or negative effects on the same trait, and QTLs for yield-related traits which showed overdominant and dominant were detected in both locations.7. The correlation coefficients between heterozygosity of the marker genotypes and trait measurements were small for all the traits and indicated that the lack of correlation between them.8. All possible two-locus combinations were tested for t 2 yield-related traits using 169 codominant markers. With P＜0.01, 1093 and 1121 combinations which were significantly affected the traits were detected in Wuhan and Jingmen, respectively. Further conformation by randomization test revealed 297 and 285 two-locus interactions in Wuhan and Jingmen, respectively, whereas only six were common in both locations. The results showed that the number of two-locus combinations significantly affected the yield-related traits were greater than that of QTLs.9. Interactions included the epistasis between QTLs and non-QTLs, and the one between non-QTLs. The later occupied the majority. The effect of individual locus of the interaction was small, whereas the significant effects to the trait were gotten when the two loci interacted. The same locus may have interaction with several other loci to affect the same trait, and the same two-locus interaction may affect several traits, like pleiotropy.10. The dominant and overdominant effects at single locus level and the epistasis effects at multiple loci level are all the important sources of the genetic basis of heterosis in B. napus.11. Ten amplified sequences for functional markers were cloned and sequenced. Comparing the sequence of cloned by us with the original one, we found that the similarity of them is average high to 98%, with the least 96%, which indicated that the designed primers can really amplify the original sequence. In addition, the sequences in both parents amplified by five functional primer pairs were randomly selected and sequenced. The comparation of the sequences between the parents showed the high similarity of the functional genes between cultivars.12. The physical localizations of Arabidopsis genes corrponding to the 107 ones of B. napus intergrated to the map were determined by the seqview tool of TAIR. Alignment between the B. napus LGs and A. thaliana chromosomes revealed that genes localized on each five Arabidopsis chromosome and covered a total of 110.95 MB of Arabidopsis genome. Syntenic regions were sought between Arabidopsis and Brassica. A total of 21 conserved regions, involved in 60 functional markers and 50 genes of B. napus and Arabidopsis, respectively, were identified. These conserved regions were located at 15 LGs of B. napus, and each conserved region in Arabidopsis corresponded to 1-7 regions ofB. napus.13. The genes which were located in the QTLs regions in B. napus were used to search Genbank by BLASTX to achieve the homologous sequence in rice, maize and wheat. The results showed that most have similar sequences. Furthermore, the homologous sequences in rice were mapped by in-silico way, and the QTLs were searched around this gene. The regions where some genes located were found the QTLs for the similar trait in rice compared to B. napus.