Gene Differential Expression Between Intersubgenomic Hybrid And Its Parents In Brassica Napus

Brassica napus(AACC,2n=38) is an amphidiploid species that originated from spontaneous hybridization between B.rapa(AA) and B.oleracea(CC).The role of cultivated B.napus as a commercial oil crop in Asia,Europe,North America and Australia,has progressively increased due to better production potential and improvement in seed quality.However,the short cultivation history and intensive breeding of this species has led to a comparatively narrow genetic base.Considerable efforts have been made to enrich the genetic diversity of B.napus by the introgression of genomic components of its related species or by developing synthetic B.napus lines by hybridization between B.rapa and B.oleracea.Several interesting changes have been observed in synthetic B.napus,such as chromosomal rearrangements,deletions of DNA sequences,variations at DNA methylation loci and the alteration of gene expression patterns.Heterosis has been widely used in agriculture to increase the seed yield,and it has been applied to a large number of crops.Research has revealed that analysis of the differences in gene expression profiles between the hybrids and their parents is an important method for elucidating the molecular basis of heterosis in crops such as wheat, riceand maize.These results are consistent with the hypothesis that multiple molecular mechanisms contribute to heterosis.In Brassica,heterosis was first reported in biomass for Sun in China.Subsequently many studies have estimated the extent of heterosis for seed yield.Divergent evolution and isolation have led to differentiation within the same genome among different species.To distinguish different subgenomes in Brassica,the genomes of the three diploid species B.rapa,B.nigra and B.oleracea were designated as A^r,Bⁿ and C^o while the genomes of the three amphidiploid species B.napus,B.juncea and B.carinata were designated as AⁿCⁿ,A^jC^j and B^cC^c,respectively.A new-type B. napus,A^r/nA^r/nCⁿCⁿ or A^r/nA^r/nC^c/nC^c/n,was created by interspecific hybridization and artificial selection.A hybrid(A^r/nAⁿCⁿCⁿ or A^r/nAⁿC^c/nCⁿ) was developed by crossing the new-type B.napus to B.napus and strong heterosis was observed in the hybrids.This typeof heterosis was considered as intersubgenomic heterosis and its existence showed that some alleles that were derived from related species could favourably contribute to increasing the seed yield of rapeseed. In this study,we compared the gene expression profiles of new-type B.napus lines with those of hybrids derived from crosses between new-type B.napus lines and B.napus cultivars,six objectives are addressed in this paper:1.Twenty-five hybrids developed between five lines of new-typed B.napus,two with A^r introgression from B.rapa and three with A^r and C^c introgression from both B. rapa and B.carinata as female parents and five cultivars of natural B.napus as male parents to hybridize each other.The average value of mid-parent heterosis(MPH) and high-parent heterosis(HPH) in hybrids was 33.3%and 17.0%,respectively.2.Three new-typed B.napus lines and their parents were chose,and cDNA-AFLP differential display technique was applied to obtain the first set of gene expression profiles between new-typed B.napus and their original parents.With 33 primer combination,we detected 1167 differential expressed TDFs.Over 50%transcript-derived fragment(TDF) altered their display pattern between new-typed B.napus and its parental natural B.napus.3.From 25 combinations,we chose nine combinations to obtain the second set of expression profiles between hybrids and their two B.napus parents by cDNA-AFLP differential display technique.With 32 primer combination,we detected 1210 differentially displayed TDFs.The type N₁⁺H^-N^- and N_t^-H^-N⁺,accounting for 51.4%of 1210 detected TDFs,were predominance among seven different patterns,followed by the dominant patterns and overdominant pattern.4.Through correlation analysis between presence or absence of single differential TDF and three types of data(HP,MPH,HPH) for six investigated agronomic traits,we found that the same differential TDF had consistent effect direction for same trait in three type heterosis data.However,for different traits,the effect direction was indefinite.For hybris performance,the negative effect TDFs were more than positive effect TDFs,they accounted for 58.1%;for MPH and HPH,the positive effect bands were much more,they accounted for 54.8%and 72.8%,respectively.5.Reused,cloned and sequenced 20 heterosis associated TDFs.We applied correlated internet servers and software to analyze these sequences information: downloading homologous sequences and assembling them into contigs.Then, EuGene'Hom was used to predict the functions of these genes with these contigs,and designed primers to confirm the assembled sequences.Based on the experience of manipulation and deeper understanding of cDNA-AFLP,we put forward a project to improve cDNA-AFLP technique. 6.Transformed some heterosis-associated TDFs into molecular marker to validate the relationship with QTLs for the yield related traits.Seven heterosis-associated TDFs were mapped in TN linkage map by 12 SSR and TRAP markers.After QTL analysis,7 markers of them developed from 4 TDFs located in the confident intervals of 23 QTL for seed yield and yield related traits in TN population.