| Yellow-seeded Brassica napus have lots of excellence, for example, thin seed coat, low fibrin content ,high protein content, high oil content and etc. So an important aim of rapeseed breeding is to select yellow-seeded rapeseed. In this study, we aimed to find the genetic factor and physical reason of seed coat color of rapeseeds, and established genetic theory for map-based cloning and provide information for yellow-seed breeding, by researching the genetic difference and difference of physical charaters related to seed coat color between yellow-seeded and black ones.Two recombinant inbred lines (RILs) were used in this study. The two populations with same femal parent GH06, which had a complete dominant main gene of yellow seed coat with 90% degree of yellow seed and the trait of yellow seed shows very steady. The male parent of RILs-I was self-inbreded from Youyan 2, and the male parent of RILs - II was Zhongyou 821.The seed coat color was analysed by using uniscanA688 scanner. The anthocyanidin, favoid, total phenol content were extracted by 5% HC1 methanol solution and then the solution was tested absorbency on 600nm, 530nm, 325nm, 280nm. The 530nm-600nm=0.1 was used as anthocyanidin unit, the favoid content was calculated by absorbency on 325nm per dry weight, the total phenol content was accounted by gallic acid standard curve. The melanin content was extracted by 2% NaOH and caculated by absorbency on 280nm per dry weight. We used Excel software record those character data, then we did variance analysis and correlated analysis by using SPSS software. On the basis of the seed coat color, related charcter analysis and the result of observational by vision, we selected ten yellow seeded lines and ten black ones from the two RILs, and contracted yellow seeded bulk and black seeded bulk. SRAP marker was used to analysis the difference display of these four bulks.From statistical analysis of seed coat color and related character we found that: seed coat color, anthocyanidin content, favoid content, total phenol content and melanin content all had transgressive segregation. The seed coat color was high negative correlated with anthocyanidin content, favoid content, total phenol content and melanin content. Favoid content was high correlated with total phenol and melanin. Total phenol was high correlated with melanin. The result of cDNA-SRAP was: 2100 amplified bands were totally obtained with 996 pairs of SRAP primer combinations with the lengh of 50-500 bp. and the repeated bands were 65.2%. Twelve steady repeatable fragments with length between 100-300 bp which were different expressed between yellow and black immature seeds were aquired. Among them, seven stable, longer fragments were sorted out, cloned and sequenced. Sequence analysis was done on http://www.ncbi.nlm.nih.gov/BLAST. The result revealed that only two of the fragments had significant homologous nucleotide sequence with Arabidopsis thaliana, one was homologize to NAD+ADP-ribosyl transferase (NM-123973),and the other to peptide transporter protein 3 (NM-122152)., the homology was 88% and 85-95% respectively. These two fragments were all appeared in the black bulks. The other five bands did not find homology sequence that function known.The results of this research showed that the black seedes have higher content of anthocyanidin, flavonoid, total phenol and melanin in seed testa. We used cDNA-SRAP to study on seed coat color was to grope for the method of differential display. The primer of SRAP marker was designed on basis of open reading frame (ORF). So the fragments acquired by this method were expressive sequence as the same as the differential display. Through the experiment we found that: the method of cDNA-SRAP was a simple operation, low cost and safty method, but low repeated ratio, lean bands character, short bands and normal steady. The two bands obtained by BSA and differential display that highly homological with NAD+ADP transferase and peptide transporter protein 3 may be related to Brassica napus seed coat color expression.
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