The Construction Of BAC Library Using The Multi-pods Material In Soybean

Soybean is one of the major oil crops in China,Due to the low yield of per unit area,the lower proportion and benefit of high quality soybean, its planting area is decreasing.The number of Soybean import increased year by year. Therefore, the soybean industry is grim.There are many factors correlate with soybean yield, including the pod number per plant, the seed number per plant, the seed number per pod, the 100-seed weight, etc. Yield character, as a quantitative trait, has many related gene-locus. The trait of the seed number per pod has a high heritability and major contribution to the soybean yield. So it's an effective way to improve the soybean yield by exploring the genes of the seed number per pod.Based on the located QTL of the seed number per pod, in this study, we constructed a BAC library by using a multi-pods family in the Recombinant Inbred Lines (RILS), which was helpful to clone the genes of the seed number per pod.The research demonstrated that the young leaves were more suitable for extracting large fragments of DNA. Because the fragments were more than 400kb, small fragments of DNA and secondary products were few. Moreover there was no protein and cytoplasmic DNA contamination. We could get the agarose embedded DNA plugs with proper concentration and less degradation. Digesting the DNA to change the concentration of enzyme within a certain time, we found that the best restriction enzyme mount of soybean genomic DNA was 3.0 U. We got the proper concentration and size of nuclear DNA fragments. Furthermore, a high efficient soybean fragments linkage system had been also constructed. The highest efficient proportion of linkage was 1:4.5.By improving the methods of desalination,which was used by Peterson, we got more transformants and increased the transformation efficiency.In this study, we constructed the BAC library including 54,912 clones.110 clones were selected randomly for testing. The result showed that the insert fragments size was between 50-200 kb and the average size was 125 kb.70% of the BAC insert fragments were more than 100kb. In the library, the total number of clones was about 5.54 times as much as the soybean haploid genome. The probability of screening any gene from this library was 99.98%.The coverage of this soybean library was high.The number of clones and the size of insert fragments were both larger.4 BAC clones were selected randomly to test. There was no loss or mutation of them after propagation through 100 generations. It indicated the BAC clones of the library were stable, the clones could conserve at least 100 generation.The coverage of the library, BAC clone insert size and the stability of clones had reached the international demands for the library construction.This is the first soybean BAC library for the yield trait.Compared with other soybean BAC libraries, although the total amount of soybean BAC clones is not particularly large, the average insert fragment of this library is larger. This library is more helpful for map-based cloning. This study provides a good physical laboratory platform. It will play an important role in soybean gene cloning, chromosome location, gene promoter isolation and analysis.