The Construction of Genomic Fosmid Library and Library PCR Screening System on Ipomoea trifida(Kunth) G. Don

doi:10.7668/hbnxb.2014.02.008

Abstract

Abstract: Ipomoea trifida (Kunth) G. Don(2n = 2x = 30) and the cultivated sweetpotato are both belonged to group B in Batata section. This species has been used as model research and improve the sweetpotato with the char- acteristics of the lower ploidy level, chromosome number and elite resistance. In order to enhance the genomics study and explore excellent genes resource of I. trifida, it is very valued to construct a genomic library. This work identi- fied I. trifida the genomic size as 531. 699 Mb by carrying out flow cytometer analysis. The I. trifida genomic DNA was isolated by low melting agarose embedding method. Then Shear the DNA by physical method and end-repair the sheared DNA to blunt,5'-phosphorylated ends. Recycle the fragment between 33 to 48 kb. Ligate the blunt-ended DNA to the Cloning-Ready CopyControl pCC1FOS vector (Epicentre) . Package the ligated DNA and transfection EPI300-T1 R cells. The genomic Fosmid library containing 101 952 clones in 106 2 96-well plates was constructed. The average size of the inserted DNA in recombinant plasmids was 35 kb. The library coverage was at least a 6. 7- fold genome equivalent and the probability of harboring any gene in the genome of the strain was 99. 88% . With 20 plates as a group, we build the PCR screen system. Every group contained 1 super-pool,20 plate-pools,12 column-pools and 8 row-pools. A positive clone would be found by at most 93 PCR reactions. 10 genes were used in this screen system to identify the efficiency of the system. The average positive clone number was 8. 2, in which the low- est was 3 and the highest was 16.

Key words: Wild sweetpotato relatives, Genomics, Fosmid library, PCR screening system

CLC Number:

LI Ang, WU Zhi-ming, ZHOU Zhi-lin, ZHAO Dong-lan, ZHANG An, MA Dai-fu, LI Ya-dong, TANG Jun, CAO Qing-he. The Construction of Genomic Fosmid Library and Library PCR Screening System on Ipomoea trifida(Kunth) G. Don[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(2): 45-50. doi: 10.7668/hbnxb.2014.02.008.

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