Rice Chloroplast Genome Assembling,Validating And Polymorphism Analysis

in the process of shot-gun sequencing the nuclear genomes of the paternal and maternal parents, Pei'ai 64S and 93-11, of super-hybrid rice "Liangyou Pei jiu" , we accumulated cytoplasm genome sequences as byproducts. Based on the published chloroplast genome sequence of Nipponbare, we drew out over 50 thousand sequences respectively from the sequence database of Pei 'ai 64s and 93-11 with the help of the software BLAST. We assembled the full chloroplast sequence of two materials with these sequences through software Phrap. In the result, we found:1. The full length of Pei 'ai 64s chloroplast is 134,559bp, while that of 93-11 i s 134, 505bp, that are di f f erent from pub! i shed sequence of Nipponbare, which is 134,525bp.2. The assembl i ng strategy and the assembl ed chl oropl ast genome sequences methods were verified through molecular biology technology.3. we analyzed the single Nucleic PolymorphismsSNPs) and poly-base inDel variation by comparison of chloroplast genomic sequences of the three rice varieties.4. Three pai rs of primers were designed to amplify three InDel sites of 69bp, 32bp and 15bp. we verified the first two variation sites, while the variation site of 15bp does not exist. The 15bp difference may be attributed to improper sequencing of Nipponbare chloplast DNA.5. Two pairs of primers were designed to amplify the different fragments in 27 rice varieties. It was found that indica and j aponi ca van' eti es coul d be di sti ngui shed by the presence and 1 ack of a 69bp fragment (except Zhai yeqi ng 8) . The pattern of 32bp fragment i s cl osel y rel ated to that of 69bp fragment. This result is in accordance with the previous conclusion, that the difference of 69bp fragment has relation to differentiation of indica and japonica rice. Besides, 32bpfragment is also related to 'differentiation of indica and japonica rice.6. The GC content of the three rice chloroplast genomes were analyzed. There is little difference in GC content among the three materials, of which 93-11 has 39.00%, Pei'ai 64s has 38.52% and Nipponbare has 38.99%. It indicates that the i ngredi ent of chl opl ast genome DMA i n vari ous ri ce vari eti es is conservative.7. 339, 332 and 328 ORFs were predicted in the three rice chloroplast genomes. Among them, 40 non-overlapping ORFs were all identified, the genomes of which the longest ORF is 4,626bp. It differs from other species.