| Sweetpotato is an important crop as food,feed,industrial materials and new-type energy.Due to its complex genetic background,the study of the sweetpotato genome was lagged off.In this study,the first sweetpotato BAC library was constructed using sweetpotato line Xu 781,the composition and structure of sweetpotato genome were surveyed through BAC-end sequences analysis,and the BAC clones containing IbMIPS genes were analyzed.The main results were shown as following:1.The BAC library contained 240,384 clones with an average insert size of 101 kb,had low organellar DNA contamination(0.48%),represented approximately 7.93-10.82 × genome equivalents based on the sweetpotato genome size of 2,200-3,000 Mb,and the likelyhood of obtaining any sweetpotato sequence of interest is greater than 99%.In order to screen the BAC library via PCR approach,626 plate pools and 223 superpools were prepared.2.Both ends of 8310 BAC clones randomly selected from the library were sequenced to generate 11,542 high-quality BAC-end sequences(BESs),with an average length of 658 bp and an accumulative length of 7,595,261 bp.Analysis of the BESs revealed that approximately 12.17%of the sweetpotato genome consists of known repetitive DNA,including 7.37%long terminal repeat(LTR)retrotransposons,1.15%Non-LTR retrotransposons and 1.42%Class ? DNA transposons;18.31%of the genome was identified as sweetpotato-unique repetitive DNA and 10.00%of the genome was predicted to be coding regions.In total,3,846 simple sequences repeats(SSRs)were identified,with a distribution of one SSR per 1.93 kb.Comparative genome analysis showed that a total of 11,229(97.29%)and 11,320(98.08%)BESs produced significant hits with the genome sequences of ?.trifida Mx23Hm and ?.trifida 0431-1,respectively.These results have showed that ?.trifida(2x)had a high close relationship with sweetpotato.Microsynteny analysis indicated that more sweetpotato BESs were matched to the whole-genome sequences of Solanum lycopersicum than those of Vitis vinifera,Theobroma cacao and Arabidopsis thaliana.3.A total of 25 BAC clones containing MIPS gene were identified based on library screening,and five IbMIPS genes,IbMIPS1,IbMIPS2,IbMIPS3,IbMIPS4 and IbMIPS5,were isolated.The DNA sequences of the 5 MIPS genes were 3,128,3,429,3,457,3,429 and 3,419 bp in length,respectively,and the sequence identity of them ranged from 96%to 99%.RT-PCR analysis showed that the expression of IbMIPS gene was induced by PEG,heat and stem nematodes.The highest expression level of IbMIPS5 was observed under PEG and stem nematodes stresses,and the highest expression level of IbMIPS1 was observed under heat stress among the five genes.Sequence alignment and phylogenetic tree analysis showed that IbMIPS was closer to MIPS in Ipomoea trifida(2x)and Ipomoea nil(2x).The 25 BAC clones were assembled into 4 contigs and one singlet using FPC software.Six BAC clones were selected for sequencing and a total of 546,131 bp sequence were obtained,the GC content was 36.62%and the repetitive sequences was 6.7%?97 genes were predicted,and some genes were related to stress response.The results of comparative genome analysis showed that the exons of IbMIPS gene and the order of the MIPS gene with its adjacent genes were highly conserved among inter-and intra-genome of investigated species.4.Of the 3,846 BES-SSRs,288 were chosen to design primers for assessing their allelic polymorphisms with 20 sweetpotato accessions.Of them,173 primer pairs(60.07%)produced polymorphic bands among the 20 tested accessions.A total of 289 SSRs were identified from the BAC full-length sequences and 284 of them were chosen for SSR primers designation.The SSR primer pairs were tested on Xushu 18,Xu 781 and 8 F1 individuals of them,and the results showed that 66 of them had produced polymorphic bands. |
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