Study on the Mitochondrial Genome of Sea Island Cotton(Gossypium barbadense) by BAC Library Screening

The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility （CMS）. Therefore, research on the cotton mitochondrial genome may provide important information for analyzing genome evolution and exploring the molecular mechanism of CMS. In this paper, we present a preliminary study on the mitochondrial genome of sea island cotton （Gossypium barbadense） based on positive clones from the bacterial artificial chromosome （BAC） library. Thirty-five primers designed with the conserved sequences of functional genes and exons of mitochondria were used to screen positive clones in the genome library of the sea island cotton variety called Pima 90-53. Ten BAC clones were obtained and verified for further study. A contig was obtained based on six overlapping clones and subsequently laid out primarily on the mitochondrial genome. One BAC clone, clone 6 harbored with the inserter of approximate 115 kb mtDNA sequence, in which more than 10 primers fragments could be amplified, was sequenced and assembled using the Solexa strategy. Fifteen mitochondrial functional genes were revealed in clone 6 by gene annotation. The characteristics of the syntenic gene/exon of the sequences and RNA editing were preliminarily predicted.

Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.

Heterologous expression of Avermectins biosynthetic gene cluster by construction of a Bacterial Artificial Chromosome library of the producers

Avermectins,a group of polyketide natural products,are widely used as anthelmintics in agriculture.Metabolic engineering and combinatorial biosynthesis were extensively employed to improve Avermectins production and create novel Avermectin derivatives,including Ivermectin and Doramectin.It is labor intensive and time cost to genetically manipulate Avermectins producer Streptomyces avermitilis in vivo.Cloning and heterologous expression of Avermectins biosynthetic gene cluster will make it possible to tailor the cluster in vitro.We constructed a Bacterial Artificial Chromosome(BAC)library of S.avermitilis ATCC 31267 with inserted DNA fragments ranged from 100 to 130 Kb.Five recombinant BAC clones which carried the Avermectins biosynthetic gene cluster ave(81 Kb in size)were screened out from the library.Then,ave was hetero-expressed in S.lividans.Three Avermectin components,A2a,B1a and A1a were detected from the cell extracts of recombinant strains.It will facilitate the development of Avermectin derivatives by polyketide synthase domain swapping and provide functional element for Avermectins synthetic biology study.

Cloning and Diversity Analysis of TaHKT2;2 in Hexaploid Wheat

In order to study the roles of members of HKT gene fanfily in wheat, TaHKT2 ; 2 was isolated by using homologous cloning strategy and screening genomic BAC library. TaHKT2; 2 genes were mapped on chromosomes 7A, 7B and 7D, named as TaHKT2; 2-7A, TaHKT2; 2-7B, and TaHKT2; 2-7D, respectively. TaHKT2 ; 2 and TaHKT2 ; 1 had the same genetic structure, composed of three exons and two introns, and formed a cluster with TaHKT2 ; 1 on the phylogenetic tree of plant HKT transporters. The coding sequences of TaHKT2 ; 2-7A, TaHKT2 ; 2-7B, and TaHKT2 ; 2-7D were 1 602, 1 602 and 1 596 bp long, respectively, but TaHKT2 ;2-7D cDNA sequence was not isolated by RT-PCR in eight wheat varieties. The natural diversity of TaHKT2 ;2 genes was analyzed by cloning and sequencing from 12 wheat varieties. The results showed that TaHKT2;2-7A was found to be more diverse than TaHKT2; 2-7B and TaHKT2; 2-7D. Only a few bases changed in the alleles of TaHKT2 ; 2 genes in wheat. No amino-acid natural variation lay in the P-loops of deduced protein sequences of all alleles of TaHKT2 ; 2 in 12 wheat varieties. The identity of coding sequences was nmch higher than that of 5＇ flanking regions of TaHKT2 ; 2 genes. TaHKT2 ; 2 nfight be selected over the comse of wheat domestication and belonged to domestication gene.

Molecular and FISH analysis of 45S rDNA on BAC molecule of Saccharina japonica

IGS is abundant in polymorphism,which is widely used in the analysis of intraspecific genetic diversity and phylogenetic relationships among geographical populations.In this study,the 45S rDNA repeat unit of Saccharina japonica was obtained for the first time by BAC clone sequencing.The total length of the 45S rDNA repeat unit of S.japonica was 8995 bp,including 5420 bp of 18S-5.8S-25S rDNA,and 3575 bp of IGS(Intergenic Spacer),with the GC content of 51.4%.The IGS was composed of a 465 bp of 3′-outer transcribed spacer(ETS),an 874 bp 5′-ETS,and a 2236 bp non-transcribed spacer(NTS),with the GC content of 50.1%.Fiber-FISH(fiber-fluorescence in situ hybridization)analysis of the distribution of 45S rDNA repeat units on the bacterial artificial chromosome illustrated that each fiber had at least five continuously moniliform hybridization signal points.This study provided a new candidate molecular marker for detecting intraspecific polymorphisms of S.japonica.In addition,the successful fiber-FISH analysis of the 45S rDNA on BAC molecule would contribute to the construction of the physical map and map-based cloning of this kelp.

A bacterial artificial chromosome-based physical map of Manihot esculenta ssp. flabellifolia

Cassava(Manihot esculenta) is known as the third most important food crop in the tropics and also used for industrial feedstock for biofuels. Two new bacterial artificial chromosome(BAC) libraries were constructed for W14(M. Esculenta ssp. flabellifolia), a wild ancestor of domesticated cassava. The libraries were constructed with EcoR I and Hind III insertion vectors, respectively.The EcoR I library has 29952 clones with an average insert size of 115 kb, while the Hind III library consists of 29952 clones with an average insert of 129 kb. The combined libraries contain a total of 59904 clones with an average insert size of 125 kb, representing approximately 10 ?haploid genome equivalents. A total of 29952 clones were fingerprinted and resulted in a cassava physical map composed of 2485 contigs with an average physical length of 336 kb and 2909 singletons, representing approximately762 Mb of the cassava genome. 5000 clones located at the ends of BAC contigs were selected and sequenced. A total of 6077 SNPs and 231 indels were identified, that covered459 gene sequences, of which 6 genes were associated with starch and sucrose metabolism. This BAC-based physical map provides valuable tools to understand the genetics and evolution of cassava.

Genome-wide screening and transcriptional profile analysis of desaturase genes in the European corn borer moth

Acyl-coenzyme A （Acyl-CoA） desaturases play a key role in the biosynthesis of female moth sex pheromones. Desaturase genes are encoded by a large multigene family, and they have been divided into five subgroups on the basis of biochemical functionality and phylogenetic affinity. In this study both copy numbers and transcriptional levels of desaturase genes in the European corn borer （ECB）, Ostrinia nubilalis, were investigated. The results from genome-wide screening of ECB bacterial artificial chromosome （BAC） library indicated there are many copies of some desaturase genes in the genome. An open reading frame （ORF） has been isolated for the novel desaturase gene ECB ezi-A11~ from ECB gland complementary DNA and its functionality has been analyzed by two yeast expression systems. No functional activities have been detected for it. The expression levels of the four desaturase genes both in the pheromone gland and fat body of ECB and Asian corn borer （ACB）, O. furnacalis, were determined by real-time polymerase chain reaction. In the ECB gland, All is the most abundant, although the amount of A14 is also considerable. In the ACB gland, A14 is the most abundant and is 100 times more abundant than all the other three combined. The results from the analysis of evolution of desaturase gene transcription in the ECB, ACB and other moths indicate that the pattern of A 11 gene transcription is significantly different from the transcriptional patterns of other desaturase genes and this difference is tied to the underlying nucleotide composition bias of the genome.