본 연구는 농촌진흥청 국립축산과학원에서 사육중인 제주재래돼지와 랜드레이스의 상호교차교배를 통해 생산된 417두의 F2집단을 대상으로 성장형질(생시체중, 3주령 체중, 10주령 체중, 20주령 체중)을 측정하고, 개체별 혈액으로부터 Illumina Porcine SNP 60k BeadChip을 이용하여 유전자형 분석을 실시하여 성장형질과 유전체 전장의 단일염기다형의 유전자형 간에 연관성을 알아보았다. 단일연기다형의 유전자형 분석은 돼지의 성염색체를 제외한 18개의 상염색체 내에 나타난 52,574개의 SNP표지인자 중다형성이 나타나지 않은 7,564개의 표지인자, 모든 개체에서 이형으로 나타난 47개 표지인자 및 결측률이 10 % 이상인 1,830개의 표지인자가 나타나 분석에 앞서 사전 제거를 실시하였으며, 남은 44,133개의 표지인자를 체중형질과 표지인자간 연관성 분석하는데 이용하였다. 체중에 영향하는 고정효과에 대해 일반선형모형을 설정하여 사전 보정을 실시하였으며, 여기서 얻어진 잔차값을 이용하여 표지인자와 월령별 체중간의 연관성 분석을 실시하였다. 분석결과 전장의 유전체 정보 중 통계적 판단의 오류를 고려하여 연관성이 강한(p <10-6)표지인자가 생시(BWB), 3주령(BW3), 10주령(BW10) 및 20주령(BW20) 체중에서 각각 657개, 846개, 49개, 122개로 추정되었다. 생시와 3주령 체중에 공통으로 유의적 영향을 하는 표지인자가 286개로 나타났으며, BWB와 BW10에서 5개, BWB와 BW20에서 8개, BW3와 BW10에서 13개, BW3과 BW20에서 11개, BW10과 BW20에서 1개로 나타났다. 또한 염색체별 성장형질에 영향하는 표지인자의 분포를 조사한 결과, 주령별 체중에 영향하는 표지인자는 전장의 유전체에 고르게 분포하는 것으로 조사되었으며, 특히 생시 및 3주령 체중에 영향하는 표지인자는 특정 염색체(SSC9)에서 고도의 통계적 유의차(p <10-15)를 나타내는 유전자 좌위가 있는 것으로 추정되었다.

Lepidoptera is one of the largest insect orders, but the phylogenetic relationships within this order, have yet to be completely described. One of the unresolved relationships includes the monophyly of Papilionoidea in relationship with the monotypic superfamily Hesperioidea. We newly sequenced five hesperid mitochondrial genomes (mitogenomes), representing four subfamilies: Pyrginae (Daimio tethys and Lobocla bifasciatus), Coeliadinae (Choaspes benjaminii), and Hesperiinae (Potanthus flavus), and Heteropterinae (Carterocephalus silvicola). Along with these newly sequenced hesperid genomes phylogenetic analysis was conducted with all available lepidopteran mitogenomes including three reported species of Hesperiidae that consisted of ~70 species in ten lepidopteran superfamilies. The test for the effect of optimization schemes, such as exclusion and inclusion of third codon position of 13 PCGs, other genes (22 tRNAs and two rRNAs), and with and without partitions also was performed. Majority of datasets consistently placed the monophyletic Hesperiidae the sister to ((Pieridae + Lycaenidae) + Nymphalidae), placing another true butterfly family Papilionidae as the basal lineage of this group, presenting the relationships (Papilionidae + (Hesperiidae + ((Pieridae + Lycaenidae) + Nymphalidae))). Consistent to previous result, Pyraloidea was placed as the sister to ((Bombycoidea + Geometroidea) + Noctuoidea), placing the Macrolepidoptera as non-monophyletic group.

The larch hawk moth, Sphinx morio, belongs to the lepidopteran family Sphingidae that has long been studied as a family of model insects in a diverse field. In this study, we describe the complete mitochondrial genome (mitogenome) sequences of the species in terms of general genomic features and characteristic short repetitive sequences found in the A+T-rich region. The 15,299-bp long genome consisted of a typical set of genes (13 protein-coding genes, two rRNA genes and 22 tRNA genes) and one major non-coding A+T-rich region, with the typical arrangement found in Lepidoptera. The 316-bp long A+T-rich region located between srRNA and tRNAMet harbored the conserved sequence blocks that are typically found in lepidopteran insects. Additionally, the A+T-rich region of S. morio contained three characteristic repeat sequences that are rarely found in Lepidoptera: two identical 12-bp repeat, three identical 5-bp long tandem repeat, and six nearly identical 5~6 bp long repeat sequences.

In the present study, the 17,694-bp long complete mitochondrial genome (mitogenome) of the dwarf honey bee, Apis florea (Hymenoptera: Apidae), is described with an emphasis on the noteworthy triplicated tRNAser(AGN) region and an extraordinary long A+T-rich region with repeat regions. The gene arrangement of A. florea mitogenome is identical to that of A. mellifera, but has triplicated tRNASer(AGN), each of which contains the precedent 44 bp-long and following another 64 bp-long repeats plus one complete first repeat abutting to tRNAMet. A total of 1,610-bp long two repeat regions in 1,987 bp-long A+T-rich region is composed of nearly identical 141 ~ 219-bp long five tandem repeats and 50 ~ 52-bp long 12 tandem repeats that are encompassed by three non-repeat sequences. One of the common interpretations for such repeat sequence is slipped-strand mispairing and unequal crossing-over events during DNA replication.

Gene arrangement in the mitochondrial genome (mitogenome) has been regarded as an important evolutionary event that is useful as a phylogenetic signal. The mountainous duskywing, Erynnis montanus, belongs to a lepidopteran family Hesperiidae. We sequenced 15,530-bp long complete mitogenome of the species. The genome has the typical gene content of animals (13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one major non-coding A+T-rich region). Further, E. montanus mitogenome also contained a high A/T content in the whole genome (81.7%) and the CGA (arginine) as the start codon for the COI gene, as typical in lepidopteran mitogenome. However, unlike other lepidopteran species, including two sequenced skippers, the E. montanus mitogenome has a unique arrangement tRNASer-tRNAAsn, instead of the tRNAAsn-tRNASer found unanimously in other lepidopteran species, providing a new gene arrangement in Lepidoptera. Such rearrangement probably was likely caused by duplication of gene block tRNASer-tRNAAsn and subsequent random loss of tRNAAsn in the first copy and tRNASer in the second copy, resulting in the arrangement tRNASer-tRNAAsn. Considering current phylogenetic relationships among available lepidopteran groups in connection with lepidopteran gene arrangement the new gene arrangement found in E. montanus seems to be apomorphy, requiring cautious interpretation as a phylogenetic signal.

Insects and animals can recognize surrounding environments by detecting thousands of chemical odorants. Olfaction is a complicated process that begins in the olfactory epithelium with the specific binding of volatile odorant molecules to dedicated olfactory receptors (ORs). OR proteins are encoded by the largest gene superfamily in the mammalian genome. We report here the whole genome analysis of the olfactory receptor genes of S. scrofa using conserved OR gene specific motifs and known OR protein sequences from diverse species. We identified 1,301 OR related sequences from the S. scrofa genome including 1,113 functional OR genes and 188 pseudogenes. OR genes were located in 46 different regions on 16 pig chromosomes. We classified the ORs into 17 families, three Class I and 14 Class II families, and further grouped them into 349 subfamilies. We also identified inter- and intra-chromosomal duplications of OR genes residing on 11 chromosomes. A significant number of pig OR genes (n=212) showed less than 60% amino acid sequence similarity to known OR genes of other species. We also performed a similar analysis on the cattle OR subgenome and identified 1,071 OR related sequences. We show that S. scrofa has one of the largest OR repertoires, suggesting an expansion of OR genes in the swine genome. Considering available information from literature, it seems that OR systems between mammals and insects possess high similarity in their action mechanisms and rapid evolutionary changes due to differences in living environments.

The application to genome study has been particularly developed with the introduction of the next-generation DNA sequencer (NGS) Roche/454 and Illumina/Solexa systems, along with bioinformation analysis technologies of whole-genome de novoassembly, expression profiling, DNA variation discovery, and genotyping. One of the advantages of the NGS systems is the cost-effectiveness to obtain the result of high-throughput DNA sequencing for genome, RNAnome, and miRNAnome studies. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing de novo assembly of novel genome sequencing data and for resequencing the samples with a reference genome DNA sequence. To construct high-quality contig consensus sequences, each DNA fragment read length is important to obtain de novo assembly with long reading sequences of the Roche/454 system. It is necessary to have DNA sequence information from a multiplatform NGS with at least 2× and 30×depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly, as hybrid assembly for novel genome sequencing would be cost-effective. In some cases, Illumina/Solexa data are used to construct scaffolds through de novo assembly with high coverage depth and large diverse fragment mate paired-end information,even though they are already participating in assembly and have made many contigs. Massive short-length reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. MAQ and CLC software are useful to both single nucleotide polymorphism discovery and genotyping through a comparison of resequencing data to a reference genome. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples, such as control and exposed tissue. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through de novo assembly in any whole-genome sequenced species. The 20× and 50× coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively,is effective to create novel expressed reference sequences. However, only an average 30× coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference expressed sequence tag sequence. In an in silicomethod, conserved miRNA and novel miRNA discovery is available on massive miRNAnome data in any species. Particularly, the discovered target genes of miRNA could be robust to approach genome biology study.

Recent years have seen the introduction of next-generation sequencing (NGS) technologies and their use in the fields of bioscience and biotechnology. Not only has the availability of NGS technologies revolutionized the way genome research is carried out, the massive sequence data produced by NGS technologies have also been driving the advancement in bioinformatics required for downstream analysis, storage, and accessibility. However, the processing of NGS data is still challenging owing to such properties as the shortness of reads, the sheer amount of data, and the low base qualities. For high-quality de novo genome sequencing, we used a hybrid approach that utilizes Sanger end sequencing of fosmid libraries for the scaffolding of NGS-derived contigs. This strategy was successfully applied to the genome sequencing of microbial cell factories requiring a complete gene list for the metabolic pathway, and a regulatory network for the design and development of industrial strains. Recently, software developments have facilitated de novo genome assembly which can produce genome scaffolds from short reads only. Automatic gap closing, which incorporates paired-end short reads into preexisting scaffolds, is also feasible. High-throughput multiplexed genome sequencing based on the Illumina platform has become a routine task for genome prospecting and the comparative genomic analysis of useful microbial strains. We also carried out the resequencing of E. coli strains generated from a combined approach of a long-term experimental evolution and proton beam-induced mutagenesis. NGS-based resequencing allows the identification of genetic variations from multiple samples at a lower cost, and the tracing of evolutionary pathways that are engraved in the genomic sequences. NGS-based transcriptome sequencing (RNA-seq), which is becoming popular as a substitute for traditional microarray experiments, also provides evidence for the identification of genes from novel genomes. Recent achievements in genome sequencing and analysis of eukaryotic microbes will also be introduced.

Since the Bovine sequencing and HapMap projects, there have been millions of genetic variations particularly including single nucleotide polymorphisms (SNPs) available throughout the cattle genome. While cataloguing substantial amounts of SNPs, copy number variation (CNV) has recently become getting great attention as an another form of genetic variation which is extensively distributed across the cattle genome. As it has been well known that CNVs can account for substantial proportions of phenotypic variance in other species including human, CNVs are highly expected to explain genetic variations of diverse economically important traits in cattle. Currently, two main approaches such as hybridization-based microarray and massively parallel sequencing-based method have been successfully applied to detect CNVs throughout the cattle genome. Although there is obvious difference of capabilities to detect genome-wide CNVs at fine scales between different platforms applied, all currently completed investigations exhibited that CNVs are extensively spread throughout the cattle genome as observed in other species. Despite these successes, there are still severe lacks of researches to identify multiple levels of CNVs from diverse cattle or multiple individuals, suggesting that there are a number of CNVs remained undiscovered. Furthermore, there are almost few investigations available to elucidate how each CNV is associated with traits of interest in cattle, so such functional study should be followed along with saturating most of potential CNVs on cattle genome.

Live offspring is obtained from in vitro production of porcine embryos, but the procedure is still associated with great inefficiencies. In mammalian oocytes, acquisition of meiotic competence coincides with a decrease in general transcriptional activity at the end of the oocyte growth phase. In this study, we investigated the expression and sub-cellular localization of positive transcription elongation factor P-TEFb (CDK9/Cyclin T1), a RNA polymerase II CTD kinase during pig oocyte growth and early embryonic development. Localization and expression of components involved in mRNA and rRNA transcription were assessed by immunocytochemistry in growing and fully-grown oocytes. In addition, meiotic resumption, germinal vesicle breakdown, nuclear transcription and embryonic genome activation (EGA) were analyzed in oocytes and embryos cultured in presence of a potent CDK9 inhibitor, flavopiridol. Our analyses, demonstrated that CDK9 became co- localized partially with phosphorylated Pol II CTD and mRNA splicing complexes. Surprisingly, CDK9 was co-localized with Pol I-specific transcription factor, UBF, and gradually localized in nucleolar peripheries at the final steps of oocyte growth. Later, CDK9 became associated with nucleolar structures at 4-cell stage. Treatment with flavopiridol resulted in arrest in meiotic resumption, germinal vesicle breakdown as well as a decline in global transcription. Flavopiridol also inhibited embryo development beyond EGA. All together, these data suggest that CDK9 has a dual role in both Pol I- and Pol II-dependent transcription in pig oocyte growth and embryonic development.

In the transcriptome surveys of Laodelphax striatellus, several cDNA sequences showed a high level of similarities to the insect picorna-like virus genomes. Interestingly, there was no sequence similarity between picorna-like virus sequences from the RSV-viruliferous and those from the non-viruliferous L. striatellus. Picorna-like virus from the non-viruliferous L. striatellus was a geographical isolate of Himetobi P virus (HiPV). The genome of the HiPV was 9,272 nt in length excluding the poly(A) tail and contained two open reading frames (ORFs), which were separated by a 176 nt intergenic region that functions as an internal ribosome entry site (IRES). The 5' ORF encodes the non-structural proteins and the 3' ORF encodes the capsid proteins. The partial genomic RNA of the picorna-like virus from the RSV-viruliferous L. striatellus, LsPV-2, was 8,769 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 1–8,535) encoding a 2,845 aa polyprotein. In terms of sequence similarity, identity, and genome organization, LsPV-2 resembled insect picornalike viruses belonging to the family Iflaviridae. A phylogenetic analysis based on RNA-dependent RNA polymerase (RdRp) sequence showed that LsPV-2 was most closely related to the deformed wing virus (DWV). The HiPV and LsPV-2 were incompatible each other in L. striatellus, suggesting that these two picorna-like viruses may have important functions in transmission of the RSV.

The Diadegma fenestrale was known as parasitoid on potato tuber moth, Phthorimaea operculella and diamondback moth, Plutella xylostella. This species, genus Diadegma are first reported from Korea. DfIV showed typical ichnovirus shape which two membranes surround virus capsids. The genome contents of DfIV consist about sixteen double-stranded DNA segments ranging 2 to 6 kb. To identify DfIV genes, whole genome sequencing based on GS-FLX was conducted using purified total viral DNA extracted from D. fenestrale calyx. About sixty ORFs were analyzed and several typical polydna virus gene family detected such as cys-motif, rep, vinnexin and vankyrin. This is the first report of DfIV and these lepidopteran host immune suppression genes will be deeply identified.

The black-veined white, Aporia crataegi (Lepidoptera: Papilionoidea), is nearly extinct in South Korea, although substantial numbers of dried specimens are available. One of the common practices for such species is to launch re-introduction program after proper amount of genetic information are analyzed from donor and donee populations. In this study, we sequenced complete mitochondrial genome (mitogenome) of A. crataegi to design species-specific primers for subsequent population works and to further understand the mitogenome evolution in lepodiopteran Papilionoidea. The 15,140-bp long A. crataegi mitogenome that has typical sets of 37 genes is smallest among true butterfly species with overall slightly smaller size in genes and regions throughout the genome. Arrangement of the genome is identical to those of other lepidopteran mitogenomes, in which tRNA cluster located between the A+T-rich region and ND2 gene is translocated into tRNAMet, tRNAIle, and tRNAGln from ancestral arrangement, tRNAIle, and tRNAGln, tRNAMet. The A/T content of the genome at 81.3% is the highest in Pieridae, but lower than that of lycaenid species (81.7% ~ 82.7%) The high A/T content in the genome is also reflected in codon usage, accounting for 41.69% of A/T-composed codons (TTA, ATT, TTT, and ATA). Unlikely the diversified or modified usage of anticodon for tRNASer(AGN) the species of Pieridae including A. crataegi all unanimously have GCT that has been hypothesized as ancestral for Lepidoptera. A total of 111 bp of non-coding sequences are dispersed in 13 regions, ranging in size from 1–49 bp. Among them relatively longer ones (≥ 16 bp) all have relatively higher sequence identity to other regions of the genome, suggesting partial duplication of the sequences during A. crataegi evolution. As has been reported in some species of Lepidoptera, the A. crataegi A+T-region also has typically found conserved sequences (e.g., poly-T stretch, ATAGA motif, ATTTA element, microsatellite-like A/T sequence, and poly-A stretch) and one tRNA-like sequence, and this feature was commonly found in true butterfly species.

Hz-2V, which belongs to nudiviridae, is a sexually transmitted insect virus of corn earworm, Helicoverpa zea. Hz-2V is transmitted during mating or mating attempt of infected individuals, and specifically replicates in the reproductive tissues to cause abnormal development of testes and ovary in the adult moths of next generations. The malformation of Hz-2V reproductive tissues started during the early pupal stage without clear sign of virus replication. The virus replication started at the late pupal stage to cause sterility of the emerged moths. Interestingly, the infected female moths showed a unique pathology, so called ‘waxy-plug’, which is filled with virus particles, and abnormal mating behavior while they produced 6~7 times more of pheromone than the normal female moths did. To investigate the factors of Hz-2V which control the physiological, and behavioral changes of the infected moths, the whole genome sequence of Hz-2V was determined. Bioinformatics analysis demonstrated that Hz-2V contains 113 putative ORFs including juvenile hormone esterase (JHE) and histone binding protein homologues, and a miRNA candidate which probably controls the expression of viral JHE.

The complete mitogenome (20,456 bp) of Challia fletcheri (Dermaptera: Pygidicranidae) as the first dermapteran insect is the longest among sequenced insects. The genome contained typical gene sets, but harbored the largest TRU among Exopterygota and Palaeoptera. The AT- and GC-skewness showed more Ts and Gs encoded on the major strand, whereas more As and Cs on the minor strand, presenting a reversal to the general pattern found in most insect mitogenomes. This pattern was explained in terms of inversion of replication origin. The gene arrangement of C. fletcheri genome is unique in insects and differs from the ancestral type found in insects by a series of gene translocations and/or inversions. We hypothesize that the markedly different gene arrangement is probably due to some unique organism-level properties, which allow relaxed selection against mitochondrial gene rearrangement. All phylogenetic analyses consistently placed Orthoptera as the sister to the group composed of a monophyletic Isoptera + Mantodea + Blattodea and a monophyletic Grylloblattodea + Mantophasmatodea + Phasmatodea, and placed Dermaptera as the sister to Plecoptera, leaving them as the most basal lineage of Polyneoptera.

The Samia cynthia ricini (Lepidoptera: Saturniidae) is a commercial silk-producing insect belonging to an insect family Saturniidae in Bombycoidea. The species that has presumably been originated in India, is distributed in India, China, and Japan. Unlikely domestic silkworm the prime host plant for the species is a castor-oil plant (Ricinus communis in Euphorbiaceae). Recently, the eri-silkworm also is reared in Korea and is expected to be utilized for a diverse purpose. In this report, we present the complete mitochondrial genome of the species with the emphasis of a few major characteristics. The 15,384-bp long S. cynthia ricini (Lepidoptera: Saturniidae) mitochondrial genome was amplified into three long overlapping fragments (from COI ~ ND4, ND5 ~ lrRNA, and lrRNA ~ COI) and subsequent several short fragments using the long fragments as temperate. The primers for both long and short fragments were designed solely for lepidopteran genomes, without any species-specific primers. As a usual the genome is composed of 37 genes: 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes, and one large non-coding region termed the A+T-rich region. Arrangement of the genome is identical to those of other lepidopteran mitochondrial genome, but this differs from the common arrangement found in a diverse insect order, by the movement of tRNAMet to a position 5’- up stream of tRNAIle. Unlikely previous report on the start codon for COI gene in Lepidoptera S. cynthia ricini COI gene starts with typical ATT codon located between tRNATyr and the beginning region of COI gene. The 22 tRNAs that are interspersed throughout the mitogenome ranged in length from 62 to 71 bp. All tRNAs but tRNASer(AGN) were shown to be folded into the expected cloverleaf secondary structures. More detailed structural and phylogenetic analyses among Bombycidae and Saturniidae in connection with other families in the Bombycoidea will be performed soon

The complete mitochondrial genome sequence of the nerippe fritillary butterfly, Argynnis nerippe, which is listed as an endangered species in Korea, is described with an emphasis on the A+T-rich region. The 15,140-bp long circular molecule consisted of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one control region, known in insect as the A+T-rich region, as found in typical metazoans. The 329-bp long A+T-rich region located between srRNA and tRNAMet possessed the highest A/T content (95.7%) than any other region of the genome. Along with the several conserved sequences found typically in the lepidopteran insects the genome contained one tRNAMet-like and tRNALeu(UUR) -like sequence in the A+T-rich region.

The differences in the immune response between body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, were regarded as primary factors determining their differential vector competence. To find any differences in genetic components in immune system between body and head lice, whole genome sequences of head lice were determined by both SBS [sequencing by synthesis, Illumina Genome Analyzer (Illumina-GA)] and pyrosequencing (Roche GS FLX), and compared with the reference genome sequences of body lice. The short DNA reads from Illumina-GA (an average mapping depth of 50-fold) were aligned first to the body louse reference genome, to which Roche GS FLX DNA reads (an average depth of 2.5-fold) were subsequently assembled to make up gaps between mapped consensus. Total consensus showed a size of 114 Mb and a coverage of 96% of the published body louse genome sequences. From this head louse genome sequences, a total of 12,651 genes were predicted and used for comparing with the 10,775 genes previously reported from the body louse genome. The homolog analysis identified 873 head louse-specific genes and 422 body lice-specific genes. Comparison of immune response genes between both louse species showed head lice have more number of immune-related genes than body lice. Head lice were determined to possess all of the 107 immune-related genes reported in the previous study (Kim et al., 2011), suggesting that there is no difference in genetic make-up in terms of the 107 immune-related genes between body and head lice.

The four genetically distinct isolates have been identified previously from Bombyx mori nucleopolyhedroviruses (BmNPVs) isolated in Korea. To further understand the complex of viruses infecting Bombyx mori, the genome of BmNPV-K1 and K4 strains was completely sequenced and analyzed in comparison with the genome of other sequenced baculoviruses including previously reported BmNPV. BmNPV-K1 consisted of 127,542 bp and 133 open reading frames (ORFs) of 150 nucleotides or longer with minimal overlap have been identified. In contrast, BmNPV-K4 consisted of 128,615 bp and 134 open reading frames (ORFs). Although gene arrangement is virtually identical, the genome of BmNPV-K4 is 1,073 bp longer than BmNPV-K1. This was related to the more existence of bro genes in BmNPV-K4. To investigate the relationship between BmNPV-K1 and K4, phylogenetic analysis with each member of the paired ORFs was performed. The sequence data suggest that BmNPVK1 and BmNPV-K4 are closely related but have diverged and evolved into two separate strains. This was study to identify highly related but separately evolving viruses in the same insect host and geographic location. We are currently comparing the differences of these BmNPV genomes to elucidate characteristics of each virus.