Molecular phylogenetic and dating analysis of pierid butterfly species using complete mitochondrial genomes.

Pieridae is a butterfly family whose evolutionary history is poorly understood. Due to the difficulties in identifying morphological synapomorphies within the group and the scarcity of the fossil records, only a few studies on higher phylogeny of Pieridae have been reported to date. In this study, we describe the complete mitochondrial genomes of four pierid butterfly species (Aporia martineti, Aporia hippia, Aporia bieti, and Mesapia peloria), in order to better characterize the pierid butterfly mitogenomes and perform the phylogenetic analyses using all available mitogenomic sequence data (13PCGs, rRNAs, and tRNAs) from the 18 pierid butterfly species comprising the three main subfamilies (Dismorphiinae, Coliadinae and Pierinae). Our analysis shows that the four new mitogenomes share similar features with other known pierid mitogenomes in gene order and organization. Phylogenetic analyses by maximum likelihood and Bayesian inference show that the pierid higher-level relationship is: Dismorphiinae + (Coliadinae + Pierinae), which corroborates the results of some previous molecular and morphological studies. However, we found that the Hebomoia and Anthocharis make a sister group, supporting the traditional tribe Anthocharidini; in addition, the Mesapia peloria was shown to be clustered within the Aporia group, suggesting that the genus Mesapia should be reduced to the taxonomic status of subgenus. Our molecular dating analysis indicates that the family Pieridae began to diverge during the Late Cretaceous about 92 million years ago (mya), while the subfamily Pierinae diverged from the Coliadinae at about 86 mya (Late Cretaceous).

Expressed sequence tag analysis of functional genes associated with adventitious rooting in Liriodendron hybrids.

Liriodendron hybrids (Liriodendron chinense x L. tulipifera) are important landscaping and afforestation hardwood trees. To date, little genomic research on adventitious rooting has been reported in these hybrids, as well as in the genus Liriodendron. In the present study, we used adventitious roots to construct the first cDNA library for Liriodendron hybrids. A total of 5176 expressed sequence tags (ESTs) were generated and clustered into 2921 unigenes. Among these unigenes, 2547 had significant homology to the non-redundant protein database representing a wide variety of putative functions. Homologs of these genes regulated many aspects of adventitious rooting, including those for auxin signal transduction and root hair development. Results of quantitative real-time polymerase chain reaction showed that AUX1, IRE, and FB1 were highly expressed in adventitious roots and the expression of AUX1, ARF1, NAC1, RHD1, and IRE increased during the development of adventitious roots. Additionally, 181 simple sequence repeats were identified from 166 ESTs and more than 91.16% of these were dinucleotide and trinucleotide repeats. To the best of our knowledge, the present study reports the identification of the genes associated with adventitious rooting in the genus Liriodendron for the first time and provides a valuable resource for future genomic studies. Expression analysis of selected genes could allow us to identify regulatory genes that may be essential for adventitious rooting.

Genetic analyses of the major and minor locus groups of bacterial wilt resistance in tobacco using a diallel cross design.

Tobacco germplasm samples with various levels of resistance to bacterial wilt were selected to construct F1 combinations of parental inbred lines and orthogonal diallel crosses using samples collected in 2009 (15 germplasms), 2010 (15 germplasms), and 2011 (16 germplasms). A total of 1/2P (P + 1) experimental materials were used for analysis. Based on the analyses of major and minor locus groups, genetic effects on the incidence rate and index of bacterial wilt in tobacco were investigated on the 15th and 25th day during the early stage. Significant effects were observed in major locus groups, but not in minor locus groups. Specifically, adjacent major locus groups (J1 = 13,056 and J1 = 13,055; J1 = 14,080 and J1 = 14,079) were detected in both the first and second analyses with considerable effects. Based on the additive effects of minor locus groups on the rate and index of bacterial wilt, the effects on the incidence rates of Yunyan 85, DB101, and RG11 as well as the effects on the disease index of the latter two germplasms reached the maximum. This was consistent with the disease resistance indicators of these tobacco varieties in the field (corresponding broad heritability >20%). Genetic homozygous dominant loci (+ +) increased the rate of bacterial wilt (susceptible), whereas homozygous recessive loci (- -) reduced the index of bacterial wilt (resistant) with considerable additive effects and low dominant effects, suggesting that the inheritance of the bacterial wilt rate and index in tobacco mainly relies on additive inheritance.

Mating system patterns of natural populations of Pinus koraiensis along its post-glacial colonization route in northeastern China.

To understand the genetic mechanisms underlying the endangerment of Pinus koraiensis, we studied the mating system of 49 families of this species in 3 natural populations along its post-glacial colonization route across ~1500 km in northeastern China using the chloroplast simple sequence repeat technique. We analyzed 11 polymorphic loci with clear and repeating bands, and we calculated the multi-locus outcrossing rate (tm), single-locus outcrossing rate, inbreeding index, and fixation index (F). Intra-population variation was not observed, but a large inter-population variation was observed in the outcrossing rate, and the tm increased from 0.767 (the south population) to 0.962 (the north population) along the post-glacial colonization route. The tm values within a population did not change with time over 2 consecutive years. The F values for the 3 populations were <0, which indicates an excess of heterozygotes. The mean effective number of alleles, Shannon diversity index, and Nei's genetic diversity index did not show a south-north pattern. The north population had the highest outcrossing rate but the lowest genetic diversity. The average genetic differentiation of P. koraiensis populations was 0.1251, which was within the average range of woody plants with outcrossing and wind pollination. This study suggests that the current endangerment of P. koraiensis is not related to its genetic structure; perhaps it is mainly caused by man-made and natural disturbances such as deforestation and fire. Therefore, reducing disturbances and enhancing habitats, rather than the genetic aspects, play more important roles in the long-term protection of P. koraiensis.

Complete mitochondrial genome sequence of Cheirotonus jansoni (Coleoptera: Scarabaeidae).

We sequenced the complete mitochondrial genome (mitogenome) of Cheirotonus jansoni (Coleoptera: Scarabaeidae), an endangered insect species from Southeast Asia. This long legged scarab is widely collected and reared for sale, although it is rare and protected in the wild. The circular genome is 17,249 bp long and contains a typical gene complement: 13 protein-coding genes, 2 rRNA genes, 22 putative tRNA genes, and a non-coding AT-rich region. Its gene order and arrangement are identical to the common type found in most insect mitogenomes. As with all other sequenced coleopteran species, a 5-bp long TAGTA motif was detected in the intergenic space sequence located between trnS(UCN) and nad1. The atypical cox1 start codon is AAC, and the putative initiation codon for the atp8 gene appears to be GTC, instead of the frequently found ATN. By sequence comparison, the 2590-bp long non-coding AT-rich region is the second longest among the coleopterans, with two tandem repeat regions: one is 10 copies of an 88-bp sequence and the other is 2 copies of a 153-bp sequence. Additionally, the A+T content (64%) of the 13 protein-coding genes is the lowest among all sequenced coleopteran species. This newly sequenced genome aids in our understanding of the comparative biology of the mitogenomes of coleopteran species and supplies important data for the conservation of this species.

Complete mitochondrial genomes of the Bright Sunbeam Curetis bulis and the Small Copper Lycaena phlaeas (Lepidoptera: Lycaenidae) and their phylogenetic implications.

In this study, the complete mitochondrial genomes of Curetis bulis and Lycaena phlaeas were determined and analyzed. The circular genomes are 15,162 bp long for C. bulis and 15,280 bp long for L. phlaeas, with a total A+T content of 82.6 and 83.1%, respectively. Both mitogenomes contain 37 genes, and their gene orders are similar to those of other lepidopterans. All protein-coding genes (PCGs) are initiated by ATN codons, except for cox1, which is started with the CGA codon; all PCGs terminate in the typical stop codon TAA, except for cox1, cox2, and nad4, which end with a single T. The codons TTA (Leu), ATT (Ile), TTT (Phe), ATA (Met), and AAT (Asn) appear the most frequently. Both of the mitogenome A+T-rich regions harbor the motif ATAGA, followed by a 19-bp poly(T) stretch, with C. bulis containing a microsatellite-like (AT)5 element next to the ATTTA motif, and L. phlaeas containing a microsatellite-like (TA)6 (AT) element next to the ATTTA motif. The phylogenetic trees of the 17 representative butterfly species, including the two species of this study, were reconstructed with the maximum likelihood and Bayesian inference methods, based on the 13 PCG nucleotide sequence data. The results of the phylogenetic analyses strongly supported the relationships of ((((Lycaenidae + Pieridae) + Nymphalidae) + Hesperiidae) + Papilionidae), which was markedly different from the traditional morphological view of the Lycaenidae and Nymphalidae considered to be sisters of each other.

Complete sequence of the mitochondrial genome of the Japanese buff-tip moth, Phalera flavescens (Lepidoptera: Notodontidae).

We sequenced the complete mitochondrial genome of Phalera flavescens. The mitogenome is 15,659 bp in length, including 13 protein-coding genes (atp6, atp8, cox1-3, nad1-6, nad4L, cob), two ribosomal RNAs (rrnS and rrnL), 22 transfer RNAs and an AT-rich region, a putative control region (D-loop). Gene order and orientation were found to be identical to those of other completely sequenced lepidopteran mitogenomes. All 13 protein-coding genes start with the common codon ATN, except for the cox1 gene, which uses CGA as the initial codon. Nine of the 13 protein-coding genes stop with codon TAA, while the cox1, cox2, nad5, and nad4 genes stop with the single nucleotide T. All tRNA genes can be folded into canonical cloverleaf secondary structure, except for trnS1, which loses the ''DHU'' arm. Six overlapping sequences totaling 20 bp (1-8 bp for each sequence) and 16 intergenic spacer sequences, totaling 276 bp (1-58 bp for each sequence) are scattered throughout the genome; the largest intergenic spacer is located between the trnQ and nad2 genes. A microsatellite-like structure (AT)(6)ACC(AT)(6) and 16-bp poly-T elements preceded by the ATTTA motif are present in the D-loop region. Additionally, unexpectedly, an extra 190-bp insertion, with unknown function, was found in the small subunit rRNA gene (rrnS); this gene is the longest known (1020 bp) among all of the Lepidoptera.