Complete Plastid Genome Sequence of the Brown Alga Undaria pinnatifida

In this study, we fully sequenced the circular plastid genome of a brown alga, Undaria pinnatifida. The genome is 130,383 base pairs (bp) in size; it contains a large single-copy (LSC, 76,598 bp) and a small single-copy region (SSC, 42,977 bp), separated by two inverted repeats (IRa and IRb: 5,404 bp). The genome contains 139 protein-coding, 28 tRNA, and 6 rRNA genes; none of these genes contains introns. Organization and gene contents of the U. pinnatifida plastid genome were similar to those of Saccharina japonica. There is a co-linear relationship between the plastid genome of U. pinnatifida and that of three previously sequenced large brown algal species. Phylogenetic analyses of 43 taxa based on 23 plastid protein-coding genes grouped all plastids into a red or green lineage. In the large brown algae branch, U. pinnatifida and S. japonica formed a sister clade with much closer relationship to Ectocarpus siliculosus than to Fucus vesiculosus. For the first time, the start codon ATT was identified in the plastid genome of large brown algae, in the atpA gene of U. pinnatifida. In addition, we found a gene-length change induced by a 3-bp repetitive DNA in ycf35 and ilvB genes of the U. pinnatifida plastid genome.     

Characterization of the mating-type genes in Leptographium procerum and Leptographium profanum

Leptographium procerum and the closely related species Leptographium profanum, are ascomycetes associated with root-infesting beetles on pines and hardwood trees, respectively. Both species occur in North America where they are apparently native. L. procerum has also been found in Europe, China New Zealand, and South Africa where it has most probably been introduced. As is true for many other Leptographium species, sexual states have never been observed in L. procerum or L. profanum. The objectives of this study were to clone and characterize the mating type loci of these fungi, and to develop markers to determine the mating types of individual isolates. To achieve this, a partial sequence of MAT1-2-1 was amplified using degenerate primers targeting the high mobility group (HMG) sequence. A complete MAT1-2 idiomorph of L. profanum was subsequently obtained by screening a genomic library using the HMG sequence as a probe. Long range PCR was used to amplify the complete MAT1-1 idiomorph of L. profanum and both the MAT1-1 and MAT1-2 idiomorphs of L. procerum. Characterization of the MAT idiomorphs suggests that the MAT genes are fully functional and that individuals of both these species are self-sterile in nature with a heterothallic mating system. Mating type markers were developed and tested on a population of L. procerum isolates from the USA, the assumed center of origin for this species. The results suggest that cryptic sexual reproduction is occurring or has recently taken place within this population.     

Comparative Assessment of 5′ A/T-Rich Overhang Sequences with Optimal and Sub-optimal Primers to Increase PCR Yields and Sensitivity

Efficient PCR amplifications require precisely designed and optimized oligonucleotide primers, components, and cycling conditions. Despite recent software development and reaction improvement, primer design can still be enhanced. The aims of this research are to understand (1) the effect on PCR efficiency and DNA yields of primer thermodynamics parameters, and (2) the incorporation of 5′ A/T-rich overhanging sequences (flaps) during primer design. Two primer sets, one optimal (ΔG = 0) and one sub-optimal (ΔG = 0.9), were designed using web interface software Primer3, BLASTn, and mFold to target a movement protein gene of Tobacco mosaic virus. The optimal primer set amplifies a product of 195 bp and supports higher PCR sensitivity and yields compared to the sub-optimal primer set, which amplifies a product of 192 bp. Greater fluorescence was obtained using optimal primers compared to that with sub-optimal primers. Primers designed with sub-optimal thermodynamics can be substantially improved by adding 5′ flaps. Results indicate that even if the performance of some primers can be improved substantially by 5′ flap addition, not all primers will be similarly improved. Optimal 5′ flap sequences are dependent on the primer sequences, and alter the primer’s T _m value. The manipulation of this feature may enhance primer’s efficiency to increase the PCR sensitivity and DNA yield.     

Cloning and characterization of hemerythrin gene from Sipuncula Phascolosoma esculenta

Hemerythrin is a non-heme respiratory protein involved in oxygen storage and transport in invertebrates. In the present study, the hemerythrin cDNA was cloned from Phascolosoma esculenta (denoted as PeHr) by PCR and rapid amplification of cDNA ends approaches. The full-length PeHr consisted of 770 bp containing of a 5′-terminal untranslated region (UTR) of 83 bp, a 3′-terminal UTR of 327 bp, and a coding domain sequence of 360 bp encoding a polypeptide of 120 amino acids with estimated molecular mass of 13.6 kDa and theoretical isoelectric point of 5.78. The expression profiles of PeHr were evaluated by real-time RT-PCR under blood loss stress. The expression level of PeHr was significantly up-regulated from 45 to 48 h, then slightly recovered to its original level. The coding sequence of the PeHr was cloned and expressed in Escherichia coli BL21 (DE3) for antibodies preparation. Western blotting analysis conformed that the generated antibodies could specifically identify not only recombinant product, but also native protein from the total protein extraction. Our results indicated that PeHr might be involved into haemocytes regeneration, and its function roles should be further investigated by the generated antibodies.     

Complete Plastid Genome of the Brown Alga Costaria costata (Laminariales,Phaeophyceae)

Costaria costata is a commercially and industrially important brown alga. In this study, we used next-generation sequencing to determine the complete plastid genome of C. costata. The genome consists of a 129,947 bp circular DNA molecule with an A+T content of 69.13% encoding a standard set of six ribosomal RNA genes, 27 transfer RNA genes, and 137 protein-coding genes with two conserved open reading frames (ORFs). The overall genome structure of C. costata is nearly the same as those of Saccharina japonica and Undaria pinnatifida. The plastid genomes of these three algal species retain a strong conservation of the GTG start codon while infrequently using TGA as a stop codon. In this regard, they differ substantially from the plastid genomes of Ectocarpus siliculosus and Fucus vesiculosus. Analysis of the nucleic acid substitution rates of the Laminariales plastid genes revealed that the petF gene has the highest substitution rate and the petN gene contains no substitution over its complete length. The variation in plastid genes between C. costata and S. japonica is lower than that between C. costata and U. pinnatifida as well as that between U. pinnatifida and S. japonica. Phylogenetic analyses demonstrated that C. costata and U. pinnatifida have a closer genetic relationship. We also identified two gene length mutations caused by the insertion or deletion of repeated sequences, which suggest a mechanism of gene length mutation that may be one of the key explanations for the genetic variation in plastid genomes.     

Isolation and characterization of a C-repeat binding factor (CBF)-like gene in cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is a tropical and subtropical plant and susceptible to chilling injury. In this research, a C-repeat binding factor (CBF)-like gene (GenBank accession number JQ339740) has been isolated from cassava, and named as MeCBF1. The full-length DNA of MeCBF1 is 1,037 base pair (bp), without intron. The 5′ untranslated region is 102 bp, the 3′ untranslated region is 239 bp, and the open reading frame is 696 bp encoding 231 amino acids. The deduced amino acid sequence of MeCBF1 contains two CBF conserved motifs of PKK(P/R)AGRxKFxETRHP and DSxWR. The MeCBF1 shows 83 % homology to the CRT/DRE binding factor 1 from Hevea brasiliensis (Accession no. AAY43213.1). However, in cassava, the MeCBF1 target genes showed low similarity to the CBF/DREB regulated genes in Arabidopsis thaliana. Quantitative real-time PCR showed that the MeCBF1 was highly expressed in stems and leaves, and lowly expressed in roots. In addition, the expression of the MeCBF1 quickly responded to low temperature stress (4 °C). These results suggest that, the MeCBF1 is functional in cassava. Further studies on the MeCBF1 might be helpful to reveal molecular mechanism of cassava’s high sensitivity to low temperature.     

Heterothallism revealed in the root rot fungi Berkeleyomyces basicola and B. rouxiae

Berkeleyomyces basicola and Berkeleyomyces rouxiae, two sister species previously treated collectively as Thielaviopsis basicola, reside in the Ceratocystidaceae (Microascales, Ascomycota). Both species are important root pathogens of many important agricultural crops and ornamental plants. Although T. basicola has been known for more than 150y, a sexual state has never been found and it has been assumed to be an asexual pathogen. The aim of this study was to determine the mating strategy of the two Berkeleyomyces species. Investigation of the genome sequences of two B. basicola isolates allowed for the complete characterization of the MATlocus, revealing that it has a typical heterothallic mating system with the MAT1-1andMAT1-2 idiomorphs occurring in different isolates. PCR amplification using mating type primers developed in this study, showed that the MAT1-1-1andMAT1-2-1 genes were also present in different isolates of B. rouxiae. Pairing of isolates representing the two mating types of both species,using a variety of techniques failed to produce sexual structures. Although we have found no direct evidence that they reproduce sexually, these fungi are clearly heterothallic with both mating types occurring in some countries suggesting that a cryptic sexual cycle could exist for them.     

Structure and function of the mating-type locus in the homothallic ascomycete, Didymella zeae-maydis

Homothallic Didymella zeae-maydis undergoes sexual reproduction by selfing. Sequence analysis of the mating type (MAT) locus from this fungus revealed that MAT carries both MAT1-1-1 and MAT1-2-1 genes found in heterothallic Dothideomycetes, separated by ～1.0 kb of noncoding DNA. To understand the mechanistic basis of homothallism in D. zeae-maydis, each of the MAT genes was deleted and the effects on selfing and on ability to cross in a heterothallic manner were determined. The strain carrying an intact MAT1-1-1 but defective MAT1-2-1 gene (MAT1-1-1;ΔMAT1-2-1) was self-sterile, however strains carrying an intact MAT1-2-1 but defective MAT1-1-1 gene (ΔMAT1-1-1;MAT1-2-1), when selfed, showed delayed production of a few ascospores. Attempts to cross the two MAT deletion strains yielded fewer ΔMAT1-1-1;MAT1-2-1 than MAT1-1-1;ΔMAT1-2-1 progeny and very few ascospores overall compared to WT selfs. This study demonstrates that, as in the other homothallic Dothideomycetes, both MAT genes are required for full fertility, but that, in contrast to other cases, the presence of a single MAT1-2-1 gene can induce homothallism, albeit inefficiently, in D. zeae-maydis.     

Illegal transposition of mating-type genes in yeast

Mating-type switching in the yeast Saccharomyces cerevisiae involves the transposition of a copy of a or α information from unexpressed “library” genes, HML or HMR, to replace the sequence at the mating type locus, MAT. In normal homothallic strains, where conversions of MAT may occur as often as every cell division, the switching of MAT alleles does not alter the alleles at HML or HMR. We have discovered that several mutations within or adjacent to MAT that impair the excision of the MAT allele permit conversions of the alleles at HML or HMR in more than 1% of the cells analyzed. The two mutations within the MAT locus (MATa-inc and MATα-inc) can transpose to HML or HMR without being lost at MAT. Thus a MATα-inc HMLα HMRa HO strain can switch to MATα-inc HMLα HMRα-inc HO. Even though the α-inc and a-inc alleles prevent their own replacement at MAT, these sequences are efficiently transposed back from HMLα-inc or HMLa-inc to replace normal MAT alleles. When these alleles reappear at MAT, they are again blocked in excision. Thus the sequences used to remove an allele from MAT must differ from those used to replicate and transpose it. Two cis-acting stk mutations adjacent to MAT that block switching of MATa to MATα also induce the conversion of HMLα to HMLa. However, we have previously shown that these events do not occur in strains carrying a recessive “switch” mutant (swi1) or in strains carrying a defective allele of the HO gene. In stk1 MATa HO strains, HMLα was converted to HMLa in approximately 4% of the subclones examined. In contrast, the HMLα-inc sequence was not converted in similar stk1 MATa HO strains. Thus the excision of the α-inc sequence seems to be prevented at both MAT and HML. These results suggest that the illegal conversions of HML and HMR occur by a mechanism similar to that used for normal conversions of MAT.     

Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.     

Identification and expression of the elongator protein 2 (Ajelp2) gene,a novel regeneration-related gene from the sea cucumber Apostichopus japonicus

Elongator proteins comprise six subunits (ELP1–ELP6) and form protein complexes. The elongator protein 2 gene (elp2) encodes a protein with a WD40 repeats domain that acts as a scaffold for complex assembly. It also plays an important role in growth and development. In this study, the full-length cDNA of elongator protein 2 (Ajelp2) was cloned from the sea cucumber Apostichopus japonicus (A. japonicus) using rapid amplification of cDNA ends PCR techniques and comprised 3,058 bp, including a 54 bp 5′ untranslated (UTR), a 526 bp 3′ UTR and a 2,478 bp open reading frame encoding a polypeptide of 825 amino acids. The Ajelp2 sequence showed high homology to 12 other species. The molecular weight and isoelectric of point the presumptive protein were 91.6 kDa and 5.84, respectively. In situ hybridization indicated that the gene is expressed in the body wall, intestine, respiratory tree and longitudinal muscle. The expression level of Ajelp2 increased in recovering of organs in sea cucumber and showed it’s the highest expression level at the 15th day in the intestine and respiratory tree. Its expression then gradually decreased to normal levels. In the body wall, the expression level of Ajelp2 was up-regulated and then down-regulated. These results indicated that Ajelp2 is involved in protein regulation during the regeneration process in the sea cucumber A. japonicus.