Genetic diversity of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariaceae,Rhodophyta) from west coast,Peninsular Malaysia based on mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 gene analysis

Mitochondrial cytochrome c oxidase subunit I (cox1) was employed to investigate the intraspecific genetic diversity of Gracilaria changii collected from various localities distributed along the west coast of Peninsular Malaysia. Gracilaria changii is an agarophyte with potential for commercialization in Malaysia as it has high yields of good quality agar with high gel strength for the production of food grade agar and agarose. The phylogeographic aspect of G. changii has not been studied despite its abundance and potential commercialization. In this study, six mitochondrial haplotypes (C1–C6) were revealed from 62 specimens varying by 0–3 bp over 923 bp. Results indicate that haplotype C1 is the common ancestor and the most widespread haplotype due to its prevalence in Morib, Gua Tanah, Middle Banks, Batu Besar, Batu Tengah, Sungai Pulai, and Kuala Sungai Merbok. In this study, Morib was suggested as contributing the highest intra-population diversity with the identification of three haplotypes. The mitochondrial marker cox1 is a highly divergent mitochondrial marker and is applicable for studies on species identification and assessment of genetic diversity of G. changii.     

Structure of reproductive apparatus of <Emphasis Type="Italic">Gracilaria/Gracilariopsis lemaneiformis</Emphasis> (Gracilariaceae,Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial development revealed the similar cytodynamic pattern followed by all the Gracilariaceae members developed from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released.     

Discovery,localization, and sequence characterization of molecular markers for the crown rust resistance genes <Emphasis Type="Italic">Pc38, Pc39</Emphasis>, and <Emphasis Type="Italic">Pc48</Emphasis> in cultivated oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L.)

Molecular markers for the crown rust resistance genes Pc38, Pc39, and Pc48 in cultivated oat (Avena sativa L.) were identified using near-isogenic lines and bulked segregant analysis. Six markers for Pc48, the closest being 6 cM away, were found in a Pendek-39 × Pendek-48 (Pendek3948) population, but none was found in a Pendek-48 × Pendek-38 (Pendek4838) population. Three markers for Pc39 were found in the Pendek3948 population, one of which cosegregated with the gene. This same marker was found to be 6 cM away from the gene in an OT328 × Dumont (OT328Du) population. Nine markers for Pc38 were found in the Pendek4838 population, eight of which are within 2 cM of the gene. One other marker for Pc38 was found in the OT328Du population; however, comparative mapping suggests that the Pc38 region in OT328Du is in a different location than that in Pendek4838. A number of markers unlinked to the genes under study formed linkage groups in both the Pendek3948 and Pendek4838 populations. Four of these show homology or homoeology to each other and to the Pc39 region in Pendek3948. Two RFLP clones closely linked to Pc38 code for a putative leucine-rich repeat transmembrane protein kinase and a cre3 resistance gene analogue. This study provides information to support molecular breeding in oat, and contributes to ongoing research into genomic regions associated with fungal pathogen resistance.     

DNA barcoding,phylogenetic relationships and speciation of snappers (genus <Emphasis Type="Italic">Lutjanus</Emphasis>)

The phylogenetic relationships of 13 snapper species from the South China Sea have been established using the combined DNA sequences of three full-length mitochondrial genes (COI, COII and CYTB) and two partial nuclear genes (RAG1, RAG2). The 13 species (genus Lutjanus) were selected after DNA barcoding 72 individuals, representing 20 species. Our study suggests that although DNA barcoding aims to develop species identification systems, it may also be useful in the construction of phylogenies by aiding the selection of taxa. Combined mitochondrial and nuclear gene data has an advantage over an individual dataset because of its higher resolving power.     

Development of SCAR markers linked to powdery mildew (<Emphasis Type="Italic">Uncinula necator</Emphasis>) resistance in grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L. and <Emphasis Type="Italic">Vitis</Emphasis> sp.)

Sequence-characterized amplified regions markers (SCARs) were developed from six randomly amplified polymorphic DNA (RAPD) markers linked to the major QTL region for powdery mildew (Uncinula necator) resistance in a test population derived from the cross of grapevine cultivars “Regent” (resistant) × “Lemberger”(susceptible). RAPD products were cloned and sequenced. Primer pairs with at least 21 nucleotides primer length were designed. All pairs were tested in the F1 progeny of “Regent” × “Lemberger”. The SCAR primers resulted in the amplification of specific bands of expected sizes and were tested in additional genetic resources of resistant and susceptible germplasm. All SCAR primer pairs resulted in the amplification of specific fragments. Two of the SCAR markers named ScORA7-760 and ScORN3-R produced amplification products predominantly in resistant individuals and were found to correlate to disease resistance. ScORA7-760, in particular, is suitable for marker-assisted selection for powdery mildew resistance and to facilitate pyramiding powdery mildew resistance genes from various sources.     

The expanded cattle <Emphasis Type="Italic">KIR</Emphasis> genes are orthologous to the conserved single-copy <Emphasis Type="Italic">KIR3DX1</Emphasis> gene of primates

Cattle are the only non-primate species for which expansion of the killer cell immunoglobulin-like receptor (KIR) genes has been reported. We analyzed cattle KIR sequences to determine their relationship to the two divergent lineages of primate KIR: one comprising the KIR3DX1 gene of unknown function, the second comprising all other primate KIR genes, which encode variable major histocompatibility complex class I receptors. Phylogenetics and analysis of repetitive elements shows that cattle KIR subdivide into the same two lineages as primate KIR. Unlike the primates, the lineage of variable and likely functional cattle KIR corresponds to the KIR3DX1 lineage of primate KIR, whereas the variable lineage of primate KIR is represented in cattle by one KIR gene and a related gene fragment.     

Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

Incongruence among mitochondrial,chloroplast and nuclear gene trees in <Emphasis Type="Italic">Pinus</Emphasis> subgenus <Emphasis Type="Italic">Strobus</Emphasis> (Pinaceae)

Introgression has been considered to be one of main factors leading to phylogenetic incongruence among different datasets at lower taxonomic levels. In the plants of Pinaceae, the mtDNA, cpDNA, and nuclear DNA (nrDNA) may have different evolutionary histories through introgression because they are inherited maternally, paternally and biparentally, respectively. We compared mtDNA, cpDNA, and two low-copy nrDNA phylogenetic trees in the genus Pinus subgenus Strobus, in order to detect unknown past introgression events in this group. nrDNA trees were mostly congruent with the cpDNA tree, and supported the recent sectional and subsectional classification system. In contrast, mtDNA trees split the members of sect. Quinquefoliae into two groups that were not observed in the other gene trees. The factors constituting incongruence may be divided into the following two categories: the different splits within subsect. Strobus, and the non-monophyly of subsect. Gerardianae. The former was hypothesized to have been caused by the past introgression of cpDNA, mtDNA or both between Eurasian and North American species through Beringia. The latter was likely caused by the chimeric structure of the mtDNA sequence of P. bungeana, which might have originated through past hybridization, or through a horizontal transfer event and subsequent recombination. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A PCR survey of <Emphasis Type="Italic">Hox</Emphasis> genes in the myzostomid <Emphasis Type="Italic">Myzostoma cirriferum</Emphasis>

Using degenerate primers, we were able to identify seven Hox genes for the myzostomid Myzostoma cirriferum. The recovered fragments belong to anterior class (Mci_lab, Mci_pb), central class (Mci_Dfd, Mci_Lox5, Mci_Antp, Mci_Lox4), and posterior class (Mci_Post2) paralog groups. Orthology assignment was verified by phylogenetic analyses and presence of diagnostic regions in the homeodomain as well as flanking regions. The presence of Lox5, Lox4, and Post2 supports the inclusion of Myzostomida within Lophotrochozoa. We found signature residues within flanking regions of Lox5, which are also found in annelids, but not in Platyhelminthes. As such the available Hox genes data of myzostomids support an annelid relationship. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of hybridization strains of <Emphasis Type="Italic">Porphyra</Emphasis> based on <Emphasis Type="Italic">rbc</Emphasis>L gene sequences

Two strains, which are the free-living conchocelis of Porphyra yezoensis Ueda and Porphyra haitanensis T. J. Chang et B. F. Zheng, and four “interspecific hybridization” strains of these two species are investigated. The rbcL genes of 11 samples were amplified and sequenced, each of which were about 1,400 bp with a good specificity. The results of pair-wise distance matrix and multi-alignment showed that pair-wise distance were small while homologous index was large between strains Y-2066, Y-k2001, and Y-H001. These two indexes showed the same level as the above between strains H-2001, Y-hyC₁, and Y-hyC₂; however, the distances were larger between three former strains and three latter strains. Cluster trees showed the same trend. Fertile strains appeared after 2 years of culture of unfertile interspecific hybridization conchocelis and were separated from that based on different colors. Our finding that these fertile strains can bear offspring is important for understanding the interspecies hybridization of Porphyra. Molecular analysis of the fertile strains based on rbcL gene showed maternal inheritance. We inferred that somatic recombination was one of the reasons making interspecific hybridization strains fertile.     

Conservation of Nuclear SSR Loci Reveals High Affinity of <Emphasis Type="Italic">Quercus infectoria</Emphasis> ssp. <Emphasis Type="Italic">veneris</Emphasis> A. Kern (Fagaceae) to Section <Emphasis Type="Italic">Robur</Emphasis>

Conservation of 16 nuclear microsatellite loci, originally developed for Quercus macrocarpa (section Albae), Q. petraea, Q. robur (section Robur), and Q. myrsinifolia, (subgenus Cyclobalanopsis) was tested in a Q. infectoria ssp. veneris population from Cyprus. All loci could be amplified successfully and displayed allele size and diversity patterns that match those of oak species belonging to the section Robur. At least in one case, limited amplification and high levels of homozygosity support the occurrence of “null alleles” caused by a possible mutation in the highly conserved primer areas, thus hindering PCR. The sampled population exhibited high levels of diversity despite the very limited distribution of this species in Cyprus and extended population fragmentation. Allele sizes of Q. infectoria at locus QpZAG9 partially match those of Q. alnifolia and Q. coccifera from neighboring populations. However, sequencing showed homoplasy, excluding a case of interspecific introgression with the latter, phylogenetically remote species. Q. infectoria ssp. veneris sequences at this locus were concordant to those of other species of section Robur, while sequences of Quercus alnifolia and Quercus coccifera were almost identical to Q. cerris.     

Molecular phylogeny of horsetails (<Emphasis Type="Italic">Equisetum</Emphasis>) including chloroplast <Emphasis Type="Italic">atpB</Emphasis> sequences

Equisetum is a genus of 15 extant species that are the sole surviving representatives of the class Sphenopsida. The generally accepted taxonomy of Equisetum recognizes two subgenera: Equisetum and Hippochaete. Two recent phylogenetical studies have independently questioned the monophyly of subgenus Equisetum. Here, I use original (atpB) and published (rbcL, trnL-trnF, rps4) sequence data to investigate the phylogeny of the genus. Analyses of atpB sequences give an unusual topology, with E. bogotense branching within Hippochaete. A Bayesian analysis based on all available sequences yields a tree with increased resolution, favoring the sister relationships of E. bogotense with subgenus Hippochaete.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Variability of the Mitochondrial SSU rDNA of <Emphasis Type="Italic">Nomuraea</Emphasis> Species and Other Entomopathogenic Fungi from Hypocreales

Hypocrealean arthropod pathogenic fungi have profound impact on the regulation of agricultural and medical pests. However, until now the genetic and phylogenetic relationships among species have not been clarified, such studies could clarify host specificity relationships and define species boundaries. Our purpose was to compare the sequences of the mitochondrial SSU rDNA fragments from several mitosporic entomopathogenic Hypocreales to infer relationships among them and to evaluate the possibility to use these sequences as species diagnostic tool in addition to the more commonly studied sequences of nuclear SSU rDNA. The SSU mt-rDNA proved to be useful to help in differentiation of species inside several genera. Clusters obtained with Parsimony, Bayesian, and Maximum Likelihood analyses were congruent with a new classification of the Clavicipitaceae (Sung et al. Stud Mycol. 2007;57:5-59) in which the anamorphic genera Nomuraea and Metarhizium species remain in the Clavicipitaceae and Isaria species sequenced here are assigned to the family Cordycipitaceae. Mitochondrial genomic information indicates the same general pattern of relationships demonstrated by nuclear gene sequences.     

Molecular phylogenetic analysis of key <Emphasis Type="Italic">Jatropha</Emphasis> species inferred from nrDNA ITS and chloroplast (<Emphasis Type="Italic">trn</Emphasis>L-F and <Emphasis Type="Italic">rbc</Emphasis>L) sequences

The genus Jatropha (Euphorbiaceae) contains species that are of significant economic and ornamental value. However, Jatropha breeding material is rather limited due to incomplete information regarding phylogenetic relationships among germplasm resources. Phylogenetic analyses were performed based on the internal transcribed spacer of nuclear ribosomal DNA (nrDNA ITS), two chloroplast regions (trnL-F and rbcL), and the combined (ITS+trnL-F+rbcL) dataset among twenty-five specimens representing six key Jatropha species. Phylogenetic relationships of Jatropha were well resolved between subgenus Curcas and subgenus Jatropha, and demonstrated the intermediate position of section Polymorphae among sections of both subgenera. Jatropha curcas and J. integerrima demonstrated a close phylogenetic relationship. The molecular data agreed with the morphological classification that recognized J. multifida and J. podagrica in sec. Peltatae. The distinct intraspecific divergence that occurred in J. curcas could be attributed to restricted gene flow caused by geographical isolation and different ecological conditions. Phylograms produced with trnL-F and rbcL sequence data suggested slow rates of sequence divergence among Jatropha spp., while the ITS gene tree had good resolution suggesting high genetic variation of ITS among Jatropha species.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.