Molecular analysis of the phylogenetic relationships among the diploid <Emphasis Type="Italic">Aegilops</Emphasis> species of the section <Emphasis Type="Italic">Sitopsis</Emphasis>

RAPD analysis was used to study the intraspecific variation and phylogenetic relationships of Sgenome diploid Aegilops species regarded as potential donors of the B genome of cultivated wheat. In total, 21 DNA specimens from six S-genome diploid species were examined. On a dendrogram, Ae. speltoides and Ae. aucheri formed the most isolated cluster. Among the other species, Ae. searsii was the most distant while Ae. longissima and Ae. sharonensis were the closest species. The maximum difference between individual accessions within one species was approximately the same (0.18–0.22) in Ae. bicornis, Ae. longissima, Ae. sharonensis, and Ae. searsii. The difference between the clusters of questionable species Ae. speltoides and Ae. aucheri corresponded to the intraspecific level; the difference between closely related Ae. longissima and Ae. sharonensis corresponded to the interspecific level.     

Differentiation of two hemp nettle species (<Emphasis Type="Italic">Galeopsis bifida</Emphasis> Boenn. and <Emphasis Type="Italic">G. tetrahit</Emphasis> L.) inferred from morphological characters and DNA markers

Two species of the genus Galeopsis L., G. tetrahit L. and G. bifida Boenn. (family Lamiaceae), are problematic to distinguish often wrongly recognized, and treated by some taxonomists as a single species. Morphological diagnostical characters of these species are variable and partly overlap. Species independence of G. tetrahit and G. bifida was evaluated and their diagnostic characters verified using ISSR and RAPD markers. A total of 57 ISSR and 28 RAPD fragments were obtained providing distinct subdivision of the accessions examined into two groups. Analysis of molecular data using the neighbor-joining method showed that the accessions studied fell into two clades in the same way as demonstrated by the analysis of 20 morphological characters using single linkage method. These results suggest that G. tetrahit and G. bifida are distinct species. The morphological characters were found to be more variable compared to the molecular markers, although the combined of these characters provided differentiation of the species.     

Genetic diversity of <Emphasis Type="Italic">avenin-like b</Emphasis> genes in <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss

Avenin-like storage proteins influence the rheological properties and processing quality in common wheat, and the discovery of new alleles will benefit wheat quality improvement. In this study, 13 avenin-like b alleles (TaALPb7D-A–M) were discovered in 108 Aegilops tauschii Coss. accessions. Ten alleles were reported for the first time, while the remaining three alleles were the same as alleles in other species. A total of 15 nucleotide changes were detected in the 13 alleles, resulting in only 11 amino acid changes because of synonymous mutations. Alleles TaALPb7D-E, TaALPb7D-G, and TaALPb7D-J encoded the same protein. These polymorphic sites existed in the N-terminus, Repetitive region (Left), Repetitive region (Right) and C-terminus domains, with no polymorphisms in the signal peptide sequence nor in those encoding the 18 conserved cysteine residues. Phylogenetic analysis divided the TaALPb7Ds into four clades. The Ae. tauschii alleles were distributed in all four clades, while the alleles derived from common wheat, TaALPb7D-G and TaALPb7D-C, belonged to clade III and IV, respectively. Alleles TaALPb7D-G and TaALPb7D-C were the most widely distributed, being present in nine and six countries, respectively. Iran and Turkey exhibited the highest genetic diversity with respect to TaALPb7D alleles, accessions from these countries carrying seven and six alleles, respectively, which implied that these countries were the centers of origin of the avenin-like b gene. The new alleles discovered and the phylogenetic analysis of avenin-like b genes will provide breeding materials and a theoretical basis for wheat quality improvement.     

Molecular diversity and genetic relationships in <Emphasis Type="Italic">Secale</Emphasis>

The objective of this study was to quantify the molecular diversity and to determine the genetic relationships among Secale spp. and among cultivars of Secale cereale using RAPDs, ISSRs and sequence analysis of six exons of ScMATE1 gene. Thirteen ryes (cultivated and wild) were genotyped using 21 RAPD and 16 ISSR primers. A total of 435 markers (242 RAPDs and 193 ISSRs) were obtained, with 293 being polymorphic (146 RAPDs and 147 ISSRs). Two RAPD and nine ISSR primers generated more than 80% of polymorphism. The ISSR markers were more polymorphic and informative than RAPDs. Further, 69% of the ISSR primers selected achieved at least 70% of DNA polymorphism. The study of six exons of the ScMATE1 gene also demonstrated a high genetic variability that subsists in Secale genus. One difference observed in exon 1 sequences from S. vavilovii seems to be correlated with Al sensitivity in this species. The genetic relationships obtained using RAPDs, ISSRs and exons of ScMATE1 gene were similar. S. ancestrale, S. kuprijanovii and S. cereale were grouped in the same cluster and S. segetale was in another cluster. S. vavilovii showed evidences of not being clearly an isolate species and having great intraspecific differences.     

Assessment of genetic diversity in <Emphasis Type="Italic">Mucuna</Emphasis> species of India using randomly amplified polymorphic DNA and inter simple sequence repeat markers

Genus Mucuna which is native to China and Eastern India comprises of perennial climbing legume with long slender branches, trifoliate leaves and bear green or brown pod covered with soft or rigid hairs that cause intense irritation. The plants of this genus are agronomically and economically important and commercially cultivated in India, China and other regions of the world. The high degrees of taxonomical confusions exist in Mucuna species that make authentic identification and classification difficult. In the present study, the genetic diversity among the 59 accessions of six species and three varieties of M. pruriens has been assessed using DNA fingerprinting based molecular markers techniques namely randomly amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and combined dataset of RAPD and ISSR. Also, genetic relationship among two endemic species of Mucuna namely M. imbricata and M. macrocarpa and two varieties namely IIHR hybrid (MHR) and Dhanwantari (MD) with other species under study was investigated by using cluster analysis and principal coordinate analysis. The cluster analysis of RAPD, ISSR and combined dataset of RAPD and ISSR clearly demonstrated the existence of high interspecific variation than intra-specific variation in genus Mucuna. The utility and efficacy of RAPD and ISSR for the study of intra species and interspecies genetic diversity was evident from AMOVA and PCoA analysis. This study demonstrates the genetic diversity in Mucuna species and indicates that these markers could be successfully used to assess genetic variation among the accessions of Mucuna species.     

Genetic Diversity and Phylogenetic Relationships of Two Closely Related Northeast China <Emphasis Type="Italic">Vicia</Emphasis> Species Revealed with RAPD and ISSR Markers

RAPD and ISSR analyses revealed genetic diversity and relationships among 11 populations of two closely related northeast China Vicia species, Vicia ramuliflora and V. unijuga. Both methods yielded similar and complementary results, showing high genetic diversity. Vicia ramuliflora had 100% polymorphic loci in both RAPD and ISSR, and V. unijuga had 100% polymorphic loci for RAPD and 98.96% for ISSR. Genetic differentiation was moderate among populations of each species. Genetic variation was distributed mainly within populations for the two species. The high level of gene flow was important for the allocation of genetic variation. The UPGMA dendrogram and principal coordinates analysis at the level of individuals and populations showed that V. ramuliflora and V. unijuga were more closely related than either of them was to the outgroup species, V. cracca. The small molecular variance of V. ramuliflora and V. unijuga supports the conclusion that these two species had a common ancestor.     

Identification of <Emphasis Type="Italic">Bacillus</Emphasis> Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, <Emphasis Type="Italic">recA</Emphasis>, <Emphasis Type="Italic">rpoB</Emphasis> Gene Sequencing and RAPD-PCR

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.     

Identification of <Emphasis Type="Italic">Pm58</Emphasis> from <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Key message

A novel powdery mildew-resistance gene, designated Pm58, was introgressed directly from Aegilops tauschii to hexaploid wheat, mapped to chromosome 2DS, and confirmed to be effective under field conditions. Selectable KASP? markers were developed for MAS.

Abstract

Powdery mildew caused by Blumeria graminis (DC.) f. sp. tritici (Bgt) remains a significant threat to wheat (Triticum aestivum L.) production. The rapid breakdown of race-specific resistance to Bgt reinforces the need to identify novel sources of resistance. The d-genome species, Aegilops tauschii, is an excellent source of disease resistance that is transferrable to T. aestivum. The powdery mildew-resistant Ae. tauschii accession TA1662 (2n?=?2x?=?DD) was crossed directly with the susceptible hard white wheat line KS05HW14 (2n?=?6x?=?AABBDD) followed by backcrossing to develop a population of 96 BC₂F₄ introgression lines (ILs). Genotyping-by-sequencing was used to develop a genome-wide genetic map that was anchored to the Ae. tauschii reference genome. A detached-leaf Bgt assay was used to screen BC₂F_4:6 ILs, and resistance was found to segregate as a single locus (χ?=?2.0, P value?=?0.157). The resistance gene, referred to as Pm58, mapped to chromosome 2DS. Pm58 was evaluated under field conditions in replicated trials in 2015 and 2016. In both years, a single QTL spanning the Pm58 locus was identified that reduced powdery mildew severity and explained 21% of field variation (P value?<?0.01). KASP? assays were developed from closely linked GBS-SNP markers, a refined genetic map was developed, and four markers that cosegregate with Pm58 were identified. This novel source of powdery mildew-resistance and closely linked genetic markers will support efforts to develop wheat varieties with powdery mildew resistance.

     

Genetic diversity of <Emphasis Type="Italic">Opuntia</Emphasis> spp. varieties assessed by classical marker tools (RAPD and ISSR)

Opuntia, commonly named “nopal” in Mexico, is an important crop for its agronomical, economical, ecological and cultural value. Furthermore, it is known for its taxonomic complexity. In this paper, we report the genetic variability of 52 Opuntia cultivars with agronomic and economic importance, classified into 12 different species using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. Ten primers, five for each marker type, were selected to assess their ability to detect polymorphisms in this plant accesions/varieties. Both marker systems generated a total of 307 bands, of which 50.8 % were polymorphic with an average of 15.6 polymorphic bands per primer. Thus, we assume that Mexican Opuntia varieties present broad genetic variation. Based on percentage of polymorphic bands; resolving power; polymorphic information content; and Marker Index, the K-12 (RAPD) and IS-06 (ISSR) primers were the most informative ones. Clusters obtained from RAPD, ISSR and a combination of both data sets did not match the actual taxonomic classification. On the other hand, the putative varieties currently classified in the same species were not located in the same cluster. Besides, the varieties included in O. ficus-indica, O. albicarpa and O. megacantha showed broad variation but were not well defined into separate clades; these cultivars possibly have common ancestry. The results presented here support our hypothesis about the existence of a smaller number of Opuntia species in accordance with those currently described, but with high intraspecific genetic variation.     

Genetic mapping of a novel recessive allele for non-glaucousness in wild diploid wheat <Emphasis Type="Italic">Aegilops tauschii</Emphasis>: implications for the evolution of common wheat

Cuticular wax on the aerial surface of plants has a protective function against many environmental stresses. The bluish–whitish appearance of wheat leaves and stems is called glaucousness. Most modern cultivars of polyploid wheat species exhibit the glaucous phenotype, while in a wild wheat progenitor, Ae. tauschii, both glaucous and non-glaucous accessions exist. Iw2, a wax inhibitor locus on the short arm of chromosome 2D, is the main contributor to this phenotypic variation in Ae. tauschii, and the glaucous/non-glaucous phenotype of Ae. tauschii is usually inherited by synthetic hexaploid wheat. However, a few synthetic lines show the glaucous phenotype although the parental Ae. tauschii accessions are non-glaucous. Molecular marker genotypes indicate that the exceptional non-glaucous Ae. tauschii accessions share the same genotype in the Iw2 chromosomal region as glaucous accessions, suggesting that these accessions have a different causal locus for their phenotype. This locus was assigned to the long arm of chromosome 3D using an F₂ mapping population and designated W4, a novel glaucous locus in Ae. tauschii. The dominant W4 allele confers glaucousness, consistent with phenotypic observation of Ae. tauschii accessions and the derived synthetic lines. These results implied that glaucous accessions of Ae. tauschii with the W2W2iw2iw2W4W4 genotype could have been the D-genome donor of common wheat.     

Low molecular weight glutenin subunit gene composition at <Emphasis Type="Italic">Glu</Emphasis>-<Emphasis Type="Italic">D3</Emphasis> loci of <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and common wheat and a further view of wheat evolution

Key message

A comprehensive comparison of LMW-GS genes between Ae. tauschii and its progeny common wheat.

Abstract

Low molecular weight glutenin subunits (LMW-GSs) are determinant of wheat flour processing quality. However, the LMW-GS gene composition in Aegilops tauschii, the wheat D genome progenitor, has not been comprehensively elucidated and the impact of allohexaploidization on the Glu-D3 locus remains elusive. In this work, using the LMW-GS gene molecular marker system and the full-length gene-cloning method, LMW-GS genes at the Glu-D3 loci of 218 Ae. tauschii and 173 common wheat (Triticum aestivum L.) were characterized. Each Ae. tauschii contained 11 LMW-GS genes, and the whole collection was divided into 25 haplotypes (AeH01–AeH25). The Glu-D3 locus in common wheat lacked the LMW-GS genes D3-417, D3-507 and D3-552, but shared eight genes of identical open reading frame (ORF) sequences when compared to that of Ae. tauschii. Therefore, the allohexaploidization induces deletions, but exerts no influence on LMW-GS gene coding sequences at the Glu-D3 locus. 92.17% Ae. tauschii had 7-9 LMW-GSs, more than the six subunits in common wheat. The haplotypes AeH16, AeH20 and AeH23 of Ae. tauschii ssp. strangulate distributed in southeastern Caspian Iran were the main putative D genome donor of common wheat. These results facilitate the utilization of the Ae. tauschii glutenin gene resources and the understanding of wheat evolution.

     

SSR-based molecular profiling of 237 persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.) germplasms using an <Emphasis Type="Italic">ASTRINGENCY</Emphasis>-linked marker

Pollination-constant non-astringent (PCNA) trait is desirable in persimmon production because it confers natural astringency loss in mature persimmon fruit. Expression of the PCNA trait requires six homozygous recessive PCNA (ast) alleles at the single ASTRINGENCY (AST) locus in hexaploid persimmon. When crossing non-PCNA accessions to breed PCNA offspring, knowledge of ast and non-PCNA (AST) allele dosage in the parental accessions is important, because more PCNA offspring can segregate from a non-PCNA parent with more ast and fewer AST alleles. Previously, we have demonstrated that a region linked to the AST locus has numerous fragment size polymorphisms with varying numbers of simple sequence repeats. Here, we reveal the polymorphisms in this region in a broad collection of persimmon germplasms. Among 237 accessions, we distinguished 21 AST- and 5 ast-linked fragments with different sizes. Based on the number of fragments detected per individual, we identified 21 non-PCNA accessions with three different ast alleles; by crossing these with a PCNA parent, we obtain PCNA offspring under autohexaploid inheritance. Furthermore, AST and ast allelic combination patterns in hexaploid persimmon were shown to be applicable to cultivar identification of non-PCNA accessions. We directly sequenced ast-linked fragments from 48 accessions with one-size peak of ast-linked fragment and found two distinctive groups of fragments based on single nucleotide polymorphisms. This result suggests that a bottleneck event occurred during ast allele development. We conclude that our fragment size profile can be used to accelerate PCNA breeding that uses non-PCNA parents and to study ast allele accumulation in persimmon.     

Allele-specific CAPS marker in a <Emphasis Type="Italic">Ve1</Emphasis> homolog of <Emphasis Type="Italic">Capsicum annuum</Emphasis> for improved selection of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> resistance

Verticillium wilt (Verticillium dahliae) is an economically important disease for many high-value crops. The pathogen is difficult to manage due to the long viability of its resting structures, wide host range, and the inability of fungicides to affect the pathogen once in the plant vascular system. In chile pepper (Capsicum annuum), breeding for resistance to Verticillium wilt is especially challenging due to the limited resistance sources. The dominant Ve locus in tomato (Solanum lycopersicum) contains two closely linked and inversely oriented genes, Ve1 and Ve2. Homologs of Ve1 have been characterized in diverse plant species, and interfamily transfer of Ve1 confers race-specific resistance. Queries in the chile pepper WGS database in NCBI with Ve1 and Ve2 sequences identified one open reading frame (ORF) with homology to the tomato Ve genes. Comparison of the candidate CaVe (Capsicum annuum Ve) gene sequences from susceptible and resistant accessions revealed 16 single nucleotide polymorphisms (SNPs) and several haplotypes. A homozygous haplotype was identified for the susceptible accessions and for resistant accessions. We developed a cleaved amplified polymorphic sequence (CAPS) molecular marker within the coding region of CaVe and screened diverse germplasm that has been previously reported as being resistant to Verticillium wilt in other regions. Based on our phenotyping using the New Mexico V. dahliae isolate, the marker could select resistance accessions with 48% accuracy. This molecular marker is a promising tool towards marker-assisted selection for Verticillium wilt resistance and has the potential to improve the efficacy of chile pepper breeding programs, but does not eliminate the need for a bioassay. Furthermore, this work provides a basis for future research in this important pathosystem.     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Polymorphism of the <Emphasis Type="Italic">LEAFY</Emphasis> Gene in <Emphasis Type="Italic">Brassica</Emphasis> Plants

The arabidopsis gene LEAFY controls the induction of flowering and maintenance of the floral meristem identity. By comparing the primary structure of LEAFY and its homologs in other Brassicaceae species and beyond this family, we singled out four clusters corresponding to three systematically remote families of angiosperms, Brassicaceae, Solanaceae, and Poaceae, and to gymnosperms. Both structural and functional distinctions of LEAFY homologs from their arabidopsis prototype expanded in the range Brassicaceae—Solanaceae—Poaceae. A LEAFY homolog from B. juncea cloned in our laboratory was used as a hybridization probe to analyze the restriction fragment length polymorphism in six Brassica species comprising diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) genomes. In this way we recognized LEAFY fragments specific of genomes A, B, and C; in contrast, the variations of the length and structure of the LEAFY intron 2 were not genome-specific. LEAFY polymorphism in the Brassica accessions comprising genome B was related to their geographic origin and apparently to the adaptation to day length.     

Discovery and characterization of two new stem rust resistance genes in <Emphasis Type="Italic">Aegilops sharonensis</Emphasis>

Key message

We identified two novel wheat stem rust resistance genes, Sr-1644-1Sh and Sr-1644-5Sh in Aegilops sharonensis that are effective against widely virulent African races of the wheat stem rust pathogen.

Abstract

Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F₆ recombinant inbred line and an F₂ population, two genes were identified that mapped to the short arm of chromosome 1S^sh, designated as Sr-1644-1Sh, and the long arm of chromosome 5S^sh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.

     

Reconstruction of molecular phylogeny of closely related <Emphasis Type="Italic">Amorphophallus</Emphasis> species of India using plastid DNA marker and fingerprinting approaches

Plastid DNA markers sequencing and DNA fingerprinting approaches were used and compared for resolving molecular phylogeny of closely related, previously unexplored Amorphophallus species of India. The utility of individual plastid markers namely rbcL, matK, trnH–psbA, trnLC–trnLD, their combined dataset and two fingerprinting techniques viz. RAPD and ISSR were tested for their efficacy to resolves Amorphophallus species into three sections specific clades namely Rhaphiophallus, Conophallus and Amorphophallus. In the present study, sequences of these four plastid DNA regions as well as RAPD and ISSR profiles of 16 Amorphophallus species together with six varieties of two species were generated and analyzed. Maximum likelihood and Bayesian Inference based construction of phylogenetic trees indicated that among the four plastid DNA regions tested individually and their combined dataset, rbcL was found best suited for resolving closely related Amorphophallus species into section specific clades. When analyzed individually, rbcL exhibited better discrimination ability than matK, trnH–psbA, trnLC–trnLD and combination of all four tested plastid markers. Among two fingerprinting techniques used, the resolution of Amorphophallus species using RAPD was better than ISSR and combination of RAPD +ISSR and in congruence with resolution based on rbcL.     

<Emphasis Type="Italic">Viola woosanensis</Emphasis>, a recurrent spontaneous hybrid between <Emphasis Type="Italic">V. ulleungdoensis</Emphasis> and <Emphasis Type="Italic">V. chaerophylloides</Emphasis> (Violaceae) endemic to Ulleung Island,Korea

Ulleung Island is an oceanic volcanic island in Korea, which has never been connected to the adjacent continent. Previous studies highlighted Ulleung Island as an excellent system to study the pattern and process of early stages of flowering plant evolutions on oceanic island. The predominant mode of speciation in flowering plants on Ulleung Island appears to be anagenesis. However, the potentially important role of hybrid speciation among incompletely reproductively isolated lineages cannot be ruled out. Viola woosanensis (Violaceae) is of purportedly hybrid origin between V. ulleungdoensis (i.e., formerly recognized as V. selkirkii in Ulleung Island) and V. chaerophylloides, based on morphology. To examine the origin of V. woosanensis, we sampled a total of 80 accessions, including V. woosanensis and its putative parental species and sequenced nrDNA ITS, and four highly variable chloroplast noncoding regions (trnL-trnF, rpl16 intron, atpF-atpH, and psbA-trnH). Representative species of Viola from Korea were also included in the phylogenetic analyses (maximum parsimony, maximum likelihood, and Bayesian inference). Additive polymorphic sites in the nrDNA ITS regions were confirmed by cloning amplicons from representative species. The molecular data strongly supported the hybrid origin of V. woosanensis, and the maternal and paternal parent were determined to be V. ulleungdoensis and V. chaerophylloides, respectively. The presence of two parental ribotypes in V. woosanensis (with the exception in one population) was confirmed by cloning, suggesting V. woosanensis is primarily the F₁ generation. No trace of backcrossing and introgression with its parents was detected due to low fertility of hybrid species. We found a multiple and unidirectional hybrid origin of V. woosanensis. Additional studies are required to determine which factors contribute to asymmetric gene flow of Viola species in Ulleung Island.     

New microsatellite markers developed from <Emphasis Type="Italic">Urochloa humidicola</Emphasis> (Poaceae) and cross amplification in different <Emphasis Type="Italic">Urochloa</Emphasis> species

Background

Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.

Conclusions

This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

     

Evaluation of genetic diversity of <Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Acanthaceaea) using RAPD,ISSR and RAMP markers

Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.