The genetic variation and relationship among the natural hybrids of Mangifera casturi Kosterm

Mangifera casturi Kosterm., a mango plant from Kalimantan Selatan, Indonesia, has limited genetic information, severely limiting the research on its genetic variation and phylogeny. We collected M. casturi’s genomic information using next-generation sequencing, developed microsatellite markers and performed Sanger sequencing for DNA barcoding analysis. These markers were used to confirm parental origin and genetic diversity of M. casturi hybrids. The clean reads of the Kasturi accession were assembled de novo, producing 259 872 scaffolds (N50 = 1 445 bp). Fourteen polymorphic microsatellite markers were developed from 11 040 microsatellite motif-containing sequences. In total, 58 alleles were produced with a mean of 4.14 alleles per locus. Microsatellite marker analysis revealed broad genetic variation in M. casturi. Phylogenetic analysis was performed using internal transcribed spacers (ITS), matK, rbcL, and trnH-psbA. The phylogenetic tree of chloroplast markers placed Kasturi, Cuban, Pelipisan, Pinari, and Hambawang in one group, with M. indica as the female ancestor. Meanwhile, the phylogenetic tree of ITS markers indicated several Mangifera species as ancestors of M. casturi. Thus, M. casturi very likely originated from the cross-hybridization of multiple ancestors. Furthermore, crossing the F1 hybrids of M. indica and M. quadrifida with other Mangifera spp. may have generated much genetic variation. The genetic information for M. casturi will be a resource for breeding improvement, and conservation studies.

Genome survey and microsatellite motif identification of Pogonophryne albipinna

The genus Pogonophryne is a speciose group that includes 28 species inhabiting the coastal or deep waters of the Antarctic Southern Ocean. The genus has been divided into five species groups, among which the P. albipinna group is the most deep-living group and is characterized by a lack of spots on the top of the head. Here, we carried out genome survey sequencing of P. albipinna using the Illumina HiSeq platform to estimate the genomic characteristics and identify genome-wide microsatellite motifs. The genome size was predicted to be ~883.8 Mb by K-mer analysis (K=25), and the heterozygosity and repeat ratio were 0.289% and 39.03%, respectively. The genome sequences were assembled into 571,624 contigs, covering a total length of ~819.3 Mb with an N50 of 2,867 bp. A total of 2,217,422 simple sequence repeat motifs were identified from the assembly data, and the number of repeats decreased as the length and number of repeats increased. These data will provide a useful foundation for the development of new molecular markers for the P. albipinna group as well as for further whole genome sequencing of P. albipinna.

Whole genome survey and microsatellite motif identification ofArtemia franciscana

Artemia is an industrially important genus used in aquaculture as a nutritious diet for fish and as an aquatic model organism for toxicity tests. However, despite the significance of Artemia, genomic research remains incomplete and knowledge on its genomic characteristics is insufficient. In particular, Artemia franciscana of North America has been widely used in fisheries of other continents, resulting in invasion of native species. Therefore, studies on population genetics and molecular marker development as well as morphological analyses are required to investigate its population structure and to discriminate closely related species. Here, we used the Illumina Hi-Seq platform to estimate the genomic characteristics of A. franciscana through genome survey sequencing (GSS). Further, simple sequence repeat (SSR) loci were identified for microsatellite marker development. The predicted genome size was ∼867 Mb using K-mer (a sequence of k characters in a string) analysis (K = 17), and heterozygosity and duplication rates were 0.655 and 0.809%, respectively. A total of 421467 SSRs were identified from the genome survey assembly, most of which were dinucleotide motifs with a frequency of 77.22%. The present study will be a useful basis in genomic and genetic research for A. franciscana.

Molecular cytogenetics of valuable Arctic and sub-Arctic pasture grass species from the Aveneae/Poeae tribe complex (Poaceae)

Background Grasslands in the Arctic tundra undergo irreversible degradation due to climatic changes and also over-exploitation and depletion of scarce resources. Comprehensive investigations of cytogenomic structures of valuable Arctic and sub-Arctic grassland species is essential for clarifying their genetic peculiarities and phylogenetic relationships, and also successful developing new forage grass cultivars with high levels of adaptation, stable productivity and longevity. We performed molecular cytogenetic characterization of insufficiently studied pasture grass species (Poaceae) from related genera representing two neighboring clades: 1) Deschampsia and Holcus; 2) Alopecurus, Arctagrostis and Beckmannia, which are the primary fodder resources in the Arctic tundra. Results We constructed the integrated schematic maps of distribution of these species in the northern, central and eastern parts of Eurasia based on the currently available data as only scattered data on their occurrence is currently available. The species karyotypes were examined with the use of DAPI-banding, multicolour FISH with 35S rDNA, 5S rDNA and the (GTT)9 microsatellite motif and also sequential rapid multocolour GISH with genomic DNAs of Deschampsia sukatschewii, Deschampsia flexuosa and Holcus lanatus belonging to one of the studied clades. Cytogenomic structures of the species were specified; peculiarities and common features of their genomes were revealed. Different chromosomal rearrangements were detected in Beckmannia syzigachne, Deschampsia cespitosa and D. flexuosa; B chromosomes with distinct DAPI-bands were observed in karyotypes of D. cespitosa and H. lanatus. Conclusions The peculiarities of distribution patterns of the examined chromosomal markers and also presence of common homologous DNA repeats in karyotypes of the studies species allowed us to verify their relationships. The obtained unique data on distribution areas and cytogenomic structures of the valuable Arctic and sub-Arctic pasture species are important for further genetic and biotechnological studies and also plant breeding progress.

Molecular cytogenetics of valuable Arctic and sub-Arctic pasture grass species from the Aveneae/Poeae tribe complex (Poaceae)

Background Grasslands in the Arctic tundra undergo irreversible degradation due to climatic changes and also over-exploitation and depletion of scarce resources. Comprehensive investigations of cytogenomic structures of valuable Arctic and sub-Arctic grassland species is essential for clarifying their genetic peculiarities and phylogenetic relationships and also successful developing new forage grass cultivars with high levels of adaptation, stable productivity and longevity. We performed molecular cytogenetic characterization of seven insufficiently studied pasture grass species from related genera Alopecurus, Arctagrostis, Beckmannia, Deschampsia and Holcus (Poaceae) which are the primary fodder resources in the Arctic tundra. Results For these species, integrated schematic habitat maps were constructed based on the available data on their distribution in Eurasia. The species karyotypes were examined with the use of DAPI-banding, fluorescence in situ hybridization with 35S rDNA, 5S rDNA and the (GTT)9 microsatellite motif and also sequential rapid genomic in situ hybridization with genomic DNAs of Deschampsia sukatschewii, Holcus lanatus and Deschampsia flexuosa. Cytogenomic structures of the studied species were specified; peculiarities and common features of their genomes were revealed. Different chromosomal rearrangements were detected in Beckmannia syzigachne, Deschampsia cespitosa and D. flexuosa; B chromosomes with distinct DAPI-bands were observed in karyotypes of D. cespitosa and H. lanatus. Conclusions The peculiarities of distribution patterns of the examined chromosomal markers and also presence of common homologous DNA repeats in karyotypes of the studies species allowed us to verify their relationships. The obtained unique data on distribution areas and cytogenomic structures of the valuable Arctic and sub-Arctic pasture species are important for further genetic and biotechnological studies and also plant breeding progress.

A novel set of microsatellite markers for the European Grapevine Moth Lobesia botrana isolated using next-generation sequencing and their utility for genetic characterization of populations from Europe and the Middle East

Using a high-throughput 454 pyrosequencing approach a novel set of microsatellite markers was developed for one of the key grapevine insect pests, the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae). 20 primer pairs flanking a microsatellite motif were designed based on the sequences obtained and were subsequently evaluated in a sample of 14 L. botrana populations from Europe and the Middle East. 11 markers showed stable and reproducible amplification patterns; however, one of the 11 markers was monomorphic in all L. botrana populations analysed. Estimated frequencies of null alleles of more than 20% were evident for two of the markers tested, but varied substantially depending on the respective L. botrana population. In 12 of the 14 L. botrana populations observed heterozygosities were lower to those expected under Hardy–Weinberg equilibrium, indicating a deficiency of heterozygotes in the respective populations. The overall FST value of 0.075 suggested a moderate but significant genetic differentiation between the L. botrana populations included in this study. In addition, a clear geographic structure was detected in the set of samples, evident through a significant isolation by distance and through results from structure analysis. In structure analysis, L. botrana populations were grouped in two clearly separated clusters according to their European (Spain, Italy, Germany) or Middle Eastern (Israel, Syria, Turkey) origin. This novel set of microsatellite markers can now be applied to study the evolutionary ecology of this species including host shifts and host adaptation as well as spread of individuals across worldwide viticulture.

Genetic Relatedness of Lentil (Lens Culinaris L.) Germplasm by using SSR Markers

Ninety six lentil accessions from different origins were collected from National Grain Legume Research Program, Rampur; Regional Agriculture Research Station, Nepalgunj and National Agriculture Genetic Resource Center, Khumaltar, Lalitpur. Among them; four lines were Nepal Local, forty two lines were Nepal Cross; forty seven lines were ICARDA Line and finally three lines were Indian Line. All ninety six accessions were analysed by DNA fingerprinting using thirty three selected polymorphic SSR markers. The characterization was performed in Biotechnology Unit, Nepal Agricultural Research Council, Khumaltar, Lalitpur by using standard protocols. Molecular variance analysis showed that 14 % genetic variation was found between population and 86 % genetic variation was found within population with estimated variance 0.23 between population and 1.35 within population. Highest genetic distance (9) was found between landrace ILL-7979 and RL-20. In the same way, highest Nei genetic distance (0.03) between population was shown by population 1 and population 4; and lowest genetic distance were observed within the same population accessions. The heterozygosity was probably due to the introgression of genes or duplication of microsatellite motif during the breeding and or the course of lentil line evolution. All the accessions included in this study displayed significant amount of genetic variability and genetic relatedness due to different center of origin and different genetic constitutions. The diversity detected in this study may constitute the new materials for future systematic lentil breeding programs.DOI: http://dx.doi.org/10.3126/ijasbt.v1i3.8608 Int J Appl Sci Biotechnol, Vol. 1(3) 2013 : 132-136

Microsatellite Marker for Homozygosity Testing of Putative Doubled Haploids and Characterization of Mimulus Species Derived by a Cross-genera Approach

A codominant marker for homozygosity testing and species discrimination needed in breeding programs was developed and applied to different Mimulus L. species and cultivars. Degenerative primers used to amplify intron 10 of topoisomerase 6 subunit B (top6B) in distant species also amplified the locus in all analyzed Mimulus species. The sequences obtained revealed the presence of a microsatellite motif and were used to design a specific microsatellite primer pair, Mim-top6B, for Mimulus species. The microsatellite marker showed a high degree of polymorphism in Mimulus species, and the heterozygous nature of most M. aurantiacus Curtis cultivars. The marker was further used to analyze putative doubled haploids of M. aurantiacus and showed that all but one was heterozygous, indicating their hybrid origin.