DNA-based Molecular Markers Research Articles

Species Identification of Five Penaeid Shrimps Using PCR-RFLP and SSCP Analyses of 16S Ribosomal DNA

DNA-based molecular markers for differentiation of five penaeid shrimps (Penaeus monodon, P. semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) were developed based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Differentiation of P. monodon, P. semisulcatus and L. vannamei can be unambiguously carried out by PCRRFLP of 16S rDNA(560) whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S rDNA(560). Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S rDNA(560) of an individual representing the most common mitotype of each species was cloned and sequenced. The new primer pair was designed and tested against the large sample sizes (312 bp product, N = 185). The amplification success was consistent across all species. PCR-RFLP of 16S rDNA(312) was as effective as that of 16S rDNA(560). Differentiation of all shrimp species were successfully carried out by SSCP analysis.

English

  Inter-simple sequence repeats (ISSR) polymorphism was used to reveal genetic variability and phylogenetic relationships within and between three haplotypes ofMayetiola species. A set of 14 ISSR primers were screened representing di-, tri, tetra and penta-nucleotide repeats out of which 10 generated scorable bands and three were able to distinguish one of three haplotypes. The consensus tree constructed using binary data from banding patterns generated by ISSR-PCR clustered the two Mayetiola species according to their mitochondrial haplotype. Moreover, genetic diversity estimated by the coefficient of variation indicates a high intra and inter-haplotypes polymorphism. Our results indicate that ISSR can be useful as DNA-based molecular markers for studying genetic diversity and phylogenetic relationships of Mayetiola haplotypes.   Keywords: Genetic diversity, ISSR markers, Mayetiola, haplotypes.

Linkage disequilibrium and association studies in higher plants: Present status and future prospects

During the last two decades, DNA-based molecular markers have been extensively utilized for a variety of studies in both plant and animal systems. One of the major uses of these markers is the construction of genome-wide molecular maps and the genetic analysis of simple and complex traits. However, these studies are generally based on linkage analysis in mapping populations, thus placing serious limitations in using molecular markers for genetic analysis in a variety of plant systems. Therefore, alternative approaches have been suggested, and one of these approaches makes use of linkage disequilibrium (LD)-based association analysis. Although this approach of association analysis has already been used for studies on genetics of complex traits (including different diseases) in humans, its use in plants has just started. In the present review, we first define and distinguish between LD and association mapping, and then briefly describe various measures of LD and the two methods of its depiction. We then give a list of different factors that affect LD without discussing them, and also discuss the current issues of LD research in plants. Later, we also describe the various uses of LD in plant genomics research and summarize the present status of LD research in different plant genomes. In the end, we discuss briefly the future prospects of LD research in plants, and give a list of softwares that are useful in LD research, which is available as electronic supplementary material (ESM).

Inter simple sequence repeat (ISSR) analysis of genetic diversity in tef [Eragrostis tef (Zucc.) Trotter

The DNA polymorphism among 92 selected tef genotypes belonging to eight origin groups was assessed using eight inter simple sequence repeat (ISSR) primers. The objectives were to examine the possibility of using ISSR markers for unravelling genetic diversity in tef, and to assess the extent and pattern of genetic diversity in the test germplasm with respect to origin groups. The eight primers were able to separate or distinguish all of the 92 tef genotypes based on a total of 110 polymorphic bands among the test lines. The Jaccard similarity coefficient among the test genotypes ranged from 0.26 to 0.86, and at about 60 % similarity level the clustering of this matrix using the unweighted pair-group method based on arithmetic average (UPGMA) resulted in the formation of six major clusters of 2 to 37 lines with further eight lines remaining ungrouped. The standardized Nei genetic distance among the eight groups of origin ranged between 0.03 and 0.32. The UPGMA clustering using the standardized genetic distance matrix resulted in the identification of three clusters of the eight groups of origin with bootstrap values ranging from 56 to 97. The overall mean Shannon Weaver diversity index of the test lines was 0.73, indicating better resolution of genetic diversity in tef with ISSR markers than with phenotypic (morphological) traits used in previous studies. This can be attributed mainly to the larger number of loci generated for evaluation with ISSR analysis as compared to the few number of phenotypic traits amenable for assessment and which are further greatly affected by environment and genotype x environment interaction. Analysis of variance of mean Shannon Weaver diversity indices revealed substantial (P < or = 0.05) variation in the level of diversity among the eight groups of origin. In conclusion, our results indicate that ISSR can be useful as DNA-based molecular markers for studying genetic diversity and phylogenetic relationships, DNA fingerprinting for the identification of varieties or cultivars, and also for genome mapping in tef.

Intravarietal Agronomic Variability inVitis viniferaL. cv. Albariño

Specimens of centuries-old <i>Vitis vinifera</i> L. cv. Albariño were collected in 1987 throughout Galicia, Spain. Eight of these uncertified clones were planted in a homogeneous plot and characterized by ampelographic traits and DNA-based molecular markers in order to identify their agronomic variability. Clone CSIC-3 had the largest grape clusters, the highest must yield per berry, the highest alcohol potential, and relatively low total must acidity. CSIC-2 had the highest total must acidity. CSIC-9 had the largest grape clusters.

Analysis of arthropod bloodmeals using molecular genetic markers.

Little is known about the transmission dynamics of human malaria and other vector-borne diseases, partly because of the limited availability and distribution of appropriate tools for quantifying human-mosquito contact rates. Recent developments in molecular biology have allowed a significant increase in the efficacy and reliability of bloodmeal identification, and DNA-based molecular markers are now being harnessed for typing arthropod bloodmeals. The extent to which these markers have been used for analysis of mosquito bloodmeals and the potential they might have for the future is discussed, and the contributions that the advent of PCR has made are examined here.

Identification of four scallop species using PCR and restriction analysis of the ribosomal DNA internal transcribed spacer region.

Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA region spanning the 5.8S RNA gene and the 2 flanking internal transcribed spacers (ITSs) was performed to establish DNA-based molecular markers for the identification of the scallops Aequipecten opercularis, Chlamys distorta, Mimachlamys varia, and Pecten maximus. Chlamys distorta was distinguished simply by ITS size. Species-specific restriction patterns were found with the restriction enzyme AluI, and also with SmaI for A. opercularis and M. varia. When ITS sizes and the RFLPs obtained with SmaI were combined, the 4 scallops were also differentiated. Additional species-specific RFLPs were revealed after ITS-2 PCR amplification and subsequent digestion with Hsp92II. Using this marker, canned scallops were identified. Thus this work provides a simple, reliable, and rapid method for the identification of scallops that can be used when species-specific morphologic characteristics are removed or when specimens are small in size.

Molecular mapping and characterization of traits controlling fiber quality in cotton

Cotton (Gossypium spp) is the world's leading natural fiber crop. Genetic manipulation continues to play a key role in the improvement of fiber quality properties. By use of DNA-based molecular markers and a polymorphic mapping population derived from an inter specific cross between TM-1 (G. hirsutum) and 3-79 (G. barbadense), thirteen quantitative trait loci (QTLs) controlling fiber quality properties were identified in 3-79, an extra long staple (ELS) cotton. Four QTLs influenced bundle fiber strength, three influenced fiber length, and six influenced fiber fineness. These QTLs were located on different chromosomes or linkage groups and collectively explained 30% to 60%of the total phenotypic variance for each fiber quality property in the F2 population. The effects and modes of action for the individual QTLs were characterized with 3-79 alleles in TM-1 genetic background. The results indicated more recessive than dominant, with much less additive effect in the gene mode. Transgressive segregation was observed for fiber fineness that could be beneficial to improvement of this trait. Molecular markers linked to fiber quality QTLs would be most effective in marker-assisted selection (MAS) of these recessive alleles in cotton breeding programs.

Molecular markers indicate two cryptic, genetically divergent populations of Russian thistle (Salsola tragus) in California

Genetic variability among accessions of Russian thistle (Salsola tragus L.) from California was investigated using allozymes and DNA-based molecular markers. Aspartate aminotransferase and 6-phosphogluconate dehydrogenase displayed two multienzyme phenotypes that were widespread in plants throughout the state. Random amplified polymorphic DNA analysis was conducted on samples of the two isoenzymic phenotypes collected throughout California, as well as additional accessions from France and Turkey and Salsola paulsenii Litv. Six primers produced 23 polymorphic bands. Analysis of the patterns of bands by calculation of simple matching coefficients and cluster analysis confirmed the genetic distinctness of the two isoenzymic phenotypes of S. tragus; S. paulsenii was markedly different from both types. Mean fruit weights from plants grown under similar conditions were different between the two types as well. These results and preliminary cytological analysis together suggest that the two types are actually two different species of Salsola, only one of which has been previously recognized. Analysis of the DNA-based markers suggests that one of the genetic entities may be closely related to Salsola found in Europe, while the area of origin of the second entity is currently obscure.Key words: allozyme, genetic diversity, RAPD assay, Salsola tragus, Salsola paulsenii.

Identification of Races of Colletotrichum lindemuthianum with the Aid of PCR-Based Molecular Markers.

Inoculation of a common bean differential series is the usual method for identification of races of Colletotrichum lindemuthianum. This procedure is extremely useful for phytopathological as well as breeding purposes, but it requires strict control of the number of spores and incubation conditions. Furthermore, this method may result in misclassifications of isolates because of the subjectivity of symptom evaluation. We propose the use of DNA-based molecular markers as an auxiliary tool to aid the classification of races of C. lindemuthianum. Specific DNA bands were identified for races 73, 65, and 64 by polymerase chain reaction (PCR) amplification of bulked DNA samples from isolates of these three races with random primers. The presence of these bands was checked on four isolates previously classified by inoculation on a differential series as belonging to races 23, 72, 79, and 585. The molecular procedure showed that two of these isolates had been misclassified, confirming the high potential of the proposed procedure to aid the identification of races of C. lindemuthianum. Amplification products obtained with 44 different primers also allowed the determination of the genetic distances among isolates from races 73, 65, and 64. These data were used to cluster the isolates into three groups that coincide with the ones obtained by inoculation.

DNA-Based Marker Systems to Determine Genetic Diversity of Weedy Species and Their Application to Biocontrol

DNA-based molecular markers may provide information about introduced weedy species that would be useful in biological weed control efforts. Chloroplast DNA restriction fragment length polymorphisms (cpDNA RFLP) and random amplified polymorphic DNA (RAPD) analysis are two DNA-based marker techniques that can provide estimates of genetic variation in native and introduced populations of weedy species. Profiles provided by these techniques could furnish the necessary information to determine the geographic origins of introduced species and provide evidence for multiple introductions. Although DNA-based markers would not necessarily identify the genetic basis for host-pest compatibility, they would enable identification of specific host genotypes. Current criteria for selecting a weedy species as a target for biological control are primarily political and economic. The importance of genetic diversity and population structure in determining the vulnerability of plant populations to insects or diseases has not been fully appreciated. Estimates of genetic diversity based on DNA marker analysis could be used as one criteria for determining which plants are targeted for biological control. The success of biological weed control efforts has been limited by the high levels of genetic diversity occurring in target weed specks and the lack of biocontrol agent and target weed compatibilities. DNA-based markers may be used to increase our understanding of these factors and contribute to the success of biological weed control by helping to target the most vulnerable species and provide more realistic expectations of the potential for success given available resources.

Association Mapping for Improvement of Quantitative Traits in Plant Breeding Populations

DNA-based molecular markers have been extensively utilized for mapping of genes and quantitative trait loci (QTL) of interest based on linkage analysis in mapping populations. This is in contrast to human genetics that use of linkage disequilibrium (LD)-based mapping for fine mapping of QTLs using single nucleotide polymorphisms. LD based association mapping (AM) has promise to be used in plants. Possible use of such approach may be for fine mapping of genes / QTLs, identifying favorable alleles for marker aided selection and cross validation of results from linkage mapping for precise location of genes / QTLs of interest. In the present review, we discuss different mapping populations, approaches, prospects and limitations of using association mapping in plant breeding populations. This is expected to create awareness in plant breeders in use of AM in crop improvement activities.Key words: Association mapping; plant breeding; DNA marker; quantitative trait lociDOI: http://dx.doi.org/10.3126/njb.v2i1.5686 Nepal Journal of Biotechnology Jan.2012, Vol.2(1): 72-89