7/1/2013 Annotated Bibliography Environmental 1) Elsey-Quirk, T., D. Seliskar and J. Gallagher. 2011. Differential Population Response of Allocation, Phenology, and Tissue Chemistry in Spartina alterniflora. Plant Ecology. 212: 1873-1885. Abstract: Phenotypic variation within species is widespread among salt marsh plants. For Spartina alterniflora, the dominant species of low intertidal wetlands across the Atlantic and Gulf coasts of the US, distinct phenological and morphological differences among populations from different latitudes have been found. To determine whether S. alterniflora plants from lower latitudes and those regenerated from Delaware tissue cultures would maintain differences from that of native plants, we conducted a field study in a natural salt marsh in Delaware, US. After two growing seasons, plant height, stem density, above- and belowground biomass, elemental composition, and nutrient resorption were measured. Natural variation in porewater salinity influenced physiological traits of Na+/K+ ratio regulation and nitrogen resorption efficiency similarly across populations. While plant height exhibited plasticity where populations tended to converge to a similar height, several other traits remained distinct. Delaware plants had a greater rate of rhizome growth than Georgia and Louisiana plants, which correlated with a greater magnitude of fall senescence. If traits such as seasonal translocation are plastic and can change with the length of the growing season, climate warming may alter belowground biomass production of S. alterniflora in wetlands of the mid-Atlantic. Extra Notes: -Geographic patterns of genetic variation exist -S. alterniflora separated into 2 distinct groups based on morphological characteristics -Distinct differences between Gulf coast and south Atlantic populations -Tissue culture technique produced plants that were phenotypically divergent -Emphasis on belowground biomass -Suggests that a natural marsh environment can exert a greater influence on height than population differences -Researchers raise questions on how populations will respond to climate change 2) Proffitt, C. E., S. E. Travis and K. R. Edwards. 2003. Genotype and Elevation Influence Spartina alterniflora Colonization and Growth in a Created Salt Marsh. Ecological Applications. 13: 180-192. Abstract: Colonization, growth, and clonal morphology differ with genotype and are influenced by elevation. Local adaptation of Spartina alterniflora to environmental conditions may lead to dominance by different suites of genotypes in different locations within a marsh. In a constructed marsh, we found reduced colonization in terms of density of clones with 7/1/2013 increasing distance from edge in a 200-ha mudflat created in 1996; however, growth in diameter was not different among three 100-m-long zones that differed in distance from site edge. Distance from edge was confounded by elevation in this comparison of natural colonization. The rate of clonal expansion in diameter was 3.1 m/yr, and clonal growth was linear over the 28 mo of the study. The area dominated by S. alterniflora in the three distance zones increased concomitantly with clonal growth. However, the lower initial clonal densities and colonization by other plant species resulted in reduced overall dominance by S. alterniflora in the two more-interior locations. Seedling recruitment was an important component of S. alterniflora colonization at all elevations and distances from edge two years after site creation. Seedlings were spatially very patchy and tended to occur near clones that probably produced them. A field experiment revealed that S. alterniflora height and total stem length varied with genotype, while stem density and flowering stem density did not. Differences between edge and center of clonal patches also occurred for some response variables, and there were also significant interactions with genotype. Differences between edge and center are interpreted as differences in clone morphology. Elevation differences over distances of a few meters influenced total stem length and flowering stem density but not other response variables. Clones that were larger in diameter also tended to have greater stem heights and total stem lengths. A number of plant morphological measures were found to vary significantly among the five genotypes and had broad-sense heritabilities ranging up to 0.71. These results indicate that S. alterniflora populations developing on new substrata colonize broadly, but growth and reproduction vary with genotype and are influenced by changes in elevation (range: 11.8 cm), and probably other environmental factors, over relatively small distances. Differences in growth and clone morphology of different genets, and the frequent occurrence of seedlings throughout the site, underscore the importance of genetic variability in natural and created populations. Extra Notes: -S. alterniflora attains dominance in low intertidal areas mainly through its ability to tolerate anoxic flooded sediments -Colonization of bare intertidal substrate occurs through dispersal of vegetative propagules and seeds and by clonal growth Methods: DNA typing to ensure that clones were identifiably different genotypes -Clonal expansion as growth in diameter -All clones responded similarly to changes in elevation by having greater growth at lower elevations -Differences in growth of the clonal plant due to heterogeneity in elevation over relatively small spatial scales and plant genotype -Clone produces the greatest growth near the outer-edge and facilitates the capture of space Maintaining genetic diversity – sexual recombination 3) He, W., R. Feagin, J. Lu, W. Liu, Q. Yan and Z. Xie. 2007. Impacts of introduced Spartina alterniflora along an elevation gradient at Jiuduansha Shoals in the Yangtze Estuary, 7/1/2013 suburban Shanghai, China. Ecological Engineering. 29: 245-248. Abstract: Although much research has focused upon the negative impacts of invasive Spartina alterniflora upon salt marshes dominated by other Spartina spp., little is known about its impacts upon native Scirpus mariqueter marshes. In 1997, S. alterniflora was introduced to the Jiu- duansha Shoals, Yangtze Estuary, China, to accelerate the formation of marsh habitat via accretionary processes, with the larger goal of drawing waterfowl away from wetlands near the Pudong International Airport, Shanghai, China. In 2000, a nature reserve was established on the Jiuduansha Shoals, making the impact upon the native S. mariqueter community a high priority for research. Our objective was to quantify the impacts of introduced S. alterniflora and Phragmites australis to the native S. mariqueter-dominated community at this site in four elevation zones, as compared with a nearby natural shoal. We found that species diversity was greater in the lower elevations with the engineering, through elimination of the natural dominance of S. mariqueter. We also found that diversity was lessened in the higher elevations, due to rapid growth and exclusion by the planted S. alterniflora in con- junction with the native P. australis. Moreover, we found that the growth of the native S. mariqueter was stimulated when S. alterniflora was planted nearby. It is quite likely that the net effect of these ecological processes will be to accelerate further accretion, leading to an eventual replacement of the S. mariqueter-dominated community in the long-term. Future management approaches should focus upon harvesting, grazing, and perimeter-ditching the S. alterniflora to avoid this situation. Extra Notes: -Objective – quantify the impacts of introducing S. alterniflora to S. mariqueter marshes along the elevation gradient at the Jiuduansha Shoals. -Highest plant diversity was found in the intertidal high zone -Study shows that the introduction of S. alterniflora may initially increase plant diversity in these marshes by shifting the dominance of S. mariqueter in the intertidal zones, contradicting to the findings in northeastern USA 4) Holmer, M., B. Gribsholt and E. Kristensen. 2002. Effects of sea level rise on growth of Spartina anglica and oxygen dynamics in rhizosphere and salt marsh sediments. Marine Ecology Progress Series. 225:197-204. Abstract: The effect of sea level rise on the growth of Spartina anglica seedlings and on key sediment biogeochemical variables (oxygen concentrations and sulfur cycling) was studied for 4 mo in a laboratory experiment. S. anglica, grown under drained and waterlogged conditions, showed no significant differences in leaf elongation and above-ground biomass between treatments. Sulfate reduction rates were not significantly different between treatments (4.1 and 5.3 mmol m–2 d–1, respectively), and although pools of reduced sulfides were high (12.1 to 14.9 mol S m–2), no dissolved sulfides were detected in the sediments. Measurements of oxygen concentrations in rhizosphere sediment done with microelectrodes revealed a distinct oxic microzone of up to 2.5 mm around the roots of S. 7/1/2013 anglica. The oxic microzone comprised 30 to 60% of the S. anglica rhizosphere sediment, suggesting that the root-mediated oxygen supply to the rhizosphere has profound effects on the microbial processes in the sediments. The sulfate reduction was probably hampered due to the root-mediated loss of oxygen from the plants. There was no difference in oxygen dynamics in the rhizosphere between treatments, indicating that S. anglica is efficient in oxidizing the sediments also under waterlogged conditions. The root-mediated loss of oxygen from S. anglica counteracts the expected changes in sediment conditions as a consequence of sea level rise, e.g. accumulation of phytotoxic compounds such as sulfides. The results suggest that possible negative impacts of sea level rise are more likely to be found for plants with less developed root systems. 5) Ravit, B., J. G. Ehrenfeld and M. M. Haggblom. 2003. A comparison of sediment microbial communities associated with Phragmites australis and Spartina alterniflora in two brackish wetlands of New Jersey. Estuaries. 26: 465-474. Abstract: The extensive spread of Phragmites australis throughout brackish marshes on the East Coast of the United States is a major factor governing management and restoration decisions because it is assumed that biogeochemical functions are altered by the invasion. Microbial activity is important in providing wetland biogeochemical functions such as carbon and nitrogen cycling, but there is little known about sediment microbial communities in Phragmites marshes. Microbial populations associated with invasive Phragmites vegetation and with native salt marsh cordgrass, Spartina alterniflora, may differ in the relative abundance of microbial taxa (community structure) and in the ability of this biota to decompose organic substrates (community biogeochemical function). This study compares sediment microbial communities associated with Phragmites and Spartina vegetation in an undisturbed brackish marsh near Tuckerton, New Jersey (MUL), and in a brackish marsh in the anthropogenically affected Hackensack Meadowlands (SMC). We use phospholipid fatty acid (PLFA) analysis and enzymatic activity to profile sediment microbial communities associated with both plants in each site. Sediment analyses include bulk density, total organic matter, and root biomass. PLFA profiles indicate that the microbial communities differ between sites with the undisturbed site exhibiting greater fatty acid richness (62 PLFA recovered from MUL versus 38 from SMC). Activity of the 5 enzymes analyzed (,-glucosidase, acid phosphatase, chitobiase, and 2 oxidases) was higher in the undisturbed site. Differences between vegetation species as measured by Principal Components Analysis were significantly greater at the undisturbed MUL site than at SMC, and patterns of enzyme activity and PLFAs did not correspond to patterns of root biomass. We suggest that in natural wetland sediments, macrophyte rhizosphere effects influence the community composition of sediment microbial populations. Physical and chemical site disturbances may impose limits on these rhizosphere effects, decreasing sediment microbial diversity and potentially, microbial biogeochemical functions. 7/1/2013 Genetics 1) Blum, M. J., K. J. Bando, M. Katz and D. R. Strong. 2007. Geographic structure, genetic diversity and source tracking of Spartina alterniflora. Journal of Biogeography. 34: 20552069. Abstract: Aim: To examine the distribution and structure of genetic variation among native Spartina alterniflora and to characterize the evolutionary mechanisms underlying the success of non-native S. alterniflora. Location: Intertidal marshes along the Atlantic, Gulf and Pacific coasts of North America. Methods: AMOVA, parsimony analysis, haplotype networks of chloroplast DNA (cpDNA) sequences, neighbour-joining analysis, Bayesian analysis of population structure, and individual assignment testing were used. Results: Low levels of gene flow and geographic patterns of genetic variation were found among native S. alterniflora from the Atlantic and Gulf coasts of North America. The distribution of cpDNA haplotypes indicates that Atlantic coast S. alterniflora are subdivided into ‘northern’ and ‘southern’ groups. Variation observed at microsatellite loci further suggests that mid-Atlantic S. alterniflora are differentiated from S. alterniflora found in southern Atlantic and New England coastal marshes. Comparisons between native populations on the Atlantic and Gulf coasts and non-native Pacific coast populations substantiate prior studies demonstrating reciprocal interspecific hybridization in San Francisco Bay. Our results corroborate historical evidence that S. alterniflora was introduced into Willapa Bay from multiple source populations. However, we found that some Willapa Bay S. alterniflora are genetically divergent from putative sources, probably as a result of admixture following secondary contact among previously allopatric native populations. We further recovered evidence in support of models suggesting that S. alterniflora has secondarily spread within Washington State, from Willapa Bay to Grays Harbor. Main conclusions: Underlying genetic structure has often been cited as a factor contributing to ecological variation of native S. alterniflora. Patterns of genetic structure within native S. alterniflora may be the result of environmental differences among biogeographical provinces, of migration barriers, or of responses to historical conditions. Interactions among these factors, rather than one single factor, may best explain the distribution of genetic variation among native S. alterniflora. Comprehensive genetic comparisons of native and introduced populations can illustrate how biological invasions may result from dramatically different underlying factors – some of which might otherwise go unrecognized. Demonstrating that invasions can result from several independent or interacting mechanisms is important for improving risk assessment and future forecasting. Further research on S. alterniflora not only may clarify what forces structure native populations, but also may improve the management of non- native populations by enabling post-introduction genetic changes and the rapid evolution of life-history traits to be more successfully exploited. 7/1/2013 Extra notes: -S. alterniflora introduced to UK, hybridization with S. maritime and led to allopolyploid speciation and spread of hybrid, S. anglica ***examine continental- scale patterns of genetic variation among native S. alterniflora using both chloroplast and nuclear molecular markers Methods: DNA Extraction - .15-.25g of leaf tissue – DNeasy plant extraction kits Chloroplast DNA sequencing and analysis – universal primers listed in Taberlet -amplified and sequenced the trnT-trnF chloroplast intergenic spacer -15μL PCR to amplify trnT-trnF region for each individual - 2sets of PCR product: trnT-trnL segment and trnL-trnF segment -Haplotypes differentiated by sequence polymorphism and idel size variation across the trnT-trnF region -Parsimony analysis using PAUP -Trees obtained by random stepwise addition with random addition sequence (RAS) and tree bisection-reconnection branch swapping Microsatellite genotyping – genotyped at 10 microsatellite loci (listed) -Calculated expected and observed heterozygosity, Nei’s unbiased estimator of genetic diversity and genotypic diversity using ARLEQUIN Results: haplotypes into 5 groups Greatest amount of sequence divergence occurs between Delaware and Texas **Nearly all sampled genets exhibited a unique microsatellite genotype (table 2), which is consistent with previous studies that have demonstrated high genotypic diversity among S. alterniflora clones within marshes Focused on the comparison of non-native S. alterniflora (invasive) to native 2) Travis, S. E., C. E. Proffitt, R. C. Lowenfeld and T. W. Mitchell. 2002. A Comparative Assessment of Genetic Diversity among Differently-Aged Populations of Spartina Alterniflora on Restored versus Natural Wetlands. Restoration Ecology. 10: 37-42. Abstract: We collected naturally recolonizing Spartina alterniflora (smooth cord grass) from each of three restored sites and one undisturbed reference site in southwestern Louisiana to assess the impact of wetland restoration on genetic diversity. We used amplified fragment length polymorphisms (AFLPs) to produce 94 polymorphic genetic markers, which were used to characterize genetic diversity as average heterozygosity <H> and the proportion of polymorphic loci <P>. Overall our findings indicate that restored populations of S. alterniflora maintain levels of genetic diversity comparable to natural populations, which should provide some measure of resistance against environmental disturbances. Diversity estimates were lowest for the natural reference site (<H> = 0.1059; <P> = 0.2763), whereas estimates for the three restored sites ranged from <H> = 0.1148 to 0.1256 and <P> = 0.3114 to 0.3202. All sites maintained sufficiently high diversity levels to suggest significant rates of outcrossing. Overall, genetic differentiation among populations was small (Weir and Cockerham’s (H) = 0.0645), with the values from each pairwise comparison among the populations increasing with the geographic distance between sites (range � 0.0490–0.1101). These values indicate an average migration rate of 3.6 migrants, 7/1/2013 either pollen or seeds, per generation. Extra Notes: -Purpose of establishing baseline estimates of genetic diversity for populations Methods: DNA extraction using CTAB-based method Characterized diversity based on amplified fragment length polymorphisms (AFLPs) 2 PCRs – a selective preamplification – adenine AND selective restriction fragment amplification (SRFA) Fixation Index Within sites- it was determined that all of the individuals determined were genetically distinct -> indicator of high levels of clonal diversity Diversity: gene flow, migration by windborne or waterborne movement of pollen/seeds, outcrossing 3) Ayres, D. R. and D. R. Strong. 2001. Origin and Genetic Diversity of Spartina anglica (Poaceae) Using Nuclear DNA Markers. America Journal of Botany. 88: 1863-1867. Abstract: Spartina alterniflora, introduced into the UK in the 1800s, was the seed parent in an interspecific hybridization with S. maritima. The sterile Fl hybrid S. Xtownsendii gave rise to the fertile allopolyploid S. anglica by chromosomal doubling. Previous chromosome, isozyme, and cpDNA surveys did not reveal notable genetic variation within either the parental or the hybrid species. We used nuclear DNA markers (random amplified polymorphic DNA ([RAPD]) and inter-simple sequence repeats(ISSR) to further explore the origin, diversity, and parentage of S. anglica. We found DNA fragments in S. xtownsendii were the aggregate of diagnostic DNA fragments from S. maritima and S. alterniflora, thus confirming its hybrid origin. The S. Xtownsendii genotype was identical to most of the S. anglica individuals analyzed, establishing the genetic concordance of these two taxa. We found widespread genetic variation within S. anglica. This could indicate that S. anglica arose several times, from different S. maritime sires. Alternatively, alleles could have been lost through recombination and/or through loss of entire chromosomes in S. anglica. Finally, all but one S. anglica individual had a S. alterniflora component that was indistinguishable from a S. alterniflora plant extant in Marchwood, UK, leaving open the possibility that this plant is the actual seed parent of S. anglica. Extra Notes: -DNA markers generated through random amplified polymorphic DNA (RAPD) and intersimple sequence repeats (ISSR) analysis can reveal more genetic variation than either the isozyme or cpDNA surveys Methods- DNA Extraction – Proteinase K-based methodology of Guidet PCR – goal to identify RAPD and ISSR primers that strongly and reproducibly amplified species-specific diagnostic DNA fragments in S. alterniflora and S. martima -band was diagnostic if it was present in only one species Genetic similarity relationships were portrayed by unweighted pair group clustering (UPGMA) ***Figure 1*** 7/1/2013 S. anglica contained species-specific DNA fragments from both S. alterniflora and S. martima 4) Baumel, A., M. L. Ainouche and J. E. Levasseur. 2001. Molecular investigations in populations of Spartina anglica C. H. Hubbard (Poaceae) invading coastal Brittany (France). Molecular Ecology. 10: 1689-1701. Abstract: Spartina anglica is a classical example of recent alloploid speciation. It arose during the end of the nineteenth century in England by hybridization between the indigenous Spartina maritima and the introduced East-American Spartina alterniflora. Duplication of the hybrid genome (Spartina x townsendii) gave rise to a vigorous allopolyploid involved in natural and artificial invasions on different continents. Spartina anglica was first recorded in France in 1906, and since then, it has spread all along the western French coast. Earlier studies revealed that native British populations display consistent morphological plasticity and lack of isozyme variation. In this paper, we use different molecular markers (randomly amplified polymorphic DNA, intersimple sequence repeats and restriction patterns from nuclear and chloroplast DNA sequences) to analyse the genetic patterns of the French populations of S. anglica. Our results show that French populations are mainly composed of one ‘major’ multilocus genotype. This genotype is identical to the first-generation hybrid S. x townsendii from England. Losses of few markers from this genotype are observed but are restricted to a few populations from Brittany; it is likely that they appeared independently, subsequent to their introduction. In southern Brittany, no hybrids between S. anglica and S. maritima have been found where the two species co-occur. All French populations of S. anglica display the same chloroplast DNA sequences as S. alterniflora, the maternal genome donor. These findings are consistent with a severe genetic bottleneck at the time of the species formation, as a con- sequence of a unique origin of the species. Both parental nuclear sequences are present in the allopolyploid populations, revealing that for the markers investigated, no extensive changes have occurred in this young species. Extra Notes: -Molecular markers to analyze the genetic patterns on S. anglica – RAPD, ISSR, and restriction patterns from nuclear and chloroplast DNA sequences (RFLP) -Ability to accumulate large volumes of tidal sediments as a pioneer species has led to its introduction in many parts of the world -S. alterniflora = maternal genome donor when hybridization occurred because shares same tRNA L intron sequence in the chloroplast genome -Asks important question: What is the level of the genetic diversity within and among populations, for molecular markers concerning noncoding portions of the genome, which are likely to be more variable than coding regions such as isozyme loci? In other words, is the lack of isozyme variation in S. anglica populations characteristic of just those loci surveyed or does it reflect a general paucity of genetic diversity over the whole genome of this species? Methods: RAPD is a PCR-based DNA fingerprint technique used for estimating genetic variation and relationships among closely related populations or species 7/1/2013 ISSR – amplified from single-primer PCR reactions, amplified markers represent the nucleotide sequence between two microsatellite sites situated n two opposite DNA strands *For RAPD and ISSR data, the presence or absence of the PCR products (bands) were scored by eye **Figure 2** cpDNA – likely to reveal multiple origins when a parental species has alternatively provided the paternal and the maternal genome **Table 4** In the ITS region, 48 nucleotide differences between S. martima and S. alterniflora were found Results: Table 4 and 5 -Weak molecular variation was encountered in the French populations of Spartina anglica analyzed, even though RAPDs usually detect high polymorphism in populations even with small sample sizes or in species that reproduce clonally. -Morphological analysis of plants from populations across the geographical and ecological distribution of the species also suggests little genetically based variation (Thompson et al. 1991a, b). Lack of genetic variation could be the result of either a unique hybridization event between S. alterniflora and S. maritima in southern England, or of multiple events involving similar parental genotypes. ---- So is there limited variation clonally due to the many hybridizations? Or does this just pertain to France? -All the chloroplast sequences investigated in the French populations of S. anglica were identical to those of S. alterniflora and S. x townsendii, in agreement with previous observations of Ferris et al. (1997) who concluded that S. alterniflora is the maternal genome donor to S. anglica. -S. anglica is a model where genetic diversity is restricted at the intergenomic level (two differentiated duplicated genomes in the same nucleus) ***5) Stiller, J. W. and A. L. Denton. 1995. One hundred years of Spartina alterniflora (Poaceae) in Willapa Bay, Washington: random amplified polymorphic DNA analysis of an invasive population. Molecular Ecology. 4: 355-363. Abstract: Spartina altemiflora (smooth cordgrass) has invaded Willapa Bay, Washington, covering bare mud flats and seagrass beds. At current expansion rates it threatens to occupy most of the intertidal habitat in the bay within 40 years. Although a major mitigation effort is underway, nothing is known about the genetic structure of the Willapa Bay S. altemiflora population or the underlying causes for the dramatic increase in seed set that has occurred in the last 15 years. Random amplified polymorphic DNA (RAPD) analysis was assessed as a method of DNA fingerprinting to analyse the genetic history and current structure of the Willapa population. The results suggest that all S. altemiflora clones in Willapa Bay are descended from a single genet. Given the limited genetic variability expected in a clonally founded population, we discuss the utility of RAPDs as genetic markers to explore the large morphological and reproductive differences reported among Willapa clones. Cluster analysis of RAPD fragments was used to show relatedness among S. altemiflora genets and may be helpful in locating the most prolific clones for mitigation efforts. Overall, RAPDs show great potential in genetic analysis of S. altemiflora, particularly in invasive 7/1/2013 populations where isozyme variability is low or nonexistent. Extra Notes: Methods: RAPD and Cluster analysis of RAPD fragments -Articles keep referencing the limited genetic variability present in the Spartina populations and that the reproductive and morphological differences among the physically distinct patches are due to environmental factors -In this paper, it assesses the use of RAPD Methods: -Sampled at 5m intervals beginning in center to assess the potential for variability of RAPD profiles within a presumed single genet -DNA extraction – young leaves using CTAB procedure -Primer and template concentration templates were run to achieve optimal RAPD patterns while minimizing artefactual bands **Table 1 and 2** -UPGMA cluster analysis – Figure 2 Results: **Note** - RAPDs are highly sensitive to PCR conditions, with proper controls they have been shown to be reliable for genotype fingerprinting and parentage analysis -No variability was seen between replicate ramets taken from the same clone -Figure 3 – gels of RAPDs generated by 3 primers -Explanation of genetic differences between center and outer portions of the alpha patch is a die-back at the center and then recolonization after at least one sexual generation **In allozyme studies of a variety of animals and plants, the proportion of polymorphic loci in a population is positively correlated with, and greater than, the proportional of heterozygous loci per individual -Phenotypic plasticity and microenvironmental differences 6) Anttila, C. K., R. A. King, C. Ferris, D. R Ayres and D. R. Strong. 2000. Reciprocal hybrid formation of Spartina in San Francisco Bay. Molecular Ecology. 9: 765-770. Abstract: Diversity in the tRNALEU1 intron of the chloroplast genome of Spartina was used to study hybridization of native California cordgrass, Spartina foliosa, with S. alterniflora, introduced to San Francisco Bay ! 25 years ago. We sequenced 544 bases of the tRNALEU1 intron and found three polymorphic sites, a pyrimidine transition at site 126 and transversions at sites 382 and 430. Spartina from outside of San Francisco Bay, where hybridization between these species is impossible, gave cpDNA genotypes of the parental species. S. foliosa had a single chloroplast haplotype, CCT, and this was unique to California cordgrass. S. alterniflora from the native range along the Atlantic coast of North America had three chloroplast haplotypes, CAT, TAA, and TAT. Hybrids were discriminated by random amplified polymorphic DNA (RAPD) phenotypes developed in a previous study. We found one hybrid that contained a cpDNA haplotype unknown in either parental species (TCT). The most significant finding was that hybridization proceeds in both directions, assuming maternal inheritance of cpDNA; 26 of the 36 hybrid Spartina plants from San Francisco Bay contained the S. foliosa haplotype, nine contained haplotypes of the 7/1/2013 invad- ing S. alterniflora, and one had the cpDNA of unknown origin. Furthermore, cpDNA of both parental species was distributed throughout the broad range of RAPD phenotypes, suggesting ongoing contributions to the hybrid swarm from both. The preponderance of S. foliosa cpDNA has entered the hybrid swarm indirectly, we propose, from F1s that backcross to S. foliosa. Flowering of the native precedes by several weeks that of the invading species, with little overlap between the two. Thus, F1 hybrids would be rare and sired by the last S. foliosa pollen upon the first S. alterniflora stigmas. The native species produces little pollen and this has low viability. An intermediate flowering time of hybrids as well as pollen that is more vigourous and abundant than that of the native species would predispose F1s to high fitness in a vast sea of native ovules. Thus, spread of hybrids to other S. foliosa marshes could be an even greater threat to the native species than introductions of alien S. alterniflora. Extra Notes: **READ for methods, not the invasive or hybrid detail** -Chloroplast DNA is strictly maternally inherited, the direction of hybridization can be deduced -discriminated parental species and hybrids according to RAPD techniques – Table 2 -PCR amplification using universal primers from Taberlet et al. (1991) -screened for variation of tRNA(LEU1) intron of chloroplast genome 7) Chelaifa, H., F. Mahé and M. Ainouche. 2010. Transcriptome divergence between the hexaploid salt-marsh sister species Spartina maritima and Spartina alterniflora (Poaceae). Molecular Ecology. 19: 2050-2063. Abstract: Invasive species are ideal model systems to investigate the evolutionary processes associated with their ecological success by comparison with closely related species. In this article, we explore transcriptome evolution following divergence between two closely related salt-marsh species, the invasive Spartina alterniflora (native to the East- American Atlantic coast, introduced in several continents) and the declining Spartina maritima (native to the Euro-African Atlantic coast). We have explored the utility of cross-species hybridization microarrays using rice (Oryza sativa) oligo-microarrays to compare leaf expression patterns between these species. Coding sequence comparisons from 10 nuclear genes (2256 bp) revealed that nucleotide divergence between Spartina and Oryza range from 8% to 14%. More than 70% of the 60-mer oligonucleotide sequences spotted on the rice microarray exhibited stable and repeatable patterns when hybridized against Spartina RNA. In total, 9353 (44.5%) genes on the array hybridized with both species S. maritima and S. alterniflora. Among these genes, 1247 genes were found to be differentially expressed between the two Spartina species, most of them (957) being up-regulated in S. alterniflora. In particular, developmental and cellular growth genes (gene ontology, biological process) were highly up-regulated in S. alterniflora and down-regulated in S. maritima, whereas genes involved in stress response were up- regulated in S. maritima. Our findings indicate the suitability of cross-species microarray hybridization between Spartina and O. sativa and reveal the extent of leaf transcriptome evolution that took place 7/1/2013 during the divergence between S. alterniflora and S. maritima. Expression patterns are consistent with the morphological differentiation and differential expansion of the two species. Extra Notes: -Microarrays – tool in the comparison of genome-wide expression patterns -EST – Expressed sequence tags doesn’t exist for Spartina Methods: include arrays of anonymous cDNA clones and the reliance on sequence conservation in homologous coding regions of model and nonmodel species -Using the Agilent 4x44K 60-mer oligo-microarrays designed from the sequenced rice genome -RNA extraction -Table 1 – RT-PCR analysis results -cDNA library was created -Lots of info on Spartina – background Continue reading for further analysis if appropriate… 8) Freshwater, D. W. 1988. Relative genome-size differences among populations of Spartina alterniflora Loisel (Poaceae) along East and Gulf Coasts of U.S.A. Journal of Experimental and Marine Biology. 120: 239-246. Abstract: Flow cytometric analysis of mithramycin-stained rhizome nuclei was used to compare relative amounts of DNA among 18 populations of Spartina alterniflora Loisel (Poaceae) from the East and Gulf Coasts of the U.S.A. The DNA amount increases significantly with increasing latitude. These findings support the general model of genome size change with latitude. Extra Notes: -Measurements of genome size in many plant species show an extensive variation in DNA content per nucleus -If variations in DNA content are of adaptive significance they would most likely occur in species with wide ranges in distribution, environment, physiological traits, and morphological features Objective: to determine if populations of S. alterniflora vary in relative DNA content along the East and Gulf Coasts -Collection and analysis of rhizome because has less variability in fluorescence peaks than leaf or root -Methods: Flow cytometric analysis of mithramycin-stained rhizome nuclei -Results indicated a positive correlation of increasing DNA amount with increasing latitude ***Figure 2 ** - gradual trend of DNA change observed in the data supports the idea of changes which proceed in small steps by the accumulation of small deletions or duplications of the genome 9) Utomo, H. S., I. Wenefrida, M. D. Materne and S. A. Harrison. 2009. Genetic diversity and 7/1/2013 population genetic structure of saltmarsh Spartina alterniflora from four coastal Louisiana basins. Aquatic Botany. 90: 30-36. Abstract: Seventy-two Spartina alterniflora accessions originating from four coastal Louisiana basins (18 accessions per basin) were used to evaluate the genetic structure of this native perennial low-intertidal plant species. The objective of this study was to determine the population genetic structure and diversity of S. alterniflora accessions originating from these four basins using amplified fragment length polymorphism (AFLP) markers. A total of 250 unambiguous and highly repeatable AFLP markers, 186 of which (74.4%) were polymorphic, were obtained using four primer combinations. Overall, pairwise similarity estimates between accessions ranged from 0.70 to 0.93 (average = 0.80) with only a small portion of alleles (0.54– 1.08%) unique to each basin. The average Hs (genetic diversity within coastal basins) was 0.20 with an Hs values of 0.19, 0.20, 0.20, and 0.21 for Mermentau, Terrebonne, Calcasieu, and Barataria-Breton basin, respectively. AMOVA analysis showed no genetic structure among basins, with the majority of genetic variation, 96.6%, residing within the basins. There was no indication of isolation by distance. Our results suggest that maintaining high levels of genetic diversity can be accomplished through the use of an adequate number of S. alterniflora samples collected within any large basin. Choosing parental lines from several Louisiana coastal basins for breeding purposes may not significantly increase genetic variability Extra Notes: -Populations of S. alterniflora in the Atlantic coast have different flowering behavior, and the differences in flowering phenology became a natural barrier for genetic exchange to occur between the Atlantic groups -DNA extraction – young piece of leaf tissue – Dneasy kit Methods: amplified fragment length polymorphism – AFLP – followed by gel analysis -Neighbor-joining method to estimate phylogeny **Figure 1** 10) Ainouche, M. L., A. Baumel, A. Salmon and G. Yannic. 2003. Hybridization, polyploidy and speciation in Spartina (Poaceae). New Phytologist. 161:165-172. Abstract: Hybridization and polyploidy are well illustrated in the genus Spartina. This paper examines how recent molecular approaches have helped our understanding of the past and recent reticulate history of species, with special focus on allopolyploid spe- ciation. Spartina species are tetraploid, hexaploid or dodecaploid perennials, most of them being native to the New World. The molecular phylogeny indicates an ancient split between the tetraploid and the hexaploid species, with S. argentinensis as sister to the hexaploid lineage. Recent hybridization and polyploidization events involved hexaploid species, resulting from introductions of the east-American S. alterniflora. In California, ongoing hybridizations with its sister species S. foliosa result in intro- gressant hybrid swarms. In Europe, hybridization with S. maritima resulted in S. × neyrautii (France) and S. × townsendii (England), with. S. alterniflora as the maternal parent. The allopolyploid S. 7/1/2013 anglica resulted from chromosome doubling of S. × townsendii. This young allopolyploid contains divergent homoeologous sub- genomes that have not undergone significant changes since their reunion. Hybridization, rather than genome duplication, appears to have shaped the allopolyploid genome at both the structural and epigenetic levels. Clonal 1) Xiao, D., L. Zhang and Z. Zhu. 2010. The range expansion patterns of Spartina alterniflora on salt marshes in the Yangtze Estuary, China. Estuarine, Coastal, and Shelf Science. 88: 99104. Abstract: The range expansion patterns of Spartina alterniflora and the roles which sexual reproduction and asexual propagation play in range expansion were investigated at the Chongming Dongtan nature reserve in the Yangtze Estuary, China. Two range expansion patterns of S. alterniflora at its advancing fronts could be found (1) S. alterniflora mudflat front (S-M) and (2) S. alterniflora-Scirpus mariqueter-mudflat front (S-S-M). One feature revealed by this study was that a flush of seedling recruitment and establishment in spring was a crucial way for S. alterniflora to colonize new habitats and achieve a fast rate of range expansion. The mean number of seedlings recruited at the S-M front was much higher than that at the S-S-M front. Once established, the survivorship of seedlings was high, both at the S-M and S-S-M fronts. The established seedlings formed new tussocks quickly by vegetative tillering and growth of rhizomes and these finally merged into dense meadows. The mean distance of range expansion of S. alterniflora, after one growing season at the S-M front, was 25.4 +/- 3.1 m yr^-1�and 2.7+/-0.5 m yr^-1 at the S-S-M front. Sexual reproduction by seedlings and asexual propagation by tillering and growth of rhizomes were the two main means by which S. alterniflora could maintain a fast rate of range expansion on the salt marshes of the Yangtze Estuary. The colonization behaviors of S. alterniflora on advancing fronts differed as a reaction to various external and internal factors. The impact of abiotic and biotic factors governing the range expansion of S. alterniflora and its implications for the spatial structure of tidal wetlands are discussed. 2) Dai, T. and R. G. Wiegert. 1996. Ramet Population Dynamics and Net Aerial Primary Productivity of Spartina Alterniflora. Ecology. 77: 276-288. Abstract: Ramet dynamics and net aerial primary productivity (NAPP) were studied in samples of Spartina alterniflora (smooth cordgrass) at Sapelo Island, Georgia. Three populations were compared: tall, short, and short with nitrogen fertilization in spring (short/ N). Tall and short S. alterniflora populations had different demographic characteristics. The short population had a shorter leaf longevity (49 vs. 72 d) and a higher leaf turnover than the tall population, which may be due to high salinity and nitrogen limitation in the high marsh. Although the average ramet longevities of tall and short populations were similar (231 and 204 d, respectively), cohorts of the tall population that emerged early in the growing season had a significantly longer average life-span than those of the short population, 7/1/2013 probably because they had more support from belowground reserves that led to a higher initial survival rate. Leaf number, leaf area, shoot density, and biomass production of the short population were greatly increased by spring nitrogen fertilization, but the longevity of leaves and ramets was little affected. Using the demographic data and phytometric equations (nondestructive method), new growth was found throughout the year in S. alterniflora populations at Sapelo Island. The average dry mass NAPP was estimated to be 1105, 2244, and 1520 g.m-2.yr-1 for the short, short/N, and tall populations, respectively. Because of its higher leaf turnover, the short population had a higher leaf to stem production ratio than the tall population. NAPP estimates obtained by nondestructive methods usually lie between overestimates and underestimates from harvest methods, indicating that non- destructive methods give accurate estimates of NAPP for salt marshes. Using the highest spring aerial production rates, the upper limits of annual dry mass total production of S. alterniflora at Sapelo Island were calculated as 2555 g/m2 for the short population and 4526 g/m2 for the tall population. These limits are lower than many previous estimates of annual total primary production for S. alterniflora. Dieback 1) Alber, M., E. M. Swenson, S. C. Adamowicz and I. A. Mendelssohn. 2008. Salt Marsh Dieback: An overview of recent events in the US. Estuarine, Costal, and Shelf Science. 80:111. Abstract: This paper provides an overview of the marsh dieback events that have been observed along the east and gulf coasts of the U.S. over the past decade. It is likely that some of the recently reported changes in marsh vegetation were affected by physical or biotic disturbances that are known to generate bare areas, such as overgrazing or wrack smothering. Other areas may be experiencing a state change such as that caused by longterm changes in sea level. However, sites in many areas are not readily explained by these causes and are considered to have experienced ‘‘sudden dieback.’’ In such cases, there are observations that the above-ground plant material thinned or browned or, in some cases, failed to re-emerge in the spring; the dieback occurred over a period of months and usually affected multiple sites within the area; and there is evidence that these events are transient (through successful transplants or natural regrowth/ recovery), although some areas take years to recover. We explored the potential linkage of dieback with drought (as characterized by the Palmer Severity Drought Index), and found that there is evidence for an association in the southeast (GA and SC) and the Gulf (LA), but not in the mid-Atlantic (DE, VA) or northeast (ME, RI, CT). We also review the evidence for potential causes of sudden dieback, including changes in soil chemistry, fungal pathogens, top–down consumer controls, and multiple stressors. There is currently no single explanation that can be applied to recent dieback. We highlight the need for the development of improved diagnostics that will allow us to better classify dieback areas and provide evidence for (or against) potential causes. 7/1/2013 2) White, S. N. and M. Alber. 2009. Drought-Associated Shifts in Spartina alterniflora and S. Cynosuroides in the Altamaha River Estuary. Wetlands. 29:215-224. Abstract: In estuarine systems, informed water resource management decisions rely, in part, on evaluating how changes in freshwater inflow and salinity affect a selected management target (i.e., habitat or organismal changes). This study examined the distribution of Spartina alterniflora and S. cynosuriodes species along the Altamaha River estuary in Georgia in association with an extensive drought, during which time freshwater inflow decreased considerably and salty water encroached into previously brackish areas. Bankside vegetation was surveyed along the length of the estuary at the beginning and end of the drought (2000, 2002), and again in 2004 after flows had increased. In addition, a removal experiment, in which one or the other plant was removed from naturally mixed communities, was conducted during the drought (2001–2002). In all surveys, S. cynosuroides densities decreased at a location that corresponded to where average high tide salinities were 14 psu, such that the downstream border shifted from approximately 3 to 6 km from the mouth of the river between 2000 and 2002 and then back to 3 km in 2004. Although the peak density of S. alterniflora also shifted upstream between 2000 and 2002, upstream densities were high in 2004 and plant distribution did not correspond with salinity. In the removal experiment, densities and relative % cover of S. alterniflora increased in all treatments (including controls), whereas densities of S. cynosuroides remained relatively constant except in treatments where it was purposely removed. We interpret these results to suggest that S. alterniflora is a strong invader that can expand into new habitat under stressful (increased salinity, low flow) conditions, and that once it is established it can co-exist with S. cynosuroides. The more rapid response of S. cynosuroides (shifts occurred within 2 years) suggests that its downstream limit is a potential indicator of changes in inflow conditions in this system. 3) Edwards, K. R., S. E. Travis and C. E. Proffitt. 2005. Genetic Effects of a Large-Scale Spartina alterniflora (Smooth Cordgrass) Dieback and Recovery in the Northern Gulf of Mexico. Estuaries. 28: 204-214. Abstract: A large-scale dieback event struck marshes along the northwestern Gulf of Mexico coast during summer 2000, in apparent response to a prolonged and severe drought. Along the Louisiana coast, large areas of the dominant marsh species, Spartina alterniflora, turned brown, followed by death of at least the aboveground structures or entire plant mortality. Key ecological and genetic measures were studied in a dieback-affected marsh in southwest Louisiana (C83 marsh, Sabine National Wildlife Refuge), for which existed predieback ecologic and genetic datasets. Effects on genetic diversity only were studied in a second set of sites in southeastern Louisiana (near BayJunop), where the dieback was more widespread. We hypothesized that stem density, live aboveground biomass, and genetic diversity would be significantly reduced compared to predieback conditions and to nearby unaffected marshes. Stem densities and biomass levels approached predieback conditions 14 months after first observance of the dieback in the Sabine marsh and were similar to or 7/1/2013 exceeded the same measures for a nearby unaffected marsh. DNA extracted from leaf samples in the Sabine and Bay Junop sites was used to construct genotype profiles using AFLPs and analyzed using the complement of Simpson's Index (1-D), the richness measure G/N, average heterozygosity <H>, and the estimated proportion of polymorphic genes <P>. Genetic diversity was relatively unaffected by the dieback at either the Sabine or Bay Junop sites. Evidence from field observations and the results of the genetic analyses suggest that seedling recruitment is an important factor in the recovery of both the Bay Junop and C83 sites, although re-growth from surviving below-ground rhizomes appeared to dominate recovery at the latter site. Survival of below-ground structures, leading to the rapid recovery observed, indicates a high level of resilience of the Sabine marsh to droughtinduced stress. Still, the genetic diversity of S. alterniflora-dominated marshes may be promoted by occasional disturbance events, which produce open areas in which seedling recruitment can occur. Microsatellites General: - Present high levels of inter – and intra – specific polymorphism -Higher variability of microsatellites is due to a higher rate of mutation compared to other neutral regions of DNA -Amplified for identification by the polymerase chain reaction (PCR) process, using the unique sequences of flanking regions as primers -Mutation in microsatellite alleles is biases in the sense that larger alleles contain more bases and are therefore likely to be mistranslated in DNA replication -Most common cause of length changes in short sequence repeats in replication slippage, caused by mismatches between DNA strands while being replicated during meiosis -Microsatellites within introns also influence phenotype 1) Rafalski, J. A. and S. T. Tingey. 1993. Genetic diagnostics in plant breeding: RAPDs, microsatellites and machines. TIG August. 9: 275-280. Abstract: No abstract. Just a review on Microsatellite repeats, RAPD, and CAPs. Extra notes: -RFLP – restriction fragment length polymorphisms – markers are codominant and provide complete genetic information at a single locus -The RAPD amplification reaction is performed on a genomic DNA template and primed by an arbitrary oligonucleotide primer, resulting in the amplification of several discrete DNA products. - Using short primers and low annealing temperatures ensures that several sites, randomly distributed in the genome, give rise to amplification products. - RAPD technology provided researchers with a quick and efficient screen for DNA sequence-based polymorphisms at a very large number of loci. 7/1/2013 ***Table 1: - Microsatellite repeats (also known as simple sequence repeats), in particular the dinucleotide repeats (AC)n, (AG)n and (AT)n, have recently been shown to be abundant and highly polymorphic in eukaryotic genomes 22-24. **Box 1, page 277 - Morgante and Olivieri30 found that simple sequence repeats are very polymorphic and that AT repeats are more numerous than AC repeats, at least among the DNA sequences in genome databases. -CAPS – Cleaved Amplified Polymorphic sequence - partial DNA sequence information for the locus of interest is used to create a set of PCR primers. 2) Selkoe, K. A. and R. J. Toonen. 2006. Microsatellites for ecologists: a practical guide to using and evaluating microsatellite markers. Ecology Letters. 9: 615-629. Abstract: Recent improvements in genetic analysis and genotyping methods have resulted in a rapid expansion of the power of molecular markers to address ecological questions. Microsatellites have emerged as the most popular and versatile marker type for ecological applications. The rise of commercial services that can isolate microsatellites for new study species and genotype samples at reasonable prices presents ecologists with the unprecedented ability to employ genetic approaches without heavy investment in specialized equipment. Nevertheless, the lack of accessible, synthesized information on the 7/1/2013 practicalities and pitfalls of using genetic tools impedes ecologists’ ability to make informed decisions on using molecular approaches and creates the risk that some will use microsatellites without understanding the steps needed to evaluate the quality of a genetic data set. The first goal of this synthesis is to provide an overview of the strengths and limitations of microsatellite markers and the risks, cost and time requirements of isolating and using microsatellites with the aid of commercial services. The second goal is to encourage the use and consistent reporting of thorough marker screening to ensure high quality data. To that end, we present a multistep screening process to evaluate candidate loci for inclusion in a genetic study that is broadly targeted to both novice and experienced geneticists alike. Extra Notes: -Microsatellites have emerged as one of the most popular choices for these studies in part because they have the potential to provide contemporary estimates of migration, have the resolving power to distinguish relatively high rates of migration from panmixia, and can estimate the relatedness of individuals. -Microsatellites are tandem repeats of 1–6 nucleotides found at high frequency in the nuclear genomes of most taxa. -Other names: simple sequence repeats (SSR), variable number tandem repeats (VNTR), and short tandem repeats (STR) -DNA surrounding a microsatellite locus is termed the flanking region -Short stretches of DNA, called oligonucleotides or primers, can be designed to bind to the flanking region and guide the amplification of a microsatellite locus with polymerase chain reaction (PCR). -Microsatellites are of particular interest to ecologists because they are one of the few molecular markers that allow researchers insight into fine-scale ecological questions. -a molecular marker must fundamentally be selectively neutral and follow Mendelian inheritance in order to be used as a tool for detecting demographic patterns, and these traits should always be confirmed for any marker type 7/1/2013 -The stability of DNA compared with enzymes allows the use of simple tissue preservatives (such as 95% ethanol) for storage. The stability of DNA compared with enzymes allows the use of simple tissue preservatives (such as 95% ethanol) for storage. - Thus, taking multiple samples of the genome by combining the results from many loci provides a more precise and statistically powerful way of comparing populations and individuals. - Although AFLP, allozymes and random amplified polymorphic DNA (RAPD) techniques are also multilocus, none of them have the resolution and power of a multilocus microsatellite study (but for distinct reasons; see Sunnucks 2000). - Microsatellites have become so popular because they are single locus, co-dominant markers for which many loci can be efficiently combined in the genotyping process to provide fast and inexpensive replicated sampling of the genome. -Drawbacks: -the region where the primer binds must be identical, with few or no mutations causing interindividual differences. - several statistics based on estimates of allele frequencies (e.g. FST and RST) rely explicitly on a mutation model -the stepwise mutational model (SMM), adds or subtracts one or more repeat units from the string of repeats at some constant rate to mimic the process of errors during DNA replication that generates mutations, creating a Gaussian-shaped allele frequency distribution -requires the assumption that all distinct alleles differ in length - Homoplasy dampens the visible allelic diversity of populations and may inflate estimates of gene flow when mutation rate is high -homoplasy is often a minimal source of bias for population genetic studies limited to populations with a ÔshallowÕ history or moderate effective population size, as the chance of homoplasy is proportional to the genetic distance of two individuals or populations -Finding a useful DNA marker locus requires identifying a region of the genome with a sufficiently high mutation rate that multiple versions (alleles) exist in a given population, and which is also located adjacent to a low mutation rate stretch of DNA that will bind PCR primers in the vast majority (approaching 100%) of individuals of the species. -There is a searchable database online for any microsatellite primers published in this journal (http://tomato.bio.trinity.edu/). The sequences themselves are archived in GenBank, and are often submitted long before their use appears in published studies. GenBank can be searched with a web-based engine run by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) - In our survey of 50 recent microsatellite studies, 28% of studies mention the importance of quality control screening but fail to report any results of statistical tests. 7/1/2013 - The most commonly reported test of loci is conformity to HWE, in which observed genotype frequencies are com- pared with the frequencies expected for an ideal population - the more loci included in a study, the more reliable the resultant data set will be. 3) Zane, L., L. Bargelloni and T. Patarnello. 2002. Strategies for microsatellites isolation: a review. Molecular Ecology. 11: 1-16. Abstract: In the last few years microsatellites have become one of the most popular molecular markers used with applications in many different fields. High polymorphism and the relative ease of scoring represent the two major features that make microsatellites of large interest for many genetic studies. The major drawback of microsatellites is that they need to be isolated de novo from species that are being examined for the first time. The aim of the present paper is to review the various methods of microsatellite isolation described in the literature with the purpose of providing useful guidelines in making appropriate choices among the large number of currently available options. In addition, we propose a fast and easy protocol which is a combination of different published methods. 4) Gupta, P. K. and R.K. Varshney. 2000. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Molecular Biology. Abstract: In recent years, a variety of molecular markers, based on microsatellites or simple sequence repeats (SSRs) have become the markers of choice, thus necessitating their development and use in a variety of plant systems. In this review, the basic principles underlying different hybridization-based (oligonucleotide fingerprinting) and PCR based approaches (STMS, MP-PCR, AMP-PCR/ ISSR/ ASSR, RAMPs/ dRAMPs, SAMPL), making use of microsatellites, have been outlined. Different methods for enrichment of genomic libraries for microsatellites have also been outlined. Relevant literature on the subject, giving a summary of results obtained using each approach, has been reviewed and critically discussed. The review also includes a discussion on literature, which deals with the use of microsatellites in genome mapping, gene tagging, DNA fingerprinting, characterization of 7/1/2013 germplasm and cytogenetics research. Special emphasis has been laid on the genome of bread wheat, where the work done in the authors’ own laboratory has also been briefly reviewed. 5) Huang, B. X., R. Peakall and P. J. Hanna. 2000. Analysis of genetic structure of blacklip abalone (Haliotis rubra) populations using RAPD, minisatellite and microsatellite marker. Marine Biology. 136: 207-216. Abstract: We investigated the utility of three polymerase chain-reaction (PCR)-based DNA molecular markers in analysing genetic structure of the populations of the blacklip abalone Haliotis rubra (Leach) of Victoria, Australia. The DNA markers included 84 randomly amplified polymorphic DNA (RAPD) bands amplified using six random primers, two minisatellites, GHR (putative growth-hormone-gene-repeat) and MIPR (putative molluscainsulin-like peptide-gene-repeat), and three microsatellites, RUBGT1 [containing (GT)n repeats], RUBCA1 [containing (CA)n repeats] and RUBGACA1 [containing (GACA)n repeats]. All three types of DNA markers revealed significant subdivision in the H. rubra populations along the coastline. This is postulated as being related to the abalone's relatively short pelagic period and limited dispersion. Further analysis revealed that a Point Cook population sampled from within the semi-enclosed Port Phillip Bay was distinct from two other central zone populations (Apollo Bay and Cape Schanck). The genotypes of microsatellites indicated excessive homozygotes across all the populations at all three microsatellite loci, and possible causes such as larval recruitment pattern and asynchronous spawning are discussed. The excessive homozygotes recorded for the three microsatellite loci contrast with those observed in the minisatellite loci GHR and MIPR, the heterozygosities of which were at Hardy±Weinberg equilibrium. 6) Biswas, M. K., L. Chai, M. H. Amar, X. Zhang, and X. Deng. 2011. Comparative analysis of genetic diversity in Citrus germplasm collection using AFLP, SSAP, SAMPL and SSR markers. Scientia Horticulturae. 129: 798-803. Abstract: In this study we evaluate the informativeness and efficiency of Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), Selectively Amplified Microsatellite Polymorphic Loci (SAMPL) and Simple Sequence Repeat (SSR) markers for genetic diversity, phylogenetic relationship among the Citrus species and mapping ability of the marker system. The SSR exhibited relatively higher level of polymorphism information content in terms of the expected heterozygosity, than that of the AFLPs, SSAPs and SAMPLs. For each marker system, average level of the discriminating potential was very close to the actual discriminating potential. Similarity matrices showed weak, yet significant correlations when Mantel’s test was applied. The highest positive (0.72) correlation was found between the AFLP and SSAP markers. The SSR and SAMPL markers were poorly correlated. The dendrogram topology among the four marker systems had high similarity. Taken together, the SSAP and SAMPL were highly efficient in detecting genetic similarity in Citrus, while the SSR may be more useful for segregation 7/1/2013 studies and genome mapping in Citrus. The SSAP and SAMPL markers could be useful for Citrus genome mapping in combination with AFLP and SSR markers. To our knowledge, this was the first detail report of a comparison of performances among AFLP, SSR and retrotrasposon based molecular marker technique on a set of samples of Citrus. Our result provides guidance for future efficient use of these molecular methods in genetic analysis of Citrus sp. and its relatives. 7) Zakaria, L., H. Kulaveraaingham, T. S. Guan, F. Abdullah, and H. Y. Wan. 2005. Random Amplified Polymorphic DNA (RAPD) and Random Amplified Micorsatellite (RAMS) of Ganoderma from Infected Oil Palm and Coconut Stumps in Malaysia. Asia Pacific Journal of Molecular Biology and Biotechnology. 13: 23-34. Abstract: RandomamplifiedpolymorphicDNA(RAPD)andrandomamplifiedmicrosatellite(RAM S)analyseswereused to determine the genetic relatedness within and between Ganoderma boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps from different locations in Malaysia. RAPD analysis using four random primers (5’ACCTGGACAC3', 5’CAGCGACAAG3', 5’AGAGGGCACA3' and 5’TGACGGCGGT3') showed variations of banding patterns within and between the isolates from oil palm and coconut stumps, indicating that they were genetically heterogeneous. There was no specific banding pattern that could differentiate between G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. RAMS analysis using four microsatellite primers, 5’BDB(ACA)5, 5’DD(CCA)5, 5’DHB(CGA)5 and 5’YHY(GT)5G, also showed variable banding patterns among the isolates from infected oil palm and coconut stumps. However, five common bands i.e. two bands (900 bp and 1200 bp) produced by primer (CGA)5, one band (1400 bp) by primer (ACA)5 and two bands (350 bp and 380 bp) by primer (CCA)5 were shown by all the G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps. Dendrograms from cluster analysis based on UPGMA of RAPD and RAMS data showed that G. boninense isolates from infected oil palm and Ganoderma sp. from coconut stumps did not cluster separately into two distinct clusters, but were clustered together, which indicated that both groups of Ganoderma are closely related. The finding that the Ganoderma isolates from coconut stumps are closely related to G. boninense isolates from infected oil palm would have an important bearing in the formulation of disease control measures and replanting procedures, especially in areas where the previous crop was coconut. 8) Simbaqueba, J., P. Sanchez, E. Sanchez, V. Zarantes, M. I. Chacon, L. S. Barrero and L. Maino-Ramirez. 2011. Development and Characterization of Microsatellite Markers for the Cape Gooseberry Physalis peruviana. PLoS One 6(10): 1-6. Abstract: Physalis peruviana, commonly known as Cape gooseberry, is an Andean Solanaceae fruit with high nutritional value and interesting medicinal properties. In the present study we report the development and characterization of microsatellite loci from a P. peruviana commercial Colombian genotype. We identified 932 imperfect and 201 perfect Simple 7/1/2013 Sequence Repeats (SSR) loci in untranslated regions (UTRs) and 304 imperfect and 83 perfect SSR loci in coding regions from the assembled Physalis peruviana leaf transcriptome. The UTR SSR loci were used for the development of 162 primers for amplification. The efficiency of these primers was tested via PCR in a panel of seven P. peruviana accessions including Colombia, Kenya and Ecuador ecotypes and one closely related species Physalis floridana. We obtained an amplification rate of 83% and a polymorphic rate of 22%. Here we report the first P. peruviana specific microsatellite set, a valuable tool for a wide variety of applications, including functional diversity, conservation and improvement of the species.
