Modeling the technology of identification of wine materials and wines by PCR analysis of microsatellite loci of grapevine chloroplast DNA

Использование полиморфных микросателлитных локусов ДНК является одним из подходов к аутентификации виноматериалов и вин. При этом SSR-маркеры хлоропластной ДНК имеют большую копийность мишени на клетку и менее подвержены деградации из-за содержания в органеллах с двойной мембраной. Целью настоящей работы являлось моделирование технологии идентификации виноматериалов и вин ПЦР-анализом микросателлитных локусов хлоропластной ДНК винограда. Подобраны условия экстракции нуклеиновых кислот, постановки ПЦР с соответствующими наборами праймеров и электрофоретической детекции, направленные на практическое воспроизведение генетического тестирования пробоподготовленного биоматериала из осаждаемого винного дебриса. Представлены наглядные результаты выравнивания частичных нуклеотидных последовательностей аллелей микросателлитных локусов хлоропластной ДНК Vitis vinifera L. Проанализирована разделяющая способность метода горизонтального электрофореза в геле «Spreadex EL 300» in silico моделированием генерируемых аллельспецифичных фрагментов, позволяющая идентифицировать известные хлоротипы винограда даже при постановке ПЦР с ограниченными наборами праймеров, нацеленных на локусы cpSSR3, cpSSR5, cpSSR10, NTCP12 и ccSSR9. The use of polymorphic microsatellite DNA loci is one of the approaches to the authentication of wine materials and wines. At the same time, SSR markers of chloroplast DNA have a large target copy number per cell and are less susceptible to degradation due to their content in organelles with a double membrane. The aim of this work was to simulate the technology of identification of wine materials and wines by PCR analysis of microsatellite loci of grapevine chloroplast DNA. The conditions for the extraction of nucleic acids, PCR with the corresponding sets of primers and electrophoretic detection were selected, aimed at the practical reproduction of genetic testing of the sample prepared biomaterial from the precipitated wine debris. Illustrative results of the alignment of partial nucleotide sequences of alleles of microsatellite loci of Vitis vinifera L. chloroplast DNA are presented. The separating ability of the method of horizontal electrophoresis in «Spreadex EL 300» gel by in silico modeling the generated allele-specific fragments, which makes it possible to identify the known chlorotypes of grapevine with a limited set of primers targeting loci (cpSSR3, cpSSR5, cpSSR10, NTCP12 and ccSSR9) has been analyzed.

Development of SSR Markers and Genetic Diversity Evaluation of Mycocentrospora Acerina Causing Round Spot of Panax Notoginseng in Yunan Province, China

Sanqi round spot, which is caused by Mycocentrospora acerina, is a destructive disease limits the production of Panax notoginseng in Yunnan province of China. However, the disease has not been studied comprehensively. In the current study, we identify M. acerina polymorphic microsatellite markers using CERVUS 3.0 and compare the genetic diversity of its isolates from P. notoginseng round spot using Simple Sequence Repeat (SSR) markers and polyacrylamide gel electrophoresis. Thirty-two SSR markers with good polymorphism were developed using MISA and CERVUS 3.0. The genetic diversity of 187 M. acerina isolates were evaluated using 14 representative SSR primers, and the polymorphic information content values of 14 sites ranged from 0.813 to 0.946, with a total of 264 alleles detected at 14 microsatellite loci. The average expected heterozygosity was 0.8967. The genetic diversity of M. acerina in Yunnan province does not reflect geographic specificity.

Using neutral and outlier loci to uncover the genetic structure of 'Mytilus galloprovincialis' populations from the eastern Adriatic coast using population genetic and seascape genetic approaches

<p>The genetic variation of the Mediterranean blue mussel, Mytilus galloprovincialis from the eastern coastline of the Adriatic Sea was investigated using polymorphic microsatellite markers. In total, 843 individuals were sampled from 18 populations representing a variety of coastal environments in Croatia, Bosnia-Herzegovina, Montenegro and Albania. Neutral loci revealed low levels of genetic structure in this continuously distributed species, while one outlier locus, MGE7, which may be under selection, provided strong evidence of genetic structure. The distribution of one of the alleles of this locus, MGE7²⁴³, was significantly correlated with latitude. A genetic seascape analysis using 9 environmental and 3 geospatial variables revealed a strong association between MGE7²⁴³ and three highly correlated environmental variables, maximum sea surface temperature, minimum salinity and maximum chlorophyll-a. This association was maintained for homozygous genotypes for the MGE7²⁴³ allele, but not for heterozygotes, providing further evidence that the locus MGE7 may be under selection or closely linked to a gene under selection. These findings highlight how previously unrecognised genetic structure can be identified through the use of genetic seascape approaches.</p>

Using neutral and outlier loci to uncover the genetic structure of 'Mytilus galloprovincialis' populations from the eastern Adriatic coast using population genetic and seascape genetic approaches

<p>The genetic variation of the Mediterranean blue mussel, Mytilus galloprovincialis from the eastern coastline of the Adriatic Sea was investigated using polymorphic microsatellite markers. In total, 843 individuals were sampled from 18 populations representing a variety of coastal environments in Croatia, Bosnia-Herzegovina, Montenegro and Albania. Neutral loci revealed low levels of genetic structure in this continuously distributed species, while one outlier locus, MGE7, which may be under selection, provided strong evidence of genetic structure. The distribution of one of the alleles of this locus, MGE7²⁴³, was significantly correlated with latitude. A genetic seascape analysis using 9 environmental and 3 geospatial variables revealed a strong association between MGE7²⁴³ and three highly correlated environmental variables, maximum sea surface temperature, minimum salinity and maximum chlorophyll-a. This association was maintained for homozygous genotypes for the MGE7²⁴³ allele, but not for heterozygotes, providing further evidence that the locus MGE7 may be under selection or closely linked to a gene under selection. These findings highlight how previously unrecognised genetic structure can be identified through the use of genetic seascape approaches.</p>

Ampelographic and molecular characterisation of grapevine varieties in the gene bank of the experimental vineyard ‘Radmilovac’ – Serbia

Characterisations of thirty grapevine varieties (Vitis vinifera L.) from the experimental vineyard ‘Radmilovac’ were conducted using a large number of OIV descriptors and eight highly polymorphic microsatellite loci. The ampelographic description contained 45 features. Molecular characterisation of selected microsatellite loci was performed using capillary electrophoresis fragment analysis. Dendrograms based on ampelographic and genetic data resulted in three groups of varieties. Qualitative ampelographic characteristics tended to manifest significant differences. The most common deviation among varieties within the group was in the characteristic OIV 051 (colouration of the upper side of a young leaf). Genetic characterisation of SSR markers through analyses of a large number of varieties contributes to better organisation of grapevine collections and simpler identification of varieties, as well as data exchange. When identifying the varieties, the results of the DNA analysis should be combined with the ampelographic descriptors, in order to select grapevine varieties with desirable viticultural and oenological traits. Integration of the obtained genetic data with the ampelographic data is of utmost importance for accurate identification of the varieties and offers a significant means for the preservation and use of the varieties.

Population Genetics of the Red Rock Lobster, Jasus edwardsii

<p>Understanding patterns of gene flow across a species range is a vital component of an effective fisheries management strategy. The advent of highly polymorphic microsatellite markers has facilitated the detection of fine-scale patterns of genetic differentiation at levels below the resolving power of earlier techniques. This has triggered the wide-spread re-examination of population structure for a number of commercially targeted species. The aims of thesis were to re-investigate patterns of gene flow of the red rock lobster Jasus edwardsii throughout New Zealand and across the Tasman Sea using novel microsatellite markers. Jasus edwardsii is a keystone species of subtidal rocky reef system and supports lucrative export markets in both Australia and New Zealand. Eight highly polymorphic microsatellite markers were developed from 454 sequence data and screened across a Wellington south coast population to obtain basic diversity indices. All loci were polymorphic with the number of alleles per locus ranging from 6-39. Observed and expected heterozygosity ranged from 0.563-0.937 and 0.583-0.961, respectively. There were no significant deviations from Hardy-Weinberg equilibrium following standard Bonferroni corrections. The loci were used in a population analysis of J. edwardsii that spanned 10 degrees of latitude and stretched 3,500 km across the South Pacific. The analysis rejected the null-hypothesis of panmixia based on earlier mDNA analysis and revealed significant population structure (FST=0.011, RST=0.028) at a wide range of scales. Stewart Island was determined to have the highest levels of genetic differentiation of all populations sampled suggesting a high degree of reproductive isolation and self-recruitment. This study also identified high levels of asymmetric gene flow from Australia to New Zealand indicating a historical source-sink relationship between the two countries. Results from the genetic analysis were consistent with results from oceanographic dispersal models and it is likely that the genetic results reflect historical and contemporary patterns of Jasus edwardsii dispersal and recruitment throughout its range.</p>

Population Genetics of the Red Rock Lobster, Jasus edwardsii

<p>Understanding patterns of gene flow across a species range is a vital component of an effective fisheries management strategy. The advent of highly polymorphic microsatellite markers has facilitated the detection of fine-scale patterns of genetic differentiation at levels below the resolving power of earlier techniques. This has triggered the wide-spread re-examination of population structure for a number of commercially targeted species. The aims of thesis were to re-investigate patterns of gene flow of the red rock lobster Jasus edwardsii throughout New Zealand and across the Tasman Sea using novel microsatellite markers. Jasus edwardsii is a keystone species of subtidal rocky reef system and supports lucrative export markets in both Australia and New Zealand. Eight highly polymorphic microsatellite markers were developed from 454 sequence data and screened across a Wellington south coast population to obtain basic diversity indices. All loci were polymorphic with the number of alleles per locus ranging from 6-39. Observed and expected heterozygosity ranged from 0.563-0.937 and 0.583-0.961, respectively. There were no significant deviations from Hardy-Weinberg equilibrium following standard Bonferroni corrections. The loci were used in a population analysis of J. edwardsii that spanned 10 degrees of latitude and stretched 3,500 km across the South Pacific. The analysis rejected the null-hypothesis of panmixia based on earlier mDNA analysis and revealed significant population structure (FST=0.011, RST=0.028) at a wide range of scales. Stewart Island was determined to have the highest levels of genetic differentiation of all populations sampled suggesting a high degree of reproductive isolation and self-recruitment. This study also identified high levels of asymmetric gene flow from Australia to New Zealand indicating a historical source-sink relationship between the two countries. Results from the genetic analysis were consistent with results from oceanographic dispersal models and it is likely that the genetic results reflect historical and contemporary patterns of Jasus edwardsii dispersal and recruitment throughout its range.</p>

Patterns of Connectivity and Isolation in Marine Populations

<p>There is ongoing debate about the levels of connectivity among marine populations and despite its importance, there is limited information on the levels of population connectivity in most geographic locations. This lack of information severely limits our ability to adequately manage the marine environment including the design and implementation of Marine Reserve (MRs) networks. The specific objectives of this thesis were to: 1) Develop polymorphic microsatellite loci for my model species, the intertidal gastropod Austrolittorina cincta; 2) Conduct population genetic studies across A.cincta populations within the Cook strait region to asses the levels of connectivity within the regional marine reserve network; 3) Determine the levels of A. cincta larval movement and settlement from an isolated source; and 4) Asses the effect of the larval abundance on settlement rates. This thesis includes laboratory studies; population genetic studies; and field surveys within New Zealand and in the Wakatobi National Park, Indonesia. Eight novel polymorphic microsatellite loci were developed for A. cincta and five of these loci were used to investigate population connectivity across seven populations within the Cook Strait region, including four marine reserves. In the population genetics study, in contrast to what was expected, I recorded low, but significant genetic differentiation between most population pairs within the Cook Strait region, over a minimum and maximum spatial scale of 55 to 300 km, including several of the MRs. In a large-scale field settlement survey on the Kapiti coast combined with the use of microsatellite markers I investigated A. cincta larval movement and settlement and found that most larvae settle within 5 km, although some larvae might travel up to 50 km. Finally, the coral settlement studies in the Wakatobi National Park revealed lower coral settlement rates at sites with low adult coral cover, suggesting an effect of the the amount of local available larvae on coral settlement rates. While it has been suggested that marine populations are demographically open, with larvae connecting populations separated over large spatial scales, this thesis shows that populations might not be as open as previously considered and localized dispersal and self-recruitment processes might be a frequent feature in marine populations. This thesis provides valuable information to managers about marine reserve networks and the importance of adequate environmental protection to ensure future viable populations.</p>

Patterns of Connectivity and Isolation in Marine Populations

<p>There is ongoing debate about the levels of connectivity among marine populations and despite its importance, there is limited information on the levels of population connectivity in most geographic locations. This lack of information severely limits our ability to adequately manage the marine environment including the design and implementation of Marine Reserve (MRs) networks. The specific objectives of this thesis were to: 1) Develop polymorphic microsatellite loci for my model species, the intertidal gastropod Austrolittorina cincta; 2) Conduct population genetic studies across A.cincta populations within the Cook strait region to asses the levels of connectivity within the regional marine reserve network; 3) Determine the levels of A. cincta larval movement and settlement from an isolated source; and 4) Asses the effect of the larval abundance on settlement rates. This thesis includes laboratory studies; population genetic studies; and field surveys within New Zealand and in the Wakatobi National Park, Indonesia. Eight novel polymorphic microsatellite loci were developed for A. cincta and five of these loci were used to investigate population connectivity across seven populations within the Cook Strait region, including four marine reserves. In the population genetics study, in contrast to what was expected, I recorded low, but significant genetic differentiation between most population pairs within the Cook Strait region, over a minimum and maximum spatial scale of 55 to 300 km, including several of the MRs. In a large-scale field settlement survey on the Kapiti coast combined with the use of microsatellite markers I investigated A. cincta larval movement and settlement and found that most larvae settle within 5 km, although some larvae might travel up to 50 km. Finally, the coral settlement studies in the Wakatobi National Park revealed lower coral settlement rates at sites with low adult coral cover, suggesting an effect of the the amount of local available larvae on coral settlement rates. While it has been suggested that marine populations are demographically open, with larvae connecting populations separated over large spatial scales, this thesis shows that populations might not be as open as previously considered and localized dispersal and self-recruitment processes might be a frequent feature in marine populations. This thesis provides valuable information to managers about marine reserve networks and the importance of adequate environmental protection to ensure future viable populations.</p>