Neighbor-joining Dendrogram Research Articles

Genetic Diversity and Relationship of Ethiopian Potato Varieties to Germplasm from North America, Europe and the International Potato Center

Potato is an increasingly important crop in Ethiopia, but the origin of local cultivars grown throughout the country is unknown. To evaluate the genetic diversity of Ethiopian potato cultivars, and to assess their relationship with germplasm from North America, Europe and the International Potato Center (CIP), 8303 SNP markers were used to characterize 44 local Ethiopian cultivars, as well as 26 CIP, 22 American and 17 European potato cultivars and advanced breeding clones. The marker data revealed that most of the local cultivars were duplicates; among the 44 cultivars tested, only 15 unique genotypes were observed. Principal component and neighbor-joining dendrogram analyses showed that American, European and CIP germplasm form three distinct clusters, with older Ethiopian cultivars overlapping the European cultivars, suggesting that the oldest local cultivars are of European descent. Local cultivars overall separated into two distinct clusters, suggesting that at least two distinct introductions gave rise to current local cultivars in Ethiopia. Ethiopian germplasm harbors comparable levels of genetic diversity to American, European, and CIP germplasm and could provide the foundation for a national potato breeding program.

Insight into the genetic variability analysis and cultivar identification of tall fescue by using SSR markers.

BackgroundGenetic diversity of 19 forage-type and 2 turf-type cultivars of tall fescue (Festuca arundinacea Schreb.) was revealed using SSR markers in an attempt to explore the genetic relationships among them, and examine potential use of SSR markers to identify cultivars by bulked samples.ResultsA total of 227 clear band was scored with 14 SSR primers and out of which 201 (88.6 %) were found polymorphic. The percentage of polymorphic bands (PPB) per primer pair varied from 62.5 to 100 % with an average of 86.9 %. The polymorphism information content (PIC) value ranged from 0.116 to 0.347 with an average of 0.257 and the highest PIC value (0.347) was noticed for primer NFA040 followed by NFA113 (0.346) whereas the highest discriminating power (D) of 1 was shown in NFA037 and LMgSSR02-01C. A Neighbor-joining dendrogram and the principal component analysis identified six major clusters and grouped the cultivars in agreement with their breeding histories. STRUCTURE analysis divided these cultivars into 3 sub-clades which correspond to distance based groupings.ConclusionThese findings indicates that SSR markers by bulking strategy are a useful tool to measure genetic diversity among tall fescue cultivars and could be used to supplement morphological data for plant variety protection.

Molecular Evaluation of Genetic Diversity in Wild-Type Mastic Tree (Pistacia lentiscus L.).

In this study, the patterns of genetic variation and phylogenetic relationships of mastic tree (Pistacia lentiscus L.) genotypes including 12 males and 12 females were evaluated using SSR, RAPD, ISSR, and ITS markers yielding 40, 703, 929 alleles, and 260-292 base pairs for ITS1 region, respectively. The average number of alleles produced from SSR, RAPD, and ISSR primers were 5.7, 14, and 18, respectively. The grouping pattern obtained from Bayesian clustering method based on each marker dataset was produced. Principal component analyses (PCA) of molecular data was investigated and neighbor joining dendrograms were subsequently created. Overall, the results indicated that ISSR and RAPD markers were the most powerful to differentiate the genotypes in comparison with other types of molecular markers used in this study. The ISSR results indicated that male and female genotypes were distinctly separated from each other. In this frame, M9 (Alaçatı) and M10 (Mesta Sakız Adası-Chios) were the closest genotypes and while F11 (Seferihisar) and F12 (Bornova/Gökdere) genotypes fall into same cluster and showing closer genetic relation. The RAPD pattern indicated that M8 (Urla) and M10 (Mesta Sakız Adası-Chios), and F10 (Mesta Sakız Adası-Chios) and F11 (Seferihisar) genotypes were the closest male and female genotypes, respectively.

Assessment of Genetic Diversity in Five Nicaraguan Populations of Cedrela odorata L. (Meliaceae) using RAPD Markers

The goal of this study was to assess the genetic diversity of Nicaraguan populations of Cedrela odorata using Random Amplified Polymorphic DNA (RAPD) markers. Thus, genomic DNA was isolated from leaf samples collected from ninety-two trees belonging to five Nicaraguan natural populations of C. odorata. The mean number of alleles per locus, effective number of alleles per locus, percentage of polymorphic loci, genetic diversity (He ) of Nei and diversity index (Ho ) of Shannon were estimated for each population assuming that the populations were in HardyWeinberg equilibrium. Total genetic diversity was partitioned in intrapopulational and interpopulational diversity using Nei’s genetic differentiation (GST) and through an Analysis of Molecular Variance (AMOVA). The ΦST matrix was used to construct a dendrogram by the neighbor-joining method. According to values of both He and Ho , Esquipulas (Deparment of Matagalpa) presented the lowest diversity level; while La Trinidad (Department of Estelí) showed the highest diversity level. Genetic differentiation was calculated obtaining a GST value of 13.36%. AMOVA also showed a similar differentiation value ΦST =13.81%). Neighbour-joining dendrogram clustered the five populations in two groups, where the group formed by La Trinidad and El Refugio (Department of Granada) presented the biggest differentiation. Correlation between genetic and geographical distances was not found.

Allele polymorphism and haplotype diversity of MICA/B in Tujia nationality of Zhangjiajie, Hunan Province, China

Previous studies indicate the distribution of major histocompatibility complex class I chain-related genes A (MICA) and B (MICB) alleles and haplotypes varies widely between different ethnic populations and geographic areas. It is meaningful to investigate allelic frequencies and establish a genetic database. In this study, we firstly reported the polymorphic variation of MICA/B in 187 healthy, unrelated Tujia individuals in Zhangjiajie region, China. Using polymerase chain reaction-sequence specific priming (PCR-SSP) and sequencing-based typing (PCR-SBT), we identified eight MICA-sequence alleles, four MICA-short tandem repeat variants, and 13 MICB variants, of which MICA∗008:04 (29.41%), MICA∗A5 (29.68%), MICA∗A5.1 (29.68%) and MICB∗005:02 (39.57%) were the most frequent. Linkage disequilibrium analysis further revealed MICB∗005:02-MICA∗019 (13.10%) and MICB∗002-MICA∗008:04 (9.89%) as the most common two-locus haplotypes. Data comparison by neighbor-joining dendrograms and principal component analysis to verify allelic frequencies in other Chinese and Asia ethnic groups showed that the Zhangjiajie Tujias were genetically closer to the Guangdong Han population, based on MICA loci variability. Our results provide new information about the MICA/B gene polymorphism in Chinese Tujia population, which will form the basis for future studies on the potential role of MICA/B in allogeneic organ transplantation and disease susceptibility in related ethnic groups.

Assessment of genetic diversity in Ethiopian cowpea [Vigna unguiculata (L.) Walp.] germplasm using simple sequence repeat markers

The genetic diversity of cowpea (Vigna unguiculata L. Walp.) in Ethiopia was analyzed using 19 uniform accessions, 62 variable accessions (yielding 185 sub-types), and two mungbean (Vigna radiata) accessions (four subtypes) as outgroup. A set of 23 polymorphic simple sequence repeat (SSR) markers was identified, and polymorphism in the various accessions was scored by determining amplicon variability. Allele frequency, genetic diversity, and polymorphism information content (PIC) were determined for each SSR marker, and a neighbor joining dendrogram was generated to show the genetic relationship among the individual accessions. A total of 75 allelic variants was defined, with the average number of alleles per locus calculated to be three. The average genetic diversity (D) was 0.47, and PIC was 0.4. Three main clusters were identified by phylogenetic analysis, and the clusters and sub-grouping were supported by STRUCTURE and principal component analysis. This grouping had a moderate fixation index value of 0.075 and gene flow (Nm) of 3.176, indicating that the accessions possess wide diversity within individuals and populations. The accessions showed no clustering by geographical origins. Three well-characterized molecular markers (SSR1, C42-2B, and 61RM2) for race specific resistance to Striga gesnerioides in the cowpea cultivar B301 were used to evaluate the accessions for their potential for use in genetic improvement against this pest. Based on this analysis, only two accessions, 222890–2 from Gambela and 286–2 from the Southern Nations, Nationalities, and Peoples (SNNP) region, were found to cluster with B301 and contain the SSR1 resistance allele. These findings will assist in germplasm conservation efforts by the Institute of Biodiversity and Conservation of Ethiopia, and contribute to future studies aimed at the genetic improvement of local germplasm for improved overall agronomic performance as well as Striga resistance in particular.

Frequency of HLA-DRB1 and HLA-DQB1 Alleles and Haplotype Association in Syrian Population

ABSTRACTThe study of Human Leukocyte Antigen (HLA) system is very important in health and diseases. As the HLA loci are the most polymorphic in the human genome, it plays a very important role in the immune responses to self and nonself antigens. In the light of the growing importance of typing the HLA alleles in transplantation, autoimmune diseases, cancer, and many other diseases, we studied 225 unrelated healthy Syrian subjects for their HLA class II genotypes in an attempt to reveal the distribution of the HLA (DRB1-DQB1) alleles in the general Syrian population. Our results revealed that the most common alleles for the DRB1 locus were DRB1*11 (26.4%), DRB1*04 (14%), and DRB1*07 (12%). However, the most frequent alleles for the DQB1 locus were DQB1*03 (40.9%) and DQB1*05 (25.1%). The frequent of two-locus haplotypes carry the most frequent alleles at these loci. The most frequently detected class II ‘‘haplotypes’’ are DRB1*11-DQB1*03 (8.9%), DRB1*01-DQB1*05 (3.6%), and DRB1*04-DQB1*03 (2.7%). Compared with other populations, our result, deduced from the analysis of genetic distances and the construction of neighbor-joining (NJ) dendrogram, and principal component analysis (PCA) indicates that Syrians are related to Middle Eastern populations. Our data about the Syrian population will aid researchers in studying the relation of HLA class II with different diseases in a Syrian population and will add to the available international literature associated with these loci.

Genetic diversity and population structure of core watermelon (Citrullus lanatus) genotypes using DArTseq-based SNPs

Watermelon [Citrullus lanatus(Thunb.) Matsum. & Nakai var.lanatus] is an economically important vegetable belonging to theCucurbitaceaefamily. Genotypes that exhibit agronomically important traits are selected for the development of elite cultivars. Understanding the genetic diversity and the genotype population structure based on molecular markers at the genome level can speed up the utilization of diverse genetic resources for varietal improvement. In the present study, we carried out an analysis of genetic diversity based on 3882 SNP markers across 37 core watermelon genotypes, including the most widely used watermelon varieties and wild watermelon. Based on the SNP genotyping data of the 37 watermelon genotypes screened, gene diversity and polymorphism information content values across chromosomes varied between 0.03–0.5 and 0.02–0.38, with averages of 0.14 and 0.13, respectively. The two wild watermelon genotypes were distinct from cultivated varieties and the remaining 35 cultivated genotypes were differentiated into three major clusters: 20 genotypes were grouped in cluster I; 11 genotypes were grouped in cluster II; three advanced breeding lines of yellow fruit flesh and genotype SW043 were grouped in cluster III. The results from neighbour-joining dendrogram, principal coordinate analysis and STRUCTURE analysis approaches were consistent, and the grouping of genotypes was generally in agreement with their origins. Here we reveal the genetic relationships among the core watermelon genotypes maintained at the Jiangsu Academy of Agricultural Sciences, China. The molecular and phenotypic characterization of the existing core watermelon genotypes, together with specific agronomic characteristics, can be utilized by researchers and breeders for future watermelon improvement.

Analysis of persistent changes to γ-aminobutyric acid receptor gene expression in Plutella xylostella subjected to sublethal amounts of spinosad.

A multi-generational approach was used to investigate the persistent effects of a sub-lethal dose of spinosad in Plutella xylostella. The susceptibility of various sub-populations of P. xylostella to spinosad and the effects of the insecticide on the gene expression of γ-aminobutyric acid receptor (GABAR) were determined. The results of a leaf dip bioassay showed that the sensitivity of P. xylostella to spinosad decreased across generations. The sub-strains had been previously selected based on a determined LC25 of spinosad. Considering that GABA-gated chloride channels are the primary targets of spinosad, the cDNA of P. xylostella was used to clone GABARα by using reverse transcription-polymerase chain reaction (RT-PCR). The mature peptide cDNA was 1477-bp long and contained a 1449-bp open reading frame encoding a protein of 483 amino acids. The resulting amino acid sequence was used to generate a neighbor-joining dendrogram, and homology search was conducted using NCBI BLAST. The protein had high similarity with the known GABAR sequence from P. xylostella. Subsequent semi-quantitative RT-PCR and real-time PCR analyses indicated that the GABAR transcript levels in the spinosad-resistant strain (RR, 145.82-fold) and in Sub1 strain (selected with LC25 spinosad for one generation) were the highest, followed by those in the spinosad-susceptible strain, the Sub10 strain (selected for ten generations), and the Sub5 strain (selected for five generations). This multi-generational study found significant correlations between spinosad susceptibility and GABAR gene expression, providing insights into the long-term effects of sub-lethal insecticide exposure and its potential to lead to the development of insecticide-resistant insect populations.

Interspecific Relationships in <I>Allium</I> subgenus <I>Melanocrommyum</I> sections <I>Acanthoprason</I> and <I>Asteroprason</I> (Amaryllidaceae) Revealed Using ISSR Markers

Abstract Allium subgenus Melanocrommyum is taxonomically one of the most complicated groups within Allium and its sectional delimitation has been changed several times. In the present study, the relationships among 136 individuals representing 28 species of the sections Acanthoprason and Asteroprason were surveyed using ISSR (inter simple sequence repeats) markers. Nine ISSR primers generated 322 polymorphic bands (on average 36 bands per primer). In the resulting neighbor joining dendrogram, most conspecific individuals were grouped into clusters, which can be regarded as evidence of the monophyly of most species. Exceptions were the positions of six single samples of A. graveolens, A. zagricum, A. shelkovnikovii, A. subakaka, and A. materculae, which were not grouped with their remaining conspecific individuals. The clusters in dendrograms were significantly correlated with morphological similarities and biogeographical distributions. The lowest molecular similarity (62%) was observed between A. shelkov...

A novel method for the identification of seahorses (genus Hippocampus) using cross-species amplifiable microsatellites

Seahorses (genus Hippocampus) are flagship species for marine conservation and evolutionary studies. Wild seahorse populations have declined sharply because of overfishing or other disrupting activities, which are attracting increasing global concern for their threatened status. For study of their population status, the identification of young seahorses by morphological examination has been controversial and remains a difficult task due to overlapping of meristic and morphological characters. Here we reported a simple and sensitive method to identify seahorse species from China based on cross-species microsatellites. The cross-species amplification success rate varied from 40.0% to 90.0% depending on the species, with a mean value of 59.6%. According to allele presence/absence of cross-species microsatellites, all the individuals representing 10 seahorse species could be accurately identified using one or more loci. In addition, we found both genome size and genetic distance between the source and target species negatively correlated with microsatellite cross-species amplification success. Moreover, a Neighbor-Joining dendrogram was constructed based on genetic similarity and suggested that the 10 seahorse species could be divided into four groups. These results demonstrate our method could improve the identification of seahorses, providing data to understand seahorse stock structure and recruitment dynamics, and therefore resources conservation.

Identification and characterization of Vitis vinifera subsp. sylvestris populations in north-western Italy

The Italian peninsula, for its favorable environmental and geo-morphological conditions, can be considered an ultimate area for survival and development of Vitis vinifera subsp. sylvestris (Gmelin) Hegi, even though severely affected by human impact. Large surveys started in early 1990 throughout the country. At the time few regions, like Piedmont located in the north-west of the country, were considered lacking of wild vinifera. More recent prospection started several years ago, leading to the discovering of five vinifera sylvestris populations plus other sites with few individuals. The sites of discovery were described for their ecological features and the identified plants were referenced and characterized by morphology (18 descriptors from the OIV list) and genetics (14 n-SSR loci). The esteemed consistency of each population ranged from 20 to 150 individuals. Morphological and biological traits (dioecious plants, females producing very small roundish black berries), as well as genetic profiles, indicated the observed plants are true vinifera sylvestris. As to the ecological requirements, plants were confirmed to be highly dependent on water availability into the soil. The neighbor-joining (NJ) dendrogram resulting from SSR allelic pattern of the individuals belonging to the five populations and to one location with isolated plants, indicated population's genetic similarity broadly reflects site's geographic distance. Considering the numerous reports in the past, spreading and consistency of wild grape germplasm from the region of Piedmont severely decreased over a period of 100-150 years. The relative short distance from wild population's sites and vineyards must also be regarded as a worrying condition because of contamination risks. All means to avoid the loss of this native Vitis germplasm must be undertaken by protection policy and proper land management.

Genetic diversity and structure of the Lesvos sheep breed

We investigated, through the use of 11 microsatellite molecular markers, the genetic diversity and structure of 350 individuals from ten flocks of the Lesvos sheep breed population (Greece). The results revealed that although the breed shows particularly high levels of observed heterozygosity (Ho=0.83), the mean number of alleles of 6.8 is somewhat lower than in other sheep breeds. FIS (−0.143±0.065) was significantly negative over loci for the five out of the ten flocks and the observed heterozygosity was consistently higher than the expected for all flocks. The low value of FST (0.021±0.003) implies that the quasi-totality of genetic variability is due to within-flock variability, suggesting a strong genetic cohesion of the total population. Furthermore, we revealed a breeding practice that comprises an important exchange of animals: the number of migrants between flocks ranged from 6.41 to 53.92. These exchanges happen both between neighboring and more distant farms as indicated by the correspondence analysis and the neighbor-joining dendrogram. This is also reflected in the clustering obtained by STRUCTURE analysis according to which all flocks had comparable proportions of membership to the five clusters detected. In conclusion, the implemented management practice confers high gene variability both at the flock level and in the total population which is very promising for the conservation of the breed as a genetic resource. However, in conjunction with the isolation of the breed on the island of Lesvos, the high gene flow among flocks may also be responsible for the rather low mean number of alleles as it decreases the overall effective size. Moreover, it is still unknown whether the present breeding practice meets adequately production objectives of the breeders, which is the other important facet of the breed management.

Analysis of genetic structure of Ranunculus baudotii in a Mediterranean wetland. Implications for selection of seeds and seedlings for conservation

Seed collection and storage of wild species in ex-situ seed banks should be continued as an integrated tool for the conservation of plants in their habitats. Although seed-bank facilities are widely used today, their seed samples often suffer low genetic diversity. Consequently, reintroduced seeds and plant material may not have the resilience to cope with future environmental stress so leading to complete wastage of seeds. Molecular techniques allow the benefit of quantification of the genetic diversity of a seed collection in comparison with that of the natural population. In this study we focus on ex-situ seed bank samples and living collections of Ranunculus peltatus subsp. baudotii. We compare their genetic diversity and structure with that of the natural population before and after undertaking a restoration project on a natural pond in the Tuscan Archipelago National Park. ISSR analyses, carried out on a total of five sampling groups, shows a relatively high level of genetic diversity for the ex-situ cultivated groups. The analysis of molecular variance, in agreement with clustering obtained in the neighbour-joining dendrogram and with the pattern from cluster analysis, suggests dividing the samples analysed into two groups: one formed by individuals sampled before the pond restoration and the other formed by the subsequent pond population. The results highlight the importance of planning mixed propagation lines which can be obtained through the use of a range of germination conditions to exploit a novel source of genetic variability which may otherwise remain hidden within the seed collection.

Chloroplast DNA analysis of Tunisian pistachio (Pistacia vera L.): Sequence variations of the intron trnL (UAA)

The trnL (UAA) intron sequences of Pistacia vera ecotypes were employed to reconstruct the phylogeny and to elucidate molecular evolution. A single band of approximately 500pb length was amplified from each cultivars. Nucleotides sequences were aligned using the ClustalW package in the Bioedit program and analyzed with MEGA program version 4. The average of GC content was 35.8% to 39.6%. High values of A+T contents were detected in data sets and may explain the high proportions of transitions founded. Sequence alignment revealed the existence of 50 informative sites and 25 different haplotypes among the 39 cultivars studied yielding to a haplotype diversity of 0.8758 and a level of nucleotide diversity of 0.018. Furthermore, an average value of pairwise nucleotide of 8.641 differences was found, showing a relatively high diversity in the chloroplast intron. The neighbor joining dendrogram shows the presence of fairly high chloroplasts diversity, structured independently from the plant's sex but structured according to geographical origin. The trnL intron mismatch distribution is multimodal; characteristic of a stable population. This was also supported by non-significant values of all neutrality test parameters (Tajima's D and Fu and Li's). Although, the level of apparent diversity is considerable, we may conclude that non-coding regions of chloroplast genome provide a new and practical opportunity to evaluate genetic diversity and to discriminate pistachio cultivars. Revealed cytoplasmic DNA markers are reliable to elaborate a molecular data base to conduct management and breeding programs on local pistachio germplasm.

Western European Wild and Landraces Hazelnuts Evaluated by SSR Markers

Hazelnut (Corylus avellana L.), a member of the family Betulaceae and the order Fagales, is native to Europe, Turkey, and the Caucasus. It has been cultivated for more than 5000 years. The majority of cultivars were selected from local wild populations over the past few centuries. Hazelnut is native to northern Portugal, with local production from landrace clonal selections. In recent years, hazelnut production has declined, and landraces are in danger of being lost. In this study, we used 16 microsatellite markers to investigate genetic diversity in wild populations, Portuguese landraces, and reference cultivars. A high level of polymorphism was found, as indicated by mean values for expected heterozygosity (0.74), observed heterozygosity (0.71), and polymorphism information content (0.78). A neighbor-joining dendrogram showed a clear separation of the wild genotypes from the landraces and reference cultivars. Bayesian analysis placed individuals in three groups, showing differentiation of wild populations, landraces, and reference cultivars. Our studies provide new insights on the origin of Portuguese landraces.

Genetic characterization of red-colored heartwood genotypes of Chinese fir using simple sequence repeat (SSR) markers.

The present study investigated the genetic characterization of red-colored heartwood Chinese fir [Cunninghamia lanceolata (Lamb.) Hook.] in Guangxi using 21 simple sequence repeat (SSR) markers and analyzes of the genetic variation (N = 149) in samples obtained from five sites in Guangxi Province, China. The number of different alleles and the Shannon's information index per locus ranged from 3 to 12 and from 0.398 to 2.258 with average values of 6 and 1.211, respectively, indicating moderate levels of genetic diversity within this germplasm collection. The observed and expected heterozygosity ranged from 0.199 to 0.827 and from 0.198 to 0.878 with an average of 0.562 and 0.584, respectively. Although, the mean fixation index was 0.044, indicative of a low level of genetic differentiation among germplasms, analysis of molecular variance revealed considerable differentiation (99%) within the samples. The neighbor-joining dendrogram revealed that the majority of red-colored Chinese fir genotypes were apparently not associated with their geographic origins. Further analysis by STRUCTURE showed that this Guangxi germplasm collection could be divided into three genetic groups comprising 76, 37, and 36 members, respectively; these were classified into mixed groups with no obvious population structure. These results were consistent with those of the cluster analysis. On the whole, our data provide a starting point for the management and conservation of the current Guangxi germplasm collection as well as for their efficient use in Chinese fir-breeding programs.

TRNALeu intron (UAA) of Ficus carica L.: genetic diversity and evolutionary patterns.

Cytoplasmic chloroplast DNA was explored to establish genetic relationships among Ficus carica cultivars and elucidate the molecular evolution of the species. The results suggest the occurrence of haplotype and nucleotide diversity. Conserved group I intron sequence motifs were detected and showed a common secondary structure, despite the presence of some mutations on their sequences. The neighbor-joining dendrogram showed a continuous diversity that characterizes local resources. The maximum parsimony tree, with an RI index of 0.507, indicated minimal homoplasy within the data set. Furthermore, our results demonstrate that the trnL intron is the seat of numerous substitutions. Herein, new insight on the mechanism involved in the evolution of the trnL intron in the fig is presented. From the study, it appears that there is an explicit rejection of the null hypothesis in F. carica. A scenario of positive selection and recent expansion of F. carica genotypes across Tunisia seems to be retained.

An assessment of tissue culture-induced genetic variability in olive (Olea europaea L.) using chloroplast rpl16 intron sequences and single primer amplification reaction (SPAR) markers

SUMMARYOlive (Olea europaea L.) is an important tree crop grown throughout the Mediterranean Basin and Iran. Propagation of true-to-type olive plants is important for olive-growing companies and farmers. For this reason, we performed a genetic analysis of 49 randomly selected tissue culture-regenerated olive plants of the cultivar ‘Koroneiki’ and compared them with the mother plant. We used the sequence of the chloroplast rpl16 intron and ten single primer amplification reaction (SPAR) markers to test for genetic fidelity vs. somaclonal variation. A Neighbor-Joining dendrogram based on the RAPD data produced four major clusters. The mother plant, along with ten regenerated plants, were placed far from the others and formed Cluster I. Members of this Cluster had the highest genetic distance from members of Cluster III (0.266), followed by plants of Cluster IV (0.177). Analysis of molecular variance (AMOVA) produced significant differences (PhiPT value = 0.37; P≤ 0.01) among these clusters. The Neighbor-Joining tree, neighbor-Net method, and Tree analysis using New Technology (TNT) based on RAPD marker and rpl16 intron sequence analyses respectively, separated the mother plant and some of the regenerated plants in a separate cluster. Tajima's test produced a diversity (D) statistic value of = −2.17 (P ≤ 0.05) based on the intron sequence data. We also identified some regenerated plants that were true-to-type with the mother plant.

Genetic relationships of the Portuguese Lidia bovine populations.

To clarify the genetic relationships among the Lidia breed lineages and two main Portuguese Lidia bovine populations, Casta Portuguesa and Brava dos Açores, 24 autosomal microsatellites were analyzed in 120 samples. Brava dos Açores showed the highest observed and expected heterozygosity (0.73 and 0.70, respectively) while Casta Portuguesa showed the lowest observed and expected heterozygosity (0.51 and 0.50, respectively). The results of this study were compared with the previous microsatellites data from the main Lidia bovine lineages. Casta Portuguesa was the most genetically isolated Lidia bovine population as revealed by the average FST genetic distance value with respect to the other lineages (32%). All the populations of Portuguese Lidia had negative FIS values. The Neighbour-joining dendrogram grouped Casta Portuguesa in the same branch with Miura, which was supported by the STRUCTURE software. The results evidenced low levels of genetic diversity and high levels of genetic differentiation in Casta Portuguesa and high levels of genetic diversity in Brava dos Açores populations, probably due to the crossbreeding of different bovine lineages at origin, and genetic flow among herds.