Genetic analysis of Canarium album in different areas of China by improved RAPD and ISSR

To lay the foundation of the classification of Canarium album (C. album), and C. album from Terminalia Chebula (T. chebula) in different areas of China, improved RAPD and ISSR analysis were performed to analyze polymorphism and genetic relationship. Ten samples were collected from different locations in China. A total of 221 fragments were detected by improved RAPD, out of which polymorphic fragments accounted for 82.3% with average amplification bands of 10.05 per primer. ISSR markers revealed a total of 147 alleles, where polymorphic fragments accounted for 83.5%, with average amplification bands of 7.35 per primer. The sizes of fragments ranged from 200 to 2500 bp and from 150 to 2000 bp in RAPD and ISSR markers, respectively. The similarity coefficient ranged from 0.46 to 0.86 with RAPD markers and 0.36 to 0.89 with ISSR markers. The results indicated that improved RAPD and ISSR methods are useful for genetic diversity study of C. album. Thus, this study provides us the theoretical basis for the breeding and classification of C. album in South and Southwest China.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Genetic analysis of Greek sheep breeds using microsatellite markers for setting conservation priorities

Ten Greek sheep breeds were analysed at 28 microsatellite markers in order to estimate their genetic diversity and differentiation. This study aims to provide information on the genetic structure of the breeds analysed and the ancestral populations, and give indications and proposals for the conservation strategies. The breeds included were the local sheep breeds raised in different regions of Greece. In total, 310 animals were sampled. Non-biased average expected heterozygosity ranged from 0.68 ± 0.134 (Skopelos breed) to 0.76 ± 0.103 (Karagouniko breed) with an average of 0.74, while the average observed heterozygosity ranged from 0.626 ± 0.132 (Skopelos) to 0.74 ± 0.135 (Kefallenias). Estimates of inbreeding coefficient (Fis) were significant for all breeds studied, except for Kefallenias and Lesvos breeds (P < 0.05). The results of the phylogenetic relationships are in accordance with the geographical location of the breeds, the history of the origin of the breeds and the breeding practices. The phylogenetic tree showed three groupings according to the bootstrapping values. Correspondence analysis showed the isolation of the Skopelos breed and the grouping of Sfakia and Anogeiano breeds in a separate cluster.     

High genetic differentiation revealed by RAPD analysis of narrowly endemic Sinocalycanthus chinensis Cheng et S.Y. Chang,an endangered species of China

Genetic diversity and genetic differentiation of narrowly endemic Sinocalycanthus chinensis Cheng et S.Y. Chang, an endangered species of China, were analyzed using random amplified polymorphic DNA (RAPD) markers. Totally, 165 stable and clearly scored RAPD bands were achieved from 12 primers. The genetic diversity of S. chinensis was high (P% = 68.84; h = 0.2421 ± 0.1978; I = 0.3615 ± 0.2789), whereas that at the population level was relatively low (P% = 14.49; h = 0.0578 ± 0.0167; I = 0.0843 ± 0.0236). High genetic differentiation among populations was detected based on Shannon's information index (0.7668), Nei's gene diversity (0.7613) and AMOVA (0.8183). This might be explained by its survival in refugia during the last glaciation in southeastern China with origin from a widespread continental progenitor. The 10 populations from different geographical sites could be clustered into two groups. Low gene flow due to mixed mating system and anthropogenic activities likely played important roles in shaping the population genetic structure of the species.     

Geometric morphometrics on Greek house mouse populations (Mus musculus domesticus) with Robertsonian and all-acrocentric chromosomal arrangements

This work aims to give the first comprehensive morphometric analysis of intraspecific variation for the different populations of the western house mouse (Mus musculus domesticus), in the Robertsonian (Rb) system of the N-NW Peloponnisos. Furthermore, we study all-acrocentric karyotype populations (2n = 40) of the species coming from several localities of Greece. We apply 2D shape analysis, i.e. landmark analysis and Elliptic Fourier Analysis, on the dorsal and ventral side of skull and the occlusal view of the first upper molar (M¹), respectively. Although significant genetic divergence between typical and Rb populations and even ongoing speciation processes have been reported for this species, this was not the case for the Greek populations studied. However, our analyses herein reveal morphologically differentiated chromosome groups in N-NW Peloponnisos Rb system and a clear geographical discrimination of the all-acrocentric (2n = 40) populations for all characters studied. We suggest that in all-acrocentric (2n = 40) karyotype mice the geographical distance drives their differentiation while within the Rb system of N-NW Peloponnisos, karyotype is the key factor that acts on their phenotypic variation.     

Morphological and molecular characterization of somaclonal variations in tissue culture-derived banana plants

In this study, 40 000 tissue culture-derived banana plants (vitroplants) at different growth stages, i.e. acclimatization, nursery and open field of banana (Musa spp.) cultivar ‘Grand Naine’ were screened for somaclonal variations using morphological investigations and molecular characterization. The total detected variants were grouped into 25 off-types (two of them died) in addition to the normal plant. Random Amplified Polymorphic DNA (RAPD) was carried out to study the differences among the normal cultivar ‘Grand Naine’ and its 23 variants using 17 arbitrary primers. Cluster analysis results revealed that ‘winged petiole’ and ‘deformed lamina’ were more related to the normal plant. However, ‘Giant plant’ and ‘weak plant’ related to each other and clustered with normal plant. According to principal coordinate analysis, most of the variants were aggregated nearly, whereas ‘variegated plant’ was separated apart from the other variants. This may reflect the genetic difference between ‘variegated plant’ and the other variants. The results obtained from both molecular and morphological analyses were in contiguous with better resolution when using the PCOORDA analysis than cluster analysis. Thus, it can be said that molecular markers can be used to eliminate the undesirable somaclonal variants from the lab without additional culture of the vitroplants in the field in order to save time and efforts.     

Analysis of genetic structure of Jamunapari goats by microsatellite markers

Genetic variation at 23 microsatellite loci, population structure, and genetic bottleneck hypothesis were examined for Jamunapari goat population found in Etawah, Uttar Pradesh, India. Estimates of genetic variability such as effective number of alleles and gene diversities revealed substantial genetic variation frequently displayed by microsatellite markers. Number of alleles observed across the microsatellite loci varied from 2 to 10 with an overall mean of 4.913 ± 1.905. Average polymorphism across the studied loci and expected gene diversity in the population were 1.066 ± 0.510 and 0.528 ± 0.237, respectively. Population was observed to be significantly differentiated into different groups, and showed fairly high level of inbreeding (f = 0.189 ± 0.049) and global heterozygote deficit. Bottleneck analysis indicated the introduction of unique/rare alleles by immigrants.     

DNA fingerprints of living fossil Ginkgo biloba by using ISSR and improved RAPD analysis

In this study, we have aimed to genetically characterize Ginkgo biloba. Nine G. biloba samples from different places of China were collected, and DNA was extracted from the leaves of these samples for inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) analysis. ISSR analysis showed high genetic variation among the nine varieties of G. biloba; the polymorphism and similarity coefficients were 87% and 0.40–0.84, respectively. RAPD analysis also showed 93% polymorphism, and the similarity coefficients ranged from 0.44 to 0.87. Persistent genetic isolation that developed for millions of years might influence the genetic variability between the samples of G. biloba. This study generates a genetic map of G. biloba, and reports the highly variable intra-species genetic characteristics of this living fossil among different geographical locations of China. Our study also suggests that ISSR and the improved RAPD markers are useful molecular tools for the genetic characterization of plants.     

Chemical and genetic diversity of two Mediterranean subspecies of Teucrium polium L.

Chemical and genetic diversity of Teucrium polium L. subsp. polium from western Algeria and T. polium L. subsp. capitatum from Corsica were investigated. Diversity within and among the two populations of subspecies was assessed according to the chemical composition of their essential oils and the genetic diversity. Chemical analysis was performed using a combination of capillary GC-RI and GC/MS after fractionation using column chromatography. Genetic structures were mapped using three polymorphic genetic markers: two chloroplast markers (RPL32-TRNL and TRNL-F) and ribosomal nuclear markers (ITS region). The statistical analysis showed that both subspecies were clearly distinguished by these chemical and genetic markers. The oil chemical compositions differed qualitatively and quantitatively between the subspecies. Both collective oils were dominated by hydrocarbon compounds however the Algerian sample oils exhibited higher amounts of hydrocarbon sesquiterpenes than those of Corsica (31.2 g/100 g vs. 4.4 g/100 g) while the latter displayed higher amounts of hydrocarbon monoterpenes than the first (59.3 g/100 g vs. 34.3 g/100 g). Neighbor-joining, Maximum likelihood and Bayesian trees constructed from chloroplast markers and nuclear ITS region sequences showed the existence of two groups associated with taxonomic and chemical characteristics. The study indicated that variation in the essential oil composition within subspecies depends on genetic background. The samples of subsp. capitatum from Corsica are a homogeneous group, in contrast to samples of subsp. polium from Algeria which were clustered in two groups. Chemical and genetic diversity of Algerian populations could be explained by geographical isolation of the populations. In addition, the morphological polymorphism observed throughout the colour of flowers could be explained by environmental parameters as well as the soil pH.     

Application of a method to determine ileal digestibility in broilers of amino acids in wheat

Growing male chickens (Ross) were fed diets of increasing wheat content (250, 500 and 750 g/kg) in which wheat was the only protein source. Other dietary ingredients were: oil (50 g/kg), a vitamin and mineral premix (50 g/kg) and a 50 : 50 mixture of pure maize starch and glucose to make diets up to 1000 g/kg. Titanium dioxide was included at 5 g/kg as an inert marker. Four wheat samples were evaluated: varieties B and H each with a low and high crude protein content (102 and 130, respectively ; 96 and 128 g CP/kg at 880 g DM/kg. Experimental diets were fed ad libitum for 3 days from 18 days of age to birds housed in pairs in cages in a fully randomised design comprising 6 replicates per diet. Samples of ileal digesta were obtained from the birds following slaughter at 21 days of age and analysed for titanium and amino acid content. For each amino acid, the amount of apparently digestible amino acid in the diet was regressed against the dietary wheat content and the linear regression thus derived extrapolated to 1000 and 0 g wheat/kg, allowing the estimation of the apparently digestible amino acid content of the wheat sample and the endogenous loss of amino acids, respectively. Dividing the former by the total amino acid content of the wheat allowed calculation of the coefficient of apparent amino acid digestibility (CAD). Addition of the intercept to the amount of apparently digestible amino acid in the wheat sample and dividing this by the total amount of amino acid in the wheat allowed the determination of the coefficient of true digestibility (CTD). Values for methionine for the four wheat samples ranged between 0.749 to 0.842 (CAD) and 0.719 to 0.892 (CTD), and for lysine from 0.531 to 0.697 (CAD) and 0.510 to 0.793 (CTD). Results revealed that an increase in crude protein in the wheat varieties resulted in a significant increase in apparently digestible amino acid content (P < 0.001), for example, the digestible cystine contents of wheat were 1.54 and 2.22 g/kg for wheat H at the low and high crude protein contents, respectively. Coefficients of digestibility also appeared to increase as the crude protein increased.     

Effect of cliff connectivity on the genetic population structure of a rock-dwelling land snail species with frequent self-fertilization

To examine the effect of habitat connectivity on the genetic differentiation in the frequently selfing gastropod Chondrina avenacea we assessed RAPD profiles in 12 subpopulations from continuous cliffs and in 12 subpopulations from small, naturally fragmented cliffs in the Swiss Jura mountains. Subpopulations of C. avenacea on continuous cliffs showed a smaller number of polymorphic RAPD loci and a lower gene diversity (Nei's h = 0.049) than subpopulations on fragmented cliffs (h = 0.078). Genetic distance was not correlated with geographical distance between subpopulations. G_ST analysis indicated strong isolation effects. Neither the number of polymorphic loci nor Nei's h was correlated with local population density. Our analysis indicates that the local population structure of C. avenacea is a combined result of frequent self-fertilization, genetic drift and low dispersal. Similar findings have been reported in studies on spatial population structure of plants. We suggest that future management actions (selective tree cutting, and controlled access of sport climbers) should also consider small, isolated limestone cliffs to preserve the genetic diversity of C. avenacea.     

SSR and morphological trait based population structure analysis of 130 diverse flax (Linum usitatissimum L.) accessions

A total of 130 flax accessions of diverse morphotypes and worldwide origin were assessed for genetic diversity and population structure using 11 morphological traits and microsatellite markers (15 gSSRs and 7 EST–SSRs). Analysis performed after classifying these accessions on the basis of plant height, branching pattern, seed size, Indian/foreign origin into six categories called sub-populations viz. fibre type exotic, fibre type indigenous, intermediate type exotic, intermediate type indigenous, linseed type exotic and linseed type indigenous. The study assessed different diversity indices, AMOVA, population structure and included a principal coordinate analysis based on different marker systems. The highest diversity was exhibited by gSSR markers (SI = 0.46; He = 0.31; P = 85.11). AMOVA based on all markers explained significant difference among fibre type, intermediate type and linseed type populations of flax. In terms of variation explained by different markers, EST-SSR markers (12%) better differentiated flax populations compared to morphological (9%) and gSSR (6%) markers at P = 0.01. The maximum Nei's unbiased genetic distance (D = 0.11) was observed between fibre type and linseed type exotic sub-populations based on EST-SSR markers. The combined structure analysis by using all markers grouped Indian fibre type accessions (63.4%) in a separate cluster along with the Indian intermediate type (48.7%), whereas Indian accessions (82.16%) of linseed type constituted an independent cluster. These findings were supported by the results of the principal coordinate analysis. Morphological markers employed in the study found complementary with microsatellite based markers in deciphering genetic diversity and population structure of the flax germplasm.     

Xanthan gum production and rheological behavior using different strains of Xanthomonas sp.

The proposal of the present study was to select and carry out the molecular characterization of strains of Xanthomonas sp. in order to correlate with gum production and determine possible genetic alterations during the study. The gums produced were also evaluated rheologically. Ten strains of Xanthomonas were used in the screening and the best ones in terms of productivity were Xanthomonas campestris pv. mangiferaeindicae 1230 (8.93 g/L), X. campestris pv. campestris 254 (9.49 g/L) and X. campestris pv. campestris 1078 (9.67 g/L). The gum produced by X. campestris pv. mangiferaeindicae presented the best apparent viscosity. The results for the profiles of the bands produced by RAPD showed considerable genetic variability amongst the evaluated strains, making not possible to neither group the strains according to pathovar or species, nor correlate the band profile with the productivity obtained. According to the RAPD analysis, no detectable mutations occurred in these bacteria during the study.     

Corynebacterium heidelbergense sp. nov., isolated from the preen glands of Egyptian geese (Alopochen aegyptiacus)

Two strains (pedersoli^T and girotti) of a new species of bacteria were isolated from the preen glands of wild Egyptian geese (Alopochen aegyptiacus) from the river Neckar in southern Germany in two subsequent years. The strains were lipophilic, fastidious, Gram-positive rods and belonged to the genus Corynebacterium. Phylogenetically, the isolates were most closely related to Corynebacterium falsenii DSM 44353^T which has been found to be associated with birds before. 16S rRNA gene sequence similarity to all known Corynebacterium spp. was significantly <97%. Corresponding values of rpoB showed low levels of similarity <87% and ANIb was <73%. G + C content of the genomic DNA was 65.0 mol% for the type strain of the goose isolates, as opposed to 63.2 mol% in Corynebacterium falsenii. MALDI-TOF MS analysis of the whole-cell proteins revealed patterns clearly different from the related species, as did biochemical tests, and polar lipid profiles. We therefore conclude that the avian isolates constitute strains of a new species, for which the name Corynebacterium heidelbergense sp. nov. is proposed. The type strain is pedersoli^T (=DSM 104638^T = LMG 30044^T).     

A Comparison of AFLP and RAPD Markers for Genetic Diversity and Cultivar Identification in Cotton

AFLP and RAPDmarkers were employed in sixteen diploid cotton (Gossypium sp) cultivars for genetic diversity estimation and cultivar identification. Polymorphism information content (PIC) and percent polymorphism were found to be more for AFLP markers as compared to RAPD markers. Average Jaccard’s genetic similarity index was found to be almost similar using either AFLP or RAPD markers. All the cultivars could be distinguished from one another using AFLP markers and also by the combined RAPD profiles. Cultivar identification indicators like resolving power, marker index and probability of chance identity of two cultivars suggested the usefulness of AFLP markers over the RAPD markers. AFLP and RAPD analyses revealed limited genetic diversity in the studied cultivars. Cluster analysis of both RAPD and AFLP data produced two clusters, one containing cultivars of G. herbaceum and another containing cultivars of G. arboreum species. Highly positive correlation between cophenetic matrices using RAPD and AFLP markers was observed. AFLP markers were found to be more efficient for genetic diversity estimation, polymorphism detection and cultivar identification.     

Genetic correlation and path analysis of transpiration efficiency for wheat flag leaves

A 4 × 4 complete diallel cross was set up with four different types of drought-resistant winter wheat varieties to define mutual relations among main quantitative traits and the contribution of each trait to the flag leaf transpiration efficiency (TE) in the grain filling phase. Through genetic correlative and path analysis, the influence of five physiological traits, net photosynthesis rate (Pn), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), transpiration rate (Tr), and leaf temperature (Tl), on flag leaf TE was observed. The results of variance analysis showed that there were very significant differences between TE and the related five traits, as well as between these five traits. The results of genetic correlation analysis showed also that the absolute values of the correlation coefficients of corresponding traits to TE should be ordered as Pn > Gs > Tl > Tr > Ci in the early grain filling stage, Ci > Tr > Pn > Gs > Tl in the middle grain filling stage, and Pn > Ci > Tl > Tr > Gs in the late grain filling stage, respectively. The results of the combined analysis of genetic correlation and path analysis showed that at the early filling stage, there existed a strong positive genetic correlation between Pn and TE, and at the same time Pn had a direct positive contribution to TE. With the progression of grain filling, the stress of high temperature and drought strengthened and the function of flag leaves weakened, so that at the late grain filling phase Ci, Tr and Tl were strongly negatively correlated with TE and all these traits conferred direct negative contributions to TE. The results of this research showed that the selection of wheat genetic materials with slow-ageing, high-photosynthesis, low-transpiration and low leaf temperature should assist breeding of wheat with more efficient use of water in north western China.     

Molecular comparative analysis of component 1 (DNA-R) of an Egyptian isolate of banana bunchy top nanovirus isolated from banana aphid (Pentalonia nigronervosa)

We are reporting a molecular comparative analysis of component 1 BBTV-DNA-R of an Egyptian isolate of (BBTV) and 30 different geographical isolates. DNA was extracted from BBTV-infected adult banana aphids collected from El-Qalubia Governorate, Egypt. Using specific primers the BBTV-DNA-R was amplified, cloned into a prokaryote vector, sequenced and a molecular comparative analysis of BBTV-DNA-R of this study and some overseas isolates of BBTV-infected banana plants was determined. Results showed that the component 1 consists of 1108 nts and contains a sequence of 69 nts representing the CR-SL of 31 nts. A CR-M (90 nts) at the position (972–1062) characterized with GC-rich sequence from nts 76 to 90 (average of 80% G + C) was found. Alignment results of BBTV DNA-R confirmed the presence of a number of conserved regions in all isolates. Large ORF of 861 nts at position 102 to 962 in the virion sense were detected. The predicted protein of this ORF consisted of 286 amino acids and had a molecular weight of 33.8 kDa. The DNA-phylogenetic analysis showed a percent identity of 98.0 and 97.9 between BBTV DNA-R and isolates of Pakistan (isolate TJ1) and Australia (isolate V1), respectively. The similarities between the gene product of Egyptian BBTV DNA-R and the 30 overseas isolates ranged from 93.7 to 99.0%. Differences in phylogenetic trees based on the entire sequence of BBTV DNA-R, CR-M and amino acid sequences confirmed the existence of two taxonomic groups of BBTV and the Egyptian isolate belongs to the south pacific group.