Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers

To meet various breeding objectives and to conserve the existing genetic resources of mulberry for future use, the present study was undertaken to investigate the amount of genetic diversity and to establish the relationships between mulberry genotypes using fluorescence-based AFLP markers. Genetic diversity was estimated in 45 mulberry accessions from different eco-geographic regions of Japan and other parts of the world. Five primer combinations amplified an average of 110 AFLP markers per primer combination, ranging in size from 35 to 500 bp. A high degree of polymorphism was revealed by these combinations that ranged from 69.7 to 82.3% across all the genotypes studied. Several rare genotype-specific bands were also identified which could be effectively utilized to distinguish different genotypes. The wide range in genetic similarity coefficients (0.58–0.99) indicated that the mulberry germplasm collection represents a genetically diverse popu-lation. The phenetic dendrogram generated by the UPGMA method grouped 45 accessions into four major clusters, which was in agreement with the results from conventional methods. Clustering of some genotypes into strictly separate groups was not readily apparent and no clear interrelationships could be depicted, in spite of their different geographic origin. In addition, AFLP analysis provided sufficient polymorphism for DNA typing and contributed additional insights into the genetic structure of the mulberry germplasm. These results will help in the formulation of appropriate strategies for conservation and variety improvement in mulberry, for which little or no knowledge of genetic diversity is currently available. Received: 30 December 1999 / Accepted: 14 March 2000     

Suitability of AFLP markers for the study of genetic relationships among Korean native dogs.

To determine the genetic relationships among domestic dog breeds, we performed both a sequence comparison of mitochondrial DNA (mtDNA) and an amplified fragment length polymorphisms (AFLP) analysis. Three of four regions of mtDNA, cytochrome b, cytochrome oxidase subunit II, and 16S rRNA genes were highly homogeneous among dog breeds, whereas the other region, the control region, showed relatively high polymorphisms with a maximum percentage difference of 3.18%. However, the control region showed extensive polymorphism even within breeds, and the relationship tree derived from the data could not clearly delimit distinct breeds. 19 EcoRI/MseI primer combinations were used to generate AFLP markers among 25 dogs from 11 breeds including three Korean native dogs. These amplification reactions allowed the detection of more than 1900 amplification products of which 408 were identified as polymorphic bands. Unrooted neighbor-joining tree based on dissimilarity values showed that the Korean native dogs were clustered together with the Asian dogs and that the Asian originated dogs were clustered separately from Western originated dogs. A consensus tree using parsimony method also showed Korean native dogs were grouped separately from the other dogs with moderate bootstrap values. Taken together, it is concluded that AFLP analysis is a more informative tool for revealing genetic relationships among dog breeds than mtDNA sequence comparison.     

The PiGMaP consortium linkage map of the pig (Sus scrofa)

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (16.5 Morgans) than the genetic map of the homogametic sex (female) (21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be 18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.     

Assessment of genetic diversity of landraces of Dendrocalamus hamiltonii using AFLP markers and association with biochemical traits

Fermented bamboo shoots are popular traditional food items of various ethnic groups of the northeastern India, especially in Manipur State. Dendrocalamus hamiltonii is an economically important bamboo species used to produce fermented bamboo shoots. We studied genetic variability of this bamboo species in Chandel and Imphal-East (commercial production districts), using AFLP molecular markers. Each of the selected primers detected polymorphisms and 1614 (95.8%) were found to be polymorphic. Cluster analysis based on Dice similarity coefficients using UPGMA differentiated the populations into two major groups. Principal coordinate analysis based on the AFLP data clearly separated the populations according to their genetic diversity and antioxidant activity. Four primers were tested through multiple regression analysis to identify marker-trait association between AFLP data and biochemical attributes, i.e., antioxidant activity and total cyanide content. The 273 bp generated by EcoRI-AAG(Joe)/MseI-CTC showed high positive correlation with antioxidant activity (r = 0.729, P < 0.01). The 396 bp generated by EcoRI-AAC(Ned)/MseI-CTG were negatively correlated with cyanide content (r = -0.694, P < 0.01). Thus, we found association of DNA markers with antioxidant activities and total cyanide content. These results could be of use for the identification of superior genotypes with desirable traits.     

Discovery of cSNPs in pig using full-length enriched cDNA libraries of the Korean native pig as a source of genetic diversity

Clones from full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We analyzed 3.210 chromatograms obtained from sequencing the 5′-ends of brainstem, liver, neocortex, and spleen clones derived from full-length enriched cDNA libraries of Korean native pigs. In addition, 50,000 pig EST sequence trace files were obtained from Genbank and combined with our sequencing information for SNP identificationin silico. For the SNP analysis, neocortex, and liver libraries were newly constructed, whereas the sequencing results from brainstem and spleen libraries were from previously constructed libraries. The putative SNPs from thein silico analysis were confirmed by genomic PCR from a group of 20 pigs of four different breeds. Using this approach, 86% of cSNPs identifiedin silico were confirmed and the SNP detection frequency was 1 SNP per 338 bp. Interestingly, we found a valine deletion at amino acid position 126 of the neuronal and endocrine protein gene in the Korean native pig. We confirmed that this deletion was caused by alternative splicing at the NAGNAG acceptors. Our study shows that large-scale EST sequencing of Korean native pigs can be effectively employed for natural polymorphism-based pig genome analysis.     

Analysis of genetic diversity and sectional relationships in Musa using AFLP markers

The AFLP technique was used to assess the genetic diversity and sectional relationships in 39 accessions representing the four main sections of the genus Musa. Eight AFLP + 3 primer pairs produced 260 polymorphic bands that were used in cluster and PCO analysis. A wide range of variability was observed among the species within the sections of the genus Musa. AFLP data was useful in separating the different sections of the genus as well as differentiating the different genomic groups of section Eumusa. Section Rhodochlamys (x = 11) appeared as a distinct entity and clustered closely with the Musa acuminata Colla complex of section Eumusa that has the same basic chromosome number. This relationship is congruent with previous studies. However, unlike previous proposals that questioned the identity of Rhodochlamys as a separate taxonomic unit, PCO analysis of the AFLP data showed that it is a distinct entity. Musa laterita Cheesman (Rhodochlamys) and Musa schizocarpa Simmonds clustered with the M. acuminata complex suggesting that they may be sources of useful genes for the improvement of the cultivated bananas. Callimusa formed a distinct unit and was closer to Australimusa than to the other sections. Although both sections share the same basic chromosome number of x = 10 these sections are genetically distinct     

Annexin A2 is involved in pig (Sus scrofa)sperm-oviduct interaction

The oviduct is a dynamic organ which modulates gamete physiology. Sperm-oviduct interaction provides the formation of a sperm storage reservoir and allows the selection of sperm with certain qualities in eutherian mammals. In sows, the oviductal sperm binding glycoprotein (SBG) has been proposed to be involved in sperm selection. In this work, based on its affinity to sperm periacrosomal membrane proteins, we isolate another pig oviductal cell protein that interacts with sperm. Peptide identification by LC/MS-MS allowed the identification of this protein as annexin A2. The presence of this annexin, as well as annexin A1 and annexin A5 in sow oviductal cells was confirmed by Western blot with specific antibodies. The three proteins were localized in sow oviduct by immunohistochemistry, showing the presence of annexin A2 at the apical surface of the oviductal epithelial cells. Based on our data and the fact that annexins have been stated as candidate receptors of bovine sperm for sperm reservoir formation, we propose that this family of proteins is involved in sperm-oviduct interaction, annexin A2 being the main sperm binding isoform in pig.     

Assessment of genetic diversity and interspecific relationships among three species of Podophyllum using AFLP markers and podophyllotoxin content

The genus Podophyllum (common name: May Apple) has high medicinal value due to the presence of anticancer molecule, podophyllotoxin. A total of 35 individuals belonging to three species of Podophyllum viz. P. hexandrum Royle, P. sikkimensis R. Chatterjee and Mukherjee both Indian species, and their American counterpart, P. peltatum L. have been investigated with a view to ascertain variation in their (1) podophyllotoxin content, and (2) molecular profiles generated through AFLP markers. The active principle content varied within the representative individuals of different populations of a species and between species; the species-wise podophyllotoxin content (% of dry wt) ranged as follows: P. hexandrum-Munsyari populations: 0.39–1.20 %, P. hexandrum-Kullu populations: 0.58–1.50 % (highest), P. peltatum: 0.50–1.30 %, and P. sikkimensis: 0.06–0.73 % (lowest). Detection of podophyllotoxin in P. sikkimensis, although at low levels, would appear to be the first report of its occurrence in this species. The genetic diversity and relationship amongst 35 sampled individuals of three species have been analyzed using 20 AFLP markers, which resulted into 1,358 loci of which 595 were polymorphic revealing 44 % polymorphism. High level of genetic diversity was observed (percent of polymorphic bands, PPB = 88.01 %; PIC = 0.813) among the species, while it was low within the individual species (PIC = 0.57 %; Marker index, MI = 4.77). Genetic similarity among the species (calculated with Euclidean coefficient) showed two major clusters. Cluster one contained all the individuals of P. peltatum (American May Apple) whereas cluster two grouped together individuals representing various populations, belonging to both the species of Indian May Apple (P. hexandrum, and P. sikkimensis). The observed paired relationship (45–50 % similarity; calculated from AFLP data) of intercontinental species in the Podophyllum group (P. hexandrum, and P. sikkimensis vs. P. peltatum) would appear to be paraphyletic. The AFLP data of the analyzed representatives have been used to examine the sister relationships among these species, and would be beneficial to find ways to strengthen the gene flow among populations to maintain the natural genetic variation within the populations of Podophyllum species.     

Assessing genetic diversity of populations of topmouth culter (Culter alburnus) in China using AFLP markers

Genetic diversity of five wild populations and a cultured population of topmouth culter (Culter alburnus) was investigated using amplified fragment length polymorphism (AFLP). A total of 373 reproducible bands amplified with seven AFLP primer combinations were obtained from 163 fish. The percentage of polymorphic loci ranged widely from 37.0% to 69.2% within distinct populations. The cultured population appeared to have a lower level of polymorphism (37.0%), gene diversity (0.121 ± 0.188) and Shannon's Information index (0.183 ± 0.263) than the wild populations. Analysis of molecular variance (AMOVA) revealed that average F_ST value overall loci was 0.2671, and the percentage of variation within population (73.29%) was larger than among populations (26.71%) (P < 0.01). The six populations were clustered into two major clades with UPGMA. The results from analysis of population pairwise gene flow indicated moderate gene flow among populations. Our study indicated that the genetic diversity of the cultured population was reduced compared with the wild populations. Geographic isolation, habitat, and artificial selection all may have played important roles in population differentiation. The information may be beneficial to future broodstock selection and defining conservation management for the different populations of topmouth culter.     

Assessment of genetic diversity among okra genotypes using SSR markers

Okra (Abelmoschus esculentus) is an important nutritious vegetable. Despite its high economic and industrial value, very little attention has been paid to assess genetic diversity of okra at molecular level. For effective conservation and proper deployment of germplasm, a study on diversity analysis of okra germplasm was conducted with DNA markers. Microsatellite/Simple sequence repeat (SSR) markers were utilized to evaluate the genetic diversity among 96 accessions of Abelmoschus, of which 92 accessions were of A. esculentus and one accession each of A. tuberculatus, A. moschatus, A. moschatus subspecies tuberosus and A. manihot. A set of 40 SSR primers were tested, of which 30 primers gave reproducible amplification which were used further for diversity analysis. With a mean of 7.1 bands per SSR, DNA amplification with 30 SSRs generated a total 213 bands, of which 60.66 % were recorded polymorphic. Polymorphic information content ranged between 0.11 and 0.80 with an average of 0.52, indicating that the majority of primers were informative. The Jaccard’s coefficient ranged from 0.107 to 0.969. The UPGMA analysis grouped Abelmoschus genotypes into three main clusters at a cut-off of 0.20. Results of present study revealed that sufficient variation exists among the studied accessions and GAO-5 which was found highly diverse can be exploited for okra improvement. The outcome of present research would assist to make use of Ablemoschus germplasm for okra breeding.     

Chromosome restriction enzyme digestion in domestic pig (Sus scrofa) constitutive heterochromatin arrangement

The bimodal karyotype of pig appears to contain two types of constitutive heterochromatin, reflecting different satellite DNA families: GC-rich heterochromatin located mainly in the centromeric regions of the biarmed chromosomes, and less-GC-rich heterochromatin in the centromeric regions of the one-armed chromosomes. In order to better discriminate this constitutive heterochromatin, we treated pig chromosome preparations with eight different restriction endonucleases, followed by C-banding. This technique allowed an expedited characterization of the constitutive heterochromatin and demonstrated its great heterogeneity in pig chromosomes. Our work allowed the detection and identification of twenty-two heterochromatin subclasses (twelve centromeric, four interstitial, five telomeric, and the Yq band). Moreover, several cryptic interstitial and telomeric bands were revealed. The work presented here is useful not only for fundamental studies of chromosome banding and constitutive heterochromatin, but also offers a new approach for pig clinical cytogenetics.     

Segmental aplasia in the uterus of a European wild pig (Sus scrofa)

This paper reports an instance of aplasia in the uterine horn of the European wild pig (Sus scrofa).     

The complete primary structure of alpha-lactalbumin isolated from pig (Sus scrofa) milk

The complete amino-acid sequence of pig alpha-lactalbumin has been determined. It was obtained by microsequencing of the native protein and the peptides derived after tryptic or cyanogen bromide cleavage. The tryptic peptides were separated by a rapid microbore HPLC method. Pig alpha-lactalbumin is 122 amino acids long and differs from the bovine homologue by 26 exchanged residues. Of the two prolines present in bovine alpha-lactalbumin, one has been deleted in the pig structure. All previously sequenced alpha-lactalbumins have shown glutamic acid at position 49, which is known to be the active site in the homologous lysozyme c structure. This residue is replaced by phenylalanine in pig alpha-lactalbumin indicating that the pig protein is the first alpha-lactalbumin with complete loss of all lysozyme functional residues.     

Characterization of Iberian pig genotypes using AFLP markers

The use of the AFLP (amplified fragment length polymorphism) technique for the characterization of highly inbred Iberian pig breed genotypes and the detection of strain-specific polymorphisms is demonstrated. Twelve different primer combinations were used on individual DNA samples from animals belonging to two black hairless Iberian pig strains, Guadyerbas and Coronado. These amplification reactions allowed the detection of more than 1700 amplification products of which 26 were identified as strain-specific markers, present in all individuals of one strain and absent in the other. Comparison of male and female amplification products within one strain also allowed the identification of 8 male-specific amplified bands. AFLP showed a great power of marker detection due to a high multiplex ratio and high reproducibility. Comparison of similarity and co-ancestry coefficient matrices also showed the usefulness of AFLP markers to estimate genetic relationships between individuals pigs.     

Evaluation of genetic diversity among Iranian accessions of Ocimum spp. using AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic, diversity of 120 Ocimum accessions belonging to five species and varieties named: Ocimum ciliatum, Ocimum minimum, Ocimum basilicum var. purpurascens, O. basilicum var. dianatnejadii and O. basilicum var. alba. Eight AFLP primer combinations revealed 150 polymorphic bands (59.5%). The highest and the lowest PIC were 0.87 and 0.81 for E-CAA/M-ACC and E-CAA/M-AGA primer combinations, respectively, with average polymorphic information content (PIC) value of 0.84 over all primer combinations. Cluster analysis based on unweighted pair group method with arithmetic Average (UPGMA) and Jaccard’s coefficient grouped the five species and varieties into two main clusters. The first cluster contained the accessions belonging to O. ciliatum and the second comprised different botanical varieties of O. basilicum and O. minimum. The means of Shannon’s diversity index and Nei’s index of genetic diversity indicated that O. basilicum had the greatest variation, while O. ciliatum showed the least variation. Nei’s genetic identity measured in five Ocimum species and botanical varieties revealed the highest identity (0.939) between O. minimum and O. basilicum var. purpurascens and the lowest genetic identity (0.611) between O. basilicum var. purpurascens and O. ciliatum. In conclusion AFLP results indicated that O. minimum should be considered as a variety of O. basilicum.     

Genetic diversity of Hibiscus tiliaceus (Malvaceae) in China assessed using AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to investigate the genetic variations within and among nine natural populations of Hibiscus tiliaceus in China. DNA from 145 individuals was amplified with eight primer pairs. No polymorphisms were found among the 20 samples of a marginal population of recent origin probably due to a founder effect. Across the other 125 individuals, 501 of 566 bands (88.5%) were polymorphic, and 125 unique AFLP phenotypes were observed. Estimates of genetic diversity agreed with life history traits of H. tiliaceus and geographical distribution. AMOVA analysis revealed that most genetic diversity resided within populations (84.8%), which corresponded to results reported for outcrossing plants. The indirect estimate of gene flow based on phiST was moderate (Nm=1.395). Long-distance dispersal of floating seeds and local environments may play an important role in shaping the genetic diversity of the population and the genetic structure of this species.     

Analysis of genetic diversity in Prunus mira Koehne ex Sargent populations using AFLP markers

Prunus mira Koehne ex Sargent (syn. Persica mira (Koehne) Kov. et Kostina), native to China, is an excellent fruit tree due to its high ecological and economical value. However, there is limited knowledge on the genetic information of P. mira. In this study, the genetic relationships of 83 P. mira accessions from five populations were assessed using amplified fragment length polymorphism (AFLP). The results showed that AFLP was a powerful tool to detect levels of genetic diversity of natural populations in P. mira. The similarity coefficient between accessions ranged from 0.12 to 0.76, with an average 0.57. 83 accessions were clustered into two major clusters at similarity coefficient of 0.225. The highest values of N _e, H and I occurred in ML population. Most of the genetic variations occur within population. There is no close relationship between geographic distance and genetic distance. At the same time, ex situ conservation needs to be established for P. mira.     

Evaluation of AFLP markers to reveal genetic diversity in Typha

We investigated to what extent DNA-markers can assist species determination in the genus Typha. A set of AFLP markers was used to discriminate samples of the species Typha latifolia and Typha angustifolia collected in Flanders (North Belgium). The T. latifolia samples formed a compact cluster while the T. angustifolia samples were divided into smaller groups. It was not clear whether interspecific hybrids or higher levels of diversity present in the T. angustifolia dataset could account for this. As in previous surveys, using isozyme and VNTR markers, AFLP markers revealed an almost complete lack of genetic variation in Flemish T. latifolia. Despite the low degree of diversity, a significant level of genetic differentiation was found between the T. latifolia samples originating from different river basins. Whether this differentiation has any ecological relevance remains to be investigated. The methodology applied was not able to detect clonal reproduction in T. latifolia. Probably, the low levels of diversity present in this species can account for this, indicating that the usefulness of the methodology applied depends on the level of diversity present in the species studied.