Specific Random Amplified Polymorphic DNA Markers Research Articles

Molecular characterization of traditional mango germplasm of coastal Andhra Pradesh using RAPD markers

A study was conducted at Horticultural research station, Venkataramannagudem for evaluating the variability of mango germplasm to conserve the elite ones and to identify the superior genotypes using molecular markers for future crop improvement. Genetic variation and relationship among 34 traditional mango germplasm were analyzed using Random Amplified Polymorphic DNA (RAPD) markers. Fifteen out of the twenty primers screened were informative and 177 amplified DNA bands with sizes ranging from 100 - 5000 bp were selected as RAPD markers. The number of amplified fragments varied from 3 (OPG 13) to 20 (OPX 04) with an average of 11.8 polymorphic fragments per marker. Specific RAPD markers for some mango germplasm were identified. Unweighted Pair Group Method using Arithmetic means (UPGMA) cluster analysis grouped all the germplasm into two clusters with a genetic similarity coefficient range of 0.67 to 0.88. The accessions within the germplasm i.e., Banganapalli -1, Banganapalli -2 and Banginapalli-3 showed genetic similarity which is in good agreement from a single ancestor with their putative pedigrees. The genotypes Panukula Mamidi and Rajamamidi were closely clustered since these two genotypes are native to the Vizianagaram district. Similarly, the genotypes Nuzividu Tiyya Mamidi and Nuzividu Rasalu occurred in the same cluster indicated that these genotypes also originated from the same geographical area, it can be concluded that they were separated into the cluster based on geographic origin. This study showed clearly that germplasm from coastal Andhra Pradesh unveiled maximum diversity and indicated the potential of RAPD markers for the identification of mango germplasm for breeding purposes. Keywords: Cluster analysis, Dendrogram, Mango, Polymorphic information content and RAPD markers

Genetic diversity and phylogenetic relationships among and within Amaranthus spp. using RAPD markers

Random Amplified Polymorphic DNA (RAPD) markers were used to investigate genetic diversity and phylogenetic relationships among 10 species belonging to the genus Amaranthus L. The results showed that the polymorphism in cultivated species was lower than that in wild ones, reflecting the selection pressures of domestication on genetic diversity in cultivated species. A specific RAPD marker was detected for each of A. powellii PI 572262, A. tricolor PI 462129, A. palmeri PI 607455, A. caudatus PI 511679 and A. quitensis PI 511744. The overall mean similarity index of amplified fragments generated by RAPD primers on genomic DNA of Amaranthus accessions indicated that A. hypochondriacus was the closest grain amaranth to A. hybridus , followed by A. caudatus and A. cruentus. A. tricolor had a maximum genetic distance from grain amaranth species, confirming its morphological classification in a distinct subgenus Albersia . Similarly, the accessions of A. palmeri were separated in a distinct cluster, supporting its classification in a distinct subgenus Acnida. A. hybridus accessions were gathered together with grain amaranth species, thereby supporting the single progenitor hypothesis for grain amaranths. A. spinosus was separated on a distinct principal coordinate axe, indicating its low correlation with other species and confirming its morphological classification in a distinct section, i.e. Centrusa .

Genetic polymorphism among seven entomopathogenic nematode species (Steinernematidae) revealed by RAPD and SRAP analyses

The strength of sequence-related amplified polymorphism (SRAP) in comparison to random amplified polymorphic DNA (RAPD) in determining the genetic polymorphism among seven species of entomopathogenic nematodes (Steinernematidae) was tested. Nine RAPD and 12 SRAP primer pair’s combinations were used. The number of polymorphic bands and the polymorphism percentages was high in SRAP analysis (97 out of 107 bands were polymorphic (90.6%)) compared to RAPD (65 out of 89 bands were polymorphic (73%)). The highest number of RAPD bands was recorded for OPE-A-07 primer (14 band) while OPE-D-20 was scored the lowest band number (7 bands). The SRAP primers Me3-em2 registered the highest number of bands (13 bands) while Me3-em3and Me2-em2 showed the lowest band number (6 bands). The genotype-specific SRAP and RAPD markers for the different ENP species were recorded. The highest number of SRAP specific markers (7 markers) was scored for Steinernema glaseri, then 4 markers for S. abbasi 2 followed by S. riobrave (3 markers), 1 marker for S. abbasi 1, S. riobrave recorded the highest number of specific RAPD marker (4 specific markers) followed by S. carpocapsae DD 136 and S. glaseri (two specific markers), then only 1 specific marker for S. abbasi 1. Based on the data obtained from RAPD and SRAP analysis, the dendrogram was created to clarify the genetic distances among different species of the studied entomopathogenic nematodes. The present study indicated that SRAP was more powerful than RAPD analysis for detecting the genetic polymorphism among closely related species of nematodes. The genotype-specific markers detected from both RAPD and SRAP can be used in future biological control programs.

Molecular Genetic Variations among Some Punjab Sheep Breeds Using RAPD Analysis

The present study was carried out on seven sheep breeds (Thalli, Lohi, Salt Range, Kajli, Sipli, Buchi and Hissradale) by Random Amplified Polymorphic DNA (RAPD) analysis using 21 random decamer primers. Blood samples were collected from different numbers of animals per breed, of both sexes. After DNA extraction PCR was carried out by using 25 µL reaction mixture containing 3 mM MgCl2, 100 µM each of dATP, dCTP, dGTP, dTTP, 0.2 µM primer, 15 ng of genomic DNA, and 5 unit of Taq polymerase, through programmable thermal cycler (Crea Con, TCY, USA).Out of 21 decamer primers 16 primers yielded easily scorable bright DNA bands while other five yielded smeared and nonspecific fragments. The genetic similarities of seven sheep breeds were high, ranging from 74.42% to 94.29 %. Thalli and Latti had greatest similarity (94.29%). The lowest genetic similarity was between Lohi and Hissardale (74.42%). The unweighted pair group method of arithmetic means (UPGMA) dendrogram obtained from the cluster analysis revealed two clusters. The study confirms that the molecular genetic techniques such as RAPD-PCR can economically and efficiently be used to establish genetic distances and similarities among and within breeds as well as to find out breed specific genetic markers. The seven sheep breeds in the study could be identified by using one breed specific RAPD marker or a combination of two or more markers. Primer GLB-08 can identify Lohi breed and primer GLA-14 can identify Hissardale. Primer GLA-19 can identify both Kajli and Sipli breeds simultaneously.

KARAKTERISASI BEBERAPA STRAIN GURAMI Osphronemus gouramy Lac. MENGGUNAKAN MARKA RAPD

Giant gouramy, Osphronemus gouramy Lac. is a popular fish species in Indonesia, especially in Java and Sumatera as this freshwater fish species has a high economic value of stable price. Fish farmers in Bogor divide giant gouramy into six strains based on egg productivity, growth rate, and maximum weight of the adult. They are soang, jepang, blue saphire, paris, bastar, and porselin. These various strains lead to the need of study on the genetic relationship among them, which can be performed by the use of RAPD (Random Amplified Polymorphic DNA) marker. This study aims to determine primers producing consistent and polymorphic RAPD markers, determine specific RAPD markers, and know the genetic relationship among several giant gouramy strains. The strains used in this study are soang, jepang, and blue saphire. Survey method was applied employing purposive random sampling technique. Total genomic DNA was isolated using Genjettm genomic DNA purification kit (Fermentas), which was then used as template to amplify RAPD markers with primers OPA-07, OPA-09, OPA-11, OPA-20, OPAH-01, OPAH-08, OPAH-09, and OPAC-14. The variables examined were patterns and numbers of specific DNA fragments as the PCR amplification products. Selected primers were determined descriptively on the basis of specific DNA bands appearing on the agarose gel. Genetic diversity was predetermined by changing qualitative band pattern into quantitative binnary data. Genetic relationship was analyzed using cladistic method with PAUP software. The results showed that only five of the eight primers produce consistent and polymorphic RAPD markers, i.e. OPA-11, OPA-20, OPAH-1, OPAH-8, and OPAH-9. Specific RAPD markers which can be used to distinguish several gouramy strains are those amplified with OPA-20 of 786 bp, OPA-20 of 1,176 bp, OPAH-8 of 1,000 bp and OPAC-14 of 1,607 bp. Nervertheless, it was found that RAPD markers cannot be used to clearly determine genetic relationship among gouramy strains.

English

Genetic variation in seven selfed and unselfed Cucurbita pepo landraces from districts in KwaZulu- Natal Province was investigated using the random amplified polymorphic DNA (RAPD). Out of 36 primers screened nine were selected, which gave 100 clear and bright fragments, out of which 94 (94%) fragments were considered polymorphic. The sizes of bands ranged from 75 to 1800 bp. The number of bands per primer ranged between nine and 14. The genetic differentiation coefficient between populations (G ST ) varied between 0.0022 and 0.0100, while the gene flow ranged between 49.4545 and 223.7226. The effective number of alleles, Nei’s gene diversity index and Shannon’s information index were the highest in the selfed population from Zululand (yellow) district (ZS) population (Ne= 1.2046; H=0.1677; I=0.3060) and the lowest in unselfed population from uThungulu (yellow) (TNS) population (Ne=1.1512; H=0.1301; I=0.2518). The production of specific RAPD markers by different primers indicated gene diversity between: Selfed and unselfed populations from the same geographic origin; populations with yellow/orange mature fruit from a population with green mature fruit; and also among different populations in general. The selfed population from uThungulu (yellow) (TS) and TNS populations, both from uThungulu district, were the highest in genetic identity (I N = 0.9996) and the closest in the genetic distance (D = 0.0004). The unselfed population from Umkhanyakude (green) (CPSP) and unselfed population from Umkhanyakude (yellow (MNS) populations as well as CPSP and TNS populations were the lowest in genetic identity (I N = 0.9985) and the furthest in genetic distance (D = 0.0015). The dendrogram mainly grouped the populations according to their mature fruit colour, and then according to their geographical origin. All genetic parameters indicated that there was plentiful genetic diversity in C. pepo landraces of northern KwaZulu-Natal, South Africa. Key words: Cucurbita pepo landraces, genetic variation, self-pollination, random amplified polymorphic DNA (RAPD) markers, northern KwaZulu-Natal.

English

The present study involved characterization of variability of qualitative morphological characters, protein patterns and random amplified polymorphic DNA (RAPD) markers in some Pisum sativum genotypes. 17 different Pisum genotypes were used in this study. They are: Seven P. sativum L. cultivars (Vectory Freezer, Master, Lincoln, Little Marvel, Montana, Local and Billinder), three wild taxa of P. sativum L., five accessions ofP. sativum L. and two accessions of P. fulvum L. The results demonstrate a high potential and resolving power of DNA-based methods (RAPD markers) for discrimination between Pisum genotypes. The two accessions of P. fulvum L. were successfully separated on the basis of RAPD markers, while the morphological characters and protein patterns could not be distinguished between them. Wild taxa collected from the north coastal region of Egypt were separated from other genotypes, suggesting different genetic pool. RAPD markers indicated that four P. sativum L. cultivars (Vectory Freezer, Master, Lincoln and Little Marvel) and two accessions P. fulvum L. are grouped in one cluster, suggesting that these cultivars may be originated from the P. fulvum L. gene pool. Data indicated 46 RAPD markers specific to ten genotypes, and one marker specific to P. fulvum L. The three wild taxa of P. sativum L. showed high number of specific RAPD markers, reflecting similarities among these taxa and their differences from other genotypes. The use of RAPD markers linked to qualitative morphological characters in breeding programs makes the selection for these characters easy and possible at the young seedling stage without reaching the maturation stage. Key words: Pisum sativum, seed storage proteins, random amplified polymorphic DNA (RAPD) markers, numerical analysis.

MOLECULAR IDENTIFICATION AND PHYLOGENETIC RELATIONSHIP AMONG LOCAL, SANGKURIANG, AND AFRICAN CATFISH BASED ON RAPD MARKER

Catfish species are commercially cultivated freshwater fish by fish farmer. Formerly, only one species of catfish had been cultivated, namely local catfish (walking catfish) Clarias batrachus. However, since 1985, local catfish was replaced by a new commodity from Africa called African catfish (C. gariepinus). In 2004, BBPBAT Sukabumi had introduced a new strain of catfish, namely Sangkuriang catfish. Taxonomic analysis needed, especially for Sangkuriang catfish using molecular marker, in order to get a clearer picture about its genetics departure from their parental, so their taxonomic status and relationships with local and African catfish is clearer.  One of the molecular markers is randomly amplified polymorphic DNA (RAPD) markers. Species status defined based on the presence and absence of specific band on each catfish. Phylogenetics relationships was inferred from phylogenetic tree which was built using UPGMA tool as implemented in NTSYS software based on the similarity of RAPD band pattern. The result showed that Sangkuriang and local catfish had specific RAPD markers, while African catfish was not. This means that all RAPD markers of African catfish were shared with those of two others catfish. Local catfish phylogenetically distantly related to African and Sakuriang catfish. Only one individual of African catfish (D4) genetically close related to Sangkuriang catfish while the rest individuals of African catfish genetically closed related to green catfish. This means that African catfish showed very divergence genetic constituent.  Key words: catfish, molecular identification, phylogenetic relationships, RAPD

Identification of sex-specific DNA markers in betel vine (Piper betle L.)

The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in male and female plants of Piper betle L. Two bulks of DNA were made drawing one each from male and female, by pooling an equal volume of DNA samples from each group of individual contributing to the bulk analysis. Fifty different random decamer primers were screened with the two bulks to identify markers associated with sex expression of which only four primers were found to be associated with sex expression. These four primers were then tested with individual plant DNA samples where sex-associated RAPD markers were identified. A ~1,400 and ~850 bp fragment from the primer OPA04 and OPN 02 respectively was found to be present in all the male individuals and absent in all the female plants. In another primer, a ~980 bp amplification product from the primer OPC 06 was present only in the female individuals. A common primer OPA 08 showed both male and female specific markers of 650 and 1,200 bp respectively. Thus, the three male- specific RAPD markers OPA041400, OPA08650 and OPN02850 and two female-specific markers OPA081200 and OPC06980 can reliably differentiate the male and female plants of P. betle L. Ploidy comparison also showed the differences in male and female plants.

Fingerprinting and assessment of genetic variability of Varroa destructor in Egypt

SummaryThis study describes the use of eleven Random Amplified Polymorphic DNA (RAPD) primers to determine the genetic variability of Varroa destructor mites in Egypt. Mites from ten different Egyptian localities were included in this study. The results indicated that 133 bands were amplified, out of a total of 923. Of these amplified bands, 95 were polymorphic (71.4%) and 38 were monomorphic (28.6%). In addition, 37 specific positive RAPD markers for V. destructor in Egypt were identified. This is the first molecular genetic study carried out with V. destructor in Egypt. The markers obtained in the present study might have potential applications mite control for the beekeeping industry in Egypt. Further detailed investigations at DNA sequence level are needed to obtain complete and accurate identification of V. destructor in Egypt.

Molecular Identification of Saccharum spp. × Erianthus fulvus Hybrids Using Sequence‐Characterized Amplified Region Markers

Identification of hybrids derived from crosses between cultivated sugarcane (Saccharum spp.) and its wild relatives, including Erianthus fulvus Nees ex Hack., is important for the development of new sugarcane cultivars. The aim of the present study was to identify true hybrids in 73 F1 progeny obtained from five crosses with cultivated sugarcane as female parents and E. fulvus as male parents. Three random 10mers were used as primers in random amplified polymorphic DNA (RAPD) reactions. A total of 10 E. fulvus–specific RAPD markers were cloned and sequenced, of which four were successfully applied as sequence‐characterized amplified region (SCAR) markers. Based on the four RAPD marker sequences, five SCAR primer pairs were designed for polymerase chain reaction amplifications. Of the 73 progeny examined, 58 produced E. fulvus–specific SCAR bands, thus representing true hybrids of cultivated sugarcane and E. fulvus These results demonstrate the first use of SCAR marker analysis as an effective tool for the identification of intergeneric sugarcane hybrids.

Molecular characterization and genetic diversity analysis of citrus cultivars by RAPD markers

Random Amplified Polymorphic DNA (RAPD) markers were used to evaluate genetic similarity and inter-relationship among 18 citrus cultivars, including 13 species and 5 hybrids. Out of 40 decamer primers screened, 25 were selected which produced 250 markers; of which 231 were polymorphic and some species or cultivar specific RAPD markers. The Jaccard coefficient was used to calculate the genetic similarity. UPGMA was used to generate the dendrogram which clearly separated Jatti-Khatti from all major clusters at a similarity coefficient of 0.61. The average genetic similarity value observed across all the genotypes was 0.63, with the 2 sweet orange cultivars, Jaffa and Blood red, showing maximum similarity (82%). The Jatti-Khatti and King Mandarin were found to be genetically most diverse. The genetic variation between cultivars was quite high and revealed their different origins.

Molecular identification of Chinese cultivated Porphyra (Bangiaceae, Rhodophyta) based on the rDNA internal transcribed spacer-1 sequence and random amplified polymorphic DNA markers1

Porphyra yezoensis Ueda and P. tenera Kjellman are two common commercial seaweeds cultivated in China, but it is difficult to identify them routinely because they are morphologically and systematically very close. The nuclear ribosomal internal transcribed spacer-1 (ITS-1) region of nine selected Porphyra cultivars from China were sequenced and determined. Combined with some ITS-1 data from GenBank, the phylogenetic analysis inferred from the neighbour-joining method indicated that the Porphyra species in this study exhibited clear taxonomic relationships and could be identified clearly to species. Based on the results, the random amplified polymorphic DNA (RAPD) technique was applied to distinguish 12 Porphyra cultivars, some of which turn out to be different lineages of the same Porphyra species. Eight specific RAPD markers were scored and used to construct a fingerprint that could distinctly identify different Porphyra cultivars. The results suggest that both the rDNA ITS-1 sequence and RAPD markers are useful methods to identify Porphyra cultivars, and may also be valid for phylogenetic and taxonomic studies.

Genetic relatedness (diversity) and cultivar identification by randomly amplified polymorphic DNA (RAPD) markers in teasle gourd (Momordica dioica Roxb.)

Genetic relationship and variation of 29 accessions of teasle gourd ( Momordica dioica Roxb.) and 1 accession of Momordica cochinchinensis Spreng. (wild relatives of teasle gourd) were examined by RAPD analysis using 44 dodecamer oligonucleotide primers. A total of 496 fragments were produced by 44 primers of which 95% bands were polymorphic. Using presence or absence of specific RAPD markers or combination of primers, 23 out of 30 accessions were identified. The genetic relatedness or genetic distance based on Nei and Li's genetic similarity varied from 0.86 to 0.65 with an average of 0.74 among 29 M. dioica accessions (when M. cochinchinensis excluded). In the phenetic dendrogram developed from cluster analysis using UPGMA method, M. cochinchinensis was out grouped as single accession, while others showing relatively weak grouping formed four groups. Clustering pattern did not demonstrate any relationship between geographical origin and genetic diversity. A DNA extraction method has been standardized. This is the first report of using RAPD techniques in teasle gourd. It was concluded that RAPD analysis is a useful tool for genotypic identification and estimation of genetic similarity in teasle gourd.

Identification ofporphyra lines (rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.

RAPD markers for discriminating tea germplasms at the inter-specific level in China

For the discrimination of tea germplasms at the inter-specific level, four tea species and varieties (Camellia sinensis, C. sinensis var. assamica, C. sinensis var. pubilimba, C. sinensis var. kucha) and their 20 wild allied species (C. sp.) preserved in the China National Germplasm Tea Repositories (CNGTR) were investigated using randomly amplified polymorphic DNA (RAPD) markers. Fifteen primers were chosen from the 61 screened for RAPD amplification. The average DNA polymorphic frequency of RAPD primers at the inter-specific level was 0.30, varying from 0.16 to 0.60, lower than that at the intra-specific level. Using the presence, sometimes absence of unique RAPD markers, it was possible to discriminate 14 of the germplasms investigated. No single primer could discriminate all the 24 germplasms. However, OPO-13 provided rich band patterns and it could discriminate 10 genotypes. The combination of two and three primers made it possible to discriminate 15 and 21 germplasms, respectively. Furthermore, the combination of band patterns or the DNA fingerprinting based on specific RAPD markers generated by OPO-13, OPO-18, OPG-12 and OPA-13 allowed the discrimination of all 24 germplasms investigated. Therefore, RAPD markers also provide a powerful tool to differentiate tea germplasms at the inter-specific level.

Identification of 27 Porphyra lines (Rhodophyta) by DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.

Molecular Discrimination of Medicinal Astragali Radix by RAPD Analysis

The randomly amplified polymorphic DNA (RAPD) analysis has been applied for estimating genetic diversity in plant populations or cultivars. To discriminate geographical origin among Astragali radix populations, RAPD analysis was carried out using 20 mer‐random primers. The similarity coefficient between the DNA of Astragali radix plants analyzed was 0.527. Although the coefficients of similarity were high, primer 7, 8 and 10 gave distinguishable bands between Korean and Chinese Astragali radix. We obtained the specific RAPD markers to discriminate between Korean and Chinese Astragali radix at a DNA level. These results suggest that this method is able to discriminate the concerned Astragali radix geographical origin species. Also, this is the first report on the genetic diversity in geographical origin among Astragali radix populations using RAPD analysis. Broader application of this approach to authenticate other morphologically similar medicinal materials is rationalized.

Gene flow from wheat (Triticum aestivum L.) to jointed goatgrass (Aegilops cylindrica Host.), as revealed by RAPD and microsatellite markers

In order to estimate the potential of gene flow between wheat (Triticum aestivum L.) and jointed goatgrass (Aegilops cylindrica Host.), we carried out mixed pollinations in experimental and natural conditions. A set of species-specific RAPD (random amplified polymorphic DNA) and microsatellite markers were used to detect the presence of parental markers in the progeny of the plants used in these experiments. No hybrids were found within the offsprings of the plants used for the greenhouse experiments, while 85 Ae. cylindrica×T. aestivum hybrids were found within 2400 analyzed F1 plants resulting from the field pollinations. The hybridization rates for individuals of different populations of the wild species differed considerably: 1% for two populations known for more than 90 years versus 7% for a newly discovered population. Most of the hybrids were completely sterile, but five of them produced 13 seeds (BC1) by backcross with Ae. cylindrica. Twelve seeds germinated and generated viable and partly fertile plants. About 25% of the wheat specific RAPD markers were found in the BC1 plants, indicating that introgression of wheat DNA into Ae. cylindrica is possible. In addition, one microsatellite marker, known to be situated on the D genome (a genome shared by both species), was also found in the BC1 plants.

Comparison of Greek breeds of horses using RAPD markers

To assess genetic markers able to distinguish between five Greek breeds of horses, the random amplified polymorphic DNA (RAPD) technique was used. A total of 40 primers were initially screened in all breeds to identify primers that consistently revealed well‐amplified, polymorphic bands. Despite the variability that was found within breeds, no specific RAPD marker for the discrimination of the breeds was detected. This is probably due to the extensive cross‐breeding that has taken place over the years, resulting in a mixing of breeds. The high phenotypic divergence of the pony of Skyros from all the other Greek breeds of horses could be explained in terms of micro‐evolutionary processes, such as natural selection owing to differential fitness. In addition, to determine the genetic affinities between breeds, all samples were screened with five primers. These primers produced a total of 93 bands, 51 of which were polymorphic. Data analysis for genetic polymorphism revealed a degree of divergence between breeds. According to the discriminant analysis (DA) classification, 100% of the individuals examined in this study could be classified correctly into their breeds. The results of this study show that RAPDs could be a powerful tool for the classification testing of individual horses, as well as for the determination of the genetic variation found among breeds, which is important for a proper management policy designed to protect this species.