Arbitrary 10-mer Research Articles

Variability among selective guava (Psidium guajava L.) varieties revealed by morphology and RAPD marker

The research was conducted for the assessment of genetic diversity using both morphological and random amplified polymorphic DNA (RAPD) analysis of twelve guava (Psidium guajava L.) varieties growing in Bangladesh. Morphological characterization of guava varieties showed a wide range of variation. The highest variability was observed between Poly and Jelly varieties.Polymerase chain reaction with 5 arbitrary 10-mer and 3 arbitrary 12- mer RAPD primers produced a total of 50 bands of which 75.23 percent were polymorphic. The highest percentage of polymorphic loci (100%) was observed for primer A and the lowest (50%) for A03 primer. The UPGMA dendrogram revealed the segregation pattern and the difference of evolutionary changes. Guava varieties were separated into two main groups, one of them was made up of Chineese, Jelly, Kazi, Apple, L-49, Local-2 and Local-3. The other one was made up of Local-1, Poly, Kashi, Thai and Bombay. The highest genetic distance between Apple and Kazi peyara indicate that these varieties might be interesting in breeding programme for improving trait of interest. This scientific information could be used for further improvement of guava.
 Jahangirnagar University J. Biol. Sci. 7(2): 89-98, 2018 (December)

Genotoxicity of monosodium glutamate

Monosodium glutamate (MSG) is one of the most widely used flavor enhancers throughout the world. The aim of this study is to investigate the genotoxic potential of MSG by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), cytokinesis-blocked micronucleus (CBMN), and random amplified polymorphic DNA-polimerase chain reaction (RAPD-PCR) in cultured human lymphocytes and alkaline comet assays in isolated human lymphocytes, which were incubated with six concentrations (250, 500, 1000, 2000, 4000 and 8000 μg/mL) of MSG. The result of this study indicated that MSG significantly and dose dependently increased the frequencies of CAs, SCE and MN in all treatments and times, compared with control. However, the replication (RI) and nuclear division indices (NDI) were not affected. In this paper, in vitro genotoxic effects of the MSG was also investigated on human peripheral lymphocytes by analysing the RAPD-PCR with arbitrary 10-mer primers. The changes occurring in RAPD profiles after MSG treatment include increase or decrease in band intensity and gain or loss of bands. In the comet assay, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1-h in vitro exposure. Our results demonstrate that MSG is genotoxic to the human peripheral blood lymphocytes in vitro.

English

Stemphylium solani isolates infect two varieties of eggplant, Solanum aethiopicum and Solanum melongena grown in Senegal (West Africa) and are morphologically quite similar. Using the random amplified polymorphic DNA (RAPD) procedure with arbitrary 10-mer primers, we were able to differentiate these S. solani isolates into two groups directly related to their plant host origin. A RAPD product of approximately 480 bases pairs obtained with OPF-20 primer were polymorphic between the two groups. Four new primers (F20F1, F20F2, F20R1 and F20R2) based on nucleotide sequence analysis of this 480 bases pairs RAPD fragment were developed. Such primers used pairwise amplified a single fragment from the DNA of S. solani isolates whatever their host origin. However, DNA extracted from Fusarium oxysporum (f. sp. vasinfectum, f. sp. elaeidis), Verticillium dahliae and Phyllosticta sp. isolates did not amplify using these primers. Our results indicate that these primers sets were good tools for specific identification of these two eggplants S. solani isolates by polymerase chain reaction (PCR). Key words: Stemphylium solani isolates, Solanum aethiopicum, Solanum melongena, random amplified polymorphic DNA (RAPD) markers, identification.

Assessment of genetic diversity and relationships among Egyptian mango (Mangifera indica L.) cultivers grown in Suez Canal and Sinai region using RAPD markers.

DNA-based RAPD (Random Amplification of Polymorphic DNA) markers have been used extensively to study genetic diversity and relationships in a number of fruit crops. In this study, 10 (7 commercial mango cultivars and 3 accessions) mango genotypes traditionally grown in Suez Canal and Sinai region of Egypt, were selected to assess genetic diversity and relatedness. Total genomic DNA was extracted and subjected to RAPD analysis using 30 arbitrary 10-mer primers. Of these, eleven primers were selected which gave 92 clear and bright fragments. A total of 72 polymorphic RAPD bands were detected out of 92 bands, generating 78% polymorphisms. The mean PIC values scores for all loci were of 0.85. This reflects a high level of discriminatory power of a marker and most of these primers produced unique band pattern for each cultivar. A dendrogram based on Nei's Genetic distance co-efficient implied a moderate degree of genetic diversity among the cultivars used for experimentation, with some differences. The hybrid which had derived from cultivar as female parent was placed together. In the cluster, the cultivars and accessions formed separate groups according to bearing habit and type of embryo and the members in each group were very closely linked. Cluster analysis clearly showed two main groups, the first consisting of indigenous to the Delta of Egypt cultivars and the second consisting of indigenous to the Suez Canal and Sinai region. From the analysis of results, it appears the majority of mango cultivars originated from a local mango genepool and were domesticated later. The results indicated the potential of RAPD markers for the identification and management of mango germplasm for breeding purposes.

Relationships between Malaysians Cultivars of Tongkat Ali (Eurycoma longifolia Jack) Obtained through RAPD Analysis

Random Amplified Polymorphic DNA (RAPD) analysis was done on Eurycoma Longifolia Jack (ELJ) cultivars originating from 7 different locations in Malaysia to determine whether the originality of ELJ cultivars can be preserved in an uncontrolled cultivation area. Out of 60 arbitrary 10-mer primers, a total of 320 DNA fragments were amplified with an average of 53.3 RAPD markers per primer. 71 (22%) of the 320 DNA-fragments was found to be monomorphic. The remaining 249 (78%) were polymorphic. The approximate size of the largest fragment produced was 3000 base pair (bp) and the easily recognizable fragment produced was 300 bp. Similarity index of RAPD banding patterns was used to investigate the relationships between ELJ cultivars. Mantel test showed that the correlation between Jaccard and Dice similarity matrices was high and significant (0.9907). The dendogram showed that ELJ cultivars can be clustered into three main clusters. Cluster 1 consisted of Sabah cultivars (East Malaysia), Cluster 2 consisted of Pahang and Terengganu cultivars (middle part of West Malaysia) while cluster 3 consisted of Kedah and Kelantan cultivars (east part of West Malaysia). The results of Principle Coordinates Analysis (PCA) were comparable to the Dendogram by UPGMA. The first two principle coordinates explained 53.62% of total variations and separated the 37 ELJ cultivars into three main clusters with a slight overlap between each other.

Morphological traits and RAPD markers for characterization and identification of minor Spanish olive cultivars from the Extremadura region

A total of 91 wild olive accessions and 31 olive cultivars growing in the Extremadura region of central-western Spain were analyzed using morphological traits and RAPD markers. We focused on three main and 16 minor Spanish olive cultivars that are recognized as native or local to the Extremadura region. The five arbitrary 10-mer primers tested on the olive cultivars gave 67 polymorphic bands, representing 91% of the total amplification products. The number of bands per primer ranged from 9 to 18, whereas the number of polymorphic bands ranged from 8 to 17. All the cultivars could be identified by a combination of three primers (OPF-6, OPA-8, and OPK-16); four cultivar-specific markers were detected. The minor local "Jariego" and "Tempranillo" cultivars showed the most distal similarities. The resulting dendrogram, using the unweighted pair-group method with arithmetic mean clustering algorithm, depicted the pattern of relationships between the local Extremadura cultivars and the cultivars from geographically connected regions. This analysis showed a correlation between most of the minor local cultivars and the geographical origin; there was no apparent clustering according to morphological traits or fruit use of olive cultivars when these parameters were used as analysis criteria.

MOLECULAR GENETIC MARKERS FOR ROOT-KNOT NEMATODE RESISTANCE IN SOME PEACH ROOTSTOCKS

Six peach rootstocks were collected according to their susceptibility to infestation of root-knot nematodes; Meloidogyne incognita and M. javanica. These rootstocks were Okinawa, Nemaguard and Nemared (nematode resistant) and Hegazy, Shami and Sultani (nematode susceptible), which screened by randomly amplified polymorphic DNA (RAPD) markers using 31 arbitrary 10-mer primers and two SSR primer pairs. Twenty molecular markers related to root-knot nematode have been detected (using 12 primers), 13 of which were negative since they appeared in susceptible root-stocks and were absent in the resistant ones, and seven were positive markers which were present in the resistant root-stocks. Dendrogram tree generated across RAPD and SSR analysis demonstrated that the highest similarity was scored between Shami and Sultani (94.1%) while the lowest similarity was scored between Okinawa and Sultani (82.3%). Because of the differences in the rootstocks traits and the molecular genetic diversity of these rootstocks, marker-assisted selection (MAS) in peach rootstocks breeding programs, under biotic stresses (Meloidogyne incognita and M. javanica), is recommended as an efficient tool for improving peach rootstocks resistance.

Application of Random Amplified Polymorphic Analysis for the Detection Genetic Variation in Tomato (Lycopersicon Esculentum Mill.)

This study was carried out to induce variations in tissue cultures of tomato (Lycopersicon esculentum Mill.) by using physical (gamma radiation) and chemical (sodium azide) mutagens. Different plant growth regulators were tested for their potentials in callus induction, and shoot and root formation. Callus initiation on hypocotyl explants was achieved on Murashige and Skoog’s (MS) medium supplemented with 0.1 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.5 mg/l 6benzyl-aminopurine (BAP). Shoot regeneration was performed using hypocotyl explants cultured on MS medium containing 0.8 mg/l indole-3-acetic acid (IAA) and 2.0 mg/l BAP. Rooting was achieved on MS medium supplemented with 0.4 mg/l IAA. Callus cultures and seedlings were irradiated with gamma radiation at six doses 5, 15, 25, 35, 45 or 55 mGy from 137 Cs source, or treated with sodium azide(SA) at four concentrations 0.5, 1.0, 2.0 or 3.0 mM for 1, 3 or 6 hours. Callus fresh weights were increased after irradiation with 25 mGy gamma rays and treatment with (0.5 or 3.0) mM SA for 1 hour. Shoot regeneration was observed in all irradiated hypocotyl explants. Shoot regeneration at (15 and 35) mGy was similar to the control. Root formation was observed on hypocotyls irradiated with low doses (5 or 15) mGy only, while SA inhibited shoot and root regeneration. Random amplified polymorphic DNA (RAPD) technique was performed todetect genetic mutation in extracted DNA samples. DNA was extracted from mother plant, callus tissue, irradiated calli with 15 or 45 mGy of gamma ray and calli treated with 0.5 and 3 mM SA. Twelve arbitrary 10-mer primers were used of which eight primers gave amplification products. The total number of amplification products was 27 bands, of which 22 bands were monomorphic bands which indicates the presence of genetic mutations. One of these primers, C-02, revealed polymorphism in callus tissues treated with 45 mGy of gamma radiation, 0.5 and 3.0 mM SA. Others primers, C-08 and C-09, revealed polymorphisms in irradiated callus tissues with 15 mGy gamma radiation. RAPD was found to be effective technique to detect genetic mutations in callus tissue.

ANALYSIS OF DONGBI LONGAN WITH ISOENZYME AND RAPD TECHNOLOGIES

Longan (Dimocarpus longan Lour.) is one of the most important subtropical fruit trees in Fujian province. Due to little genetic background, the development of longan resources study was restricted. Random amplified polymorphic DNA (RAPD) and POD isoenzyme techniques were used to study the genetic polymorphism and relationship among strains of Dongbi longan. Twenty two arbitrary 10mer primers were used in RAPD analysis of 13 Dongbi longan strains. A total of 124 bands were amplified from 13 samples, each primer pair produces 3 (S299) to 9 (S60) bands, and the average bands were 5.64. The RAPD results showed that the genetic comparability was significantly high and there was high genetic stability among 13 strains, but genes varied with the time and environment. Compared to RAPD results, there was no significant difference in POD isoenzyme among 13 samples from POD isoenzyme gel. According to the results, we concluded that RAPD technology is more suitable than POD isoenzyme technology for longan germplasm resources analysis.

Genetic diversity of Austrian pine (Pinus nigra Arnold) populations in Serbia revealed by RAPD

The ability of random amplified polymorphic DNA (RAPD) to distinguish among populations of Austrian pine from Serbia was evaluated. Ten arbitrary 10mer primers employed in the analysis produced 113 fragments of which 100 were polymorphic (88.5%). All populations had specific RAPD phenotypes. Certain rare and genotype specific bands were identified which could be effectively used to distinguish the populations. Polymorphism in RAPD markers among P. nigra population was high and sufficient to distinguish each of the populations. The results obtained suggest that RAPD markers are valuable for the genetic diversity estimation in Pinus nigra and for the study of the divergence among populations.

Genetic diversity assessment in Portugal accessions of Olea europaea by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.

Analysis of genetic diversity among Indian niger [Guizotia abyssinica (L. f.) Cass.] cultivars based on randomly amplified polymorphic DNA markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 18 cultivars of niger from India. Total genomic DNA was extracted and subjected to RAPD analysis using 80 arbitrary 10-mer primers; 17 primers were selected, which yielded a total of 124 bands, 41.20% of them polymorphic. None of the primers produced unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidean Distance matrix which revealed a minimum genetic distance between cultivars JNC-6 and N-48 and a maximum distance between IGP-76 and JN-30. Based on the distance matrix, a cluster analysis was done using a minimum variance algorithm. The dendrogram generated, based on Ward's method, grouped 18 niger cultivars into two major clusters. The first cluster consisted of early maturing cultivars ( e.g. N-129 and N-134; 80-90 days), and the second of late maturing cultivars ( e.g. GA-8 and GA-9; 135-145 days). The present study shows that there is high diversity among the niger cultivars tested and indicates the potential of RAPD markers for identification and maintenance of niger germplasm for crop improvement purposes.

PCR fingerprinting of Blastocystis isolated from symptomatic and asymptomatic human hosts

Genomic DNA from 16 Blastocystis hominis isolates comprising of eight asymptomatic isolates (A1-A8) and eight symptomatic isolates (S1-S8) was amplified by arbitrarily primed polymerase chain reaction (AP-PCR) using 38 arbitrary 10-mer primers. Six primers (A10, B5, C20, D1, F6, and F10) generated reproducible DNA fingerprints. AP-PCR amplification revealed similar DNA fingerprints among all symptomatic isolates (S1-S8) with common bands at 850 bp using primer A10, 920 bp using primer B5, and 1.3 kbp using primer D1. Isolates A1, A3, A4, A5, A6, and A7 showed similar DNA banding patterns and all asymptomatic isolates (A1-A8) shared a major band at 1 kbp using primer B5. Isolates A2 and A8 showed distinct DNA banding patterns that differed from the remainder of the isolates. The results of the phylogenetic analyses showed that all symptomatic isolates (S1-S8) formed a clade with >70% similarity among the isolates and which were clearly separate from asymptomatic isolates A1, A3, A4, A5, A6, and A7. Asymptomatic isolates A2 and A8 formed two distinct and separate clades. AP-PCR revealed higher genetic variability within the asymptomatic isolates than within the symptomatic isolates. The present study suggests that AP-PCR can be a valuable method for differentiating between isolates of B. hominis and our results support the hypothesis that our asymptomatic and symptomatic B. hominis isolates may represent two different strains/species with varying pathogenic potential.

Analysis of genetic diversity among Indian short-day onion (Allium cepa L.) cultivars using RAPD markers.

SummaryRandomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 24 cultivars of short-day onions. Total genomic DNA was extracted and subjected to RAPD analysis using 90 arbitrary 10-mer primers. Of these, 15 primers were selected which yielded 137 bands, 91.24% of which were polymorphic. None of the primers produced a unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidian Distance matrix which revealed a minimum genetic distance between cultivars ‘AFLR-722’ and ‘PBR-140’, and a maximum genetic distance between cultivars ‘PBR-139’ and ‘A.Kalyan’, and ‘MS-48’ and ‘A.Kalyan’. Based on the distance matrix, cluster analysis was done using a minimum variance algorithm.The dendrogram thus generated, based on Ward’s method, grouped the 24 onion cultivars into two major clusters. The first cluster consisted of cultivars from the northern region, and the second of cultivars from the southern region of India. The present study shows that there is high diversity among the onion cultivars selected and indicates the potential of RAPD markers for identification and maintenance of onion germplasm for crop improvement purposes.

Identification and Development of Sex Specific DNA Markers in the Ostrich Using Polymerase Chain Reaction

The objective of this study was to identify and develop DNA markers that can be used for sex diagnosis in the ostrich (Struthio camelus) using polymerase chain reaction (PCR) technology. DNA was isolated from 15 male and 15 female, year old, crossbred ostriches. Two bulked DNA samples were prepared by grouping sexes. Random amplified polymorphic DNA (RAPD) analysis was used to screen 1400 arbitrary 10-mer primers for polymorphic markers between the bulks. Potential W-linked markers generated with the female bulked DNA sample were confirmed by individual screening of the 30 samples. Five W-linked RAPD markers, UBC388431, UBC4111300, UBC656,120, UBC793687, and OPL 1 7750 were identified by primers UBC388, UBC411, UBC656, UBC793, and OPL-17. Four RAPD markers were chosen for sequence analysis after further screening using 15 male and 12 female, 14-mo-old, birds sexed at slaughter. The sequence data were used to design pairs of specific primers, 18-24 bp, for PCR amplification of individual genomic DNA from 71 birds of varying age (33 males and 38 females). Primers generated reliable female specific sequence characterized amplified regions (SCARS). Four SCARS, ST793677, ST793665, ST4111245, and ST6561023, were developed, which can be used for sex identification in the ostrich. Cross amplification tests were done using male and female genomic DNA from turkeys, domestic animals (cattle, pigs, sheep, horses, and mink), and human. The PCR primers did not generate the sex specific markers in these species, which indicated that the W-linked markers amplified from ostrich genome may be species specific markers. These results demonstrate that RAPD analysis using bulked DNA samples provides an efficient means for the detection of W-chromosome markers in avian species. The SCARS developed can be used for identifying sex in immature ostriches.

Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.

PENGGUNAAN PENANDA MOLEKULER RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) UNTUK ANALISIS KERAGAMAN GENETIK KELAPA WEST AFRICAN TALL (WAT)

ABSTRACT Lengkong, E.F. and S.D. Runtunuwu. 2005. Use of Molecular Marker Random Amplified Polymorphic DNA (RAPD) to Genetic Diversity Analysis of West African Tall (WAT) Coconut. Eugenia 11 (3):210-217. Information on genetic diversity of crop gemplasm has several important implications for plant breeder. Among others is to help the breeders to decide what sources to cross so as to making new genetic combination. The use of molecular marker to genetic diversity analysis on DNA level is usefull, because it provides an opportunity to more precisely measure genetic relationships as well is not affected by the environment. The objective of this research was to analyze genetic diversity of West African Tall (WAT) using molecular marker RAPD. Five arbitrary 10-mer primers were used to amplify total DNA genom, and to generate 44 band DNA with 26 band or 59 % were polymorphic band. It was revealed that genetic diversity within population of WAT coconut was 14 %. Based on cluster analysis, at the genetic similarity 85% or genetic diversity 15 % the population was separated on three clusters. This research concluded that the population of WAT coconut grown from open pollinated seeds has different genotype one each others. Keywords: West African Tall (WAT), Coconut, RAPD

Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori

The sex chromosomes of the silkworm, Bombyxmori, are designated ZW(XY) for females and ZZ(XX) for males. The W chromosome of B. mori does not recombine with the Z chromosome and autosomes and no genes for morphological characters have been mapped to the W chromosome as yet. Furthermore, femaleness is determined by the presence of a single W chromosome, regardless of the number of autosomes or Z chromosomes. To understand these interesting features of the W chromosome, it is necessary to analyze the W chromosome at the molecular biology level. Initially to isolate DNA sequences specific for the W chromosome as randomly amplified polymorphic DNA (RAPD) markers, we compared the genomic DNAs between males and females by PCR with arbitrary 10-mer primers. To the present, we have identified 12 W-specific RAPD markers, and with the exception of one RAPD marker, all of the deduced amino acid sequences of these W-specific RAPD markers show similarity to previously reported amino acid sequences of retrotransposable elements from various organisms. After constructing a genomic DNA lambda phage library of B. mori we obtained two lambda phage clones, one containing the W-Kabuki RAPD sequence and one containing the W-Samurai RAPD sequence and found that these DNA sequences comprised nested structures of many retrotransposable elements. To further analyze the W chromosome, we obtained 14 W-specific bacterial artificial chromosome (BAC) clones from three BAC libraries and subjected these clones to shotgun sequencing. The resulting assembly of sequences did not produce a single contiguous sequence due to the presence of many retrotransposable elements. Therefore, we coupled PCR with shotgun sequencing. Through these analyses, we found that many long terminal repeat (LTR) and non-LTR retrotransposons, retroposons, DNA transposons and their derivatives, have accumulated on the W chromosome as strata. These results strongly indicate that retrotransposable elements are the main structural component of the W chromosome.

Factors influencing in vitro regeneration from protoplasts of wild cotton (G. klotzschianum A) and RAPD analysis of regenerated plantlets

Plants of a diploid wild cotton species (G. klotzschianum A.) were efficiently regenerated from protoplasts isolated from immature somatic embryos and suspension cultures by studying various factors affecting regeneration. Purified protoplasts were cultured with the density of 2–10×105 ml−1, and the medium was k3 inorganic salts with modified KM8P organic compositions, supplemented with several combinations of PGRs. Calluses were formed from protoplasts of suspension cultures and immature somatic embryos. The influences of carbon sources and GA3 on callus differentiation and somatic embryo germination were analyzed. Somatic embryos germinated normally and formed regenerated plantlets. Regenerated plantlets were transferred to the soil and seeds were obtained. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed 23 primers that gave 74 clear reproducible bands, with amplification products being monomorphic for 14 tested plantlets. A total of 1036 bands obtained exhibited no aberration in RAPD banding patterns in the 14 plants. Plants regenerated via somatic embryogenesis from the diploid cotton protoplasts have genetic homogeneity.

Characterization of a RAPD fragment unique to species with hairy fruit skin in the genusActinidia

To develop a SCAR primer related to the hairy-fruit trait in the genusActinidia, we took a PCR-RAPD approach using arbitrary 10-mer primers. PCR with the UBC 376 primer generated specific fragments from three species with hairy fruit skin. Those fragments were then cloned to determine their nucleotide sequences. Two SCAR primers were designed from the UBC 376 primer and nucleotide sequences were obtained from the PCR fragments. A SCAR primer, OKC385, specifically amplified a 385-bp fragment from one clone ofActinidia eriantha, four ofActinidia chinensis, and four ofActinidia deliciosa. Deduced amino acid sequences of this fragment showed high sequence homology with plant cellulose synthases, which are involved in the biosynthesis of cellulose, a major cell wall component. The 385-bp fragment was specifically detected only in the seriesPerfectae C.F. Liang of sectionStellatae Li. This type has many hairs on the leaves, fruits, and stems, suggesting that the gene containing the PCR fragment is involved in hair formation in this phylogenetic group. Taken together, our results suggest that the SCAR primer, OKC385, can be used as a specific primer for early selection of the non-hair trait in breeding of the genusActinidia.