Screening and Cloning of RAPD Marker of Fluoride Tolerance Gene in Silkworm,Bombyx mori

In this study,silkworm strain T6,tolerant to fluoride,and silkworm strain 733xin,highly sensitive to fluoride,were used to construct the near-isogenic lines.300 random primers were used in RAPD amplification to DNAs of these lines.A molecular marker named S207 was found linked to the fluoride tolerance gene.Examination to F 2 segregated individuals of the above lines verified that this molecular marker was reliable.Subsequently,the molecular marker was cloned into a T vector (pUCm-T) for sequencing.Comparing with sequences available in the GenBank showed that this molecular marker was novel.We plan to convert it into a SCAR marker to facilitate establishment of a molecular marker assisted breeding system.

Development of RAPD Markers and SCAR Markers Linked to Bentazon Susceptible Lethality Gene in Rice

Rice cultivar Norin 8 and its mutant Norin 8m harbour bentazon resistance trait and bentazon susceptibility trait respectively. A total of 360 arbitrary 10-mer oligonucleotide primers were screened on the genomic DNA of Norin 8 and Norin 8m with RAPD technique. Among which, five primers produced seven polymorphic RAPD bands between Norin 8 and Norin 8m. Amplified RAPD polymorphic products were cloned and sequenced. The sequences were used to design primers for PCR. Five SCAR markers, SCAR/G18/883, SCAR/G18/890, SCAR/G18/919/948, SCAR/D10/1237 and SCAR/F03/1186, were developed from OPG18/943, OPG18/972, OPD10/1248 and OPF03/1198. F-2 progeny of 320 individuals was analyzed to map SCAR markers in relationship to ben or Ben genes. SCAR markers of SCAR/G18/883, SCAR/G18/890, SCAR/G18/919/948 were shown to cosegregate with ben or Ben genes, and SCAR/D10/1237 to be linked of Ben gene with a distance of (14.8 +/- 2.1) cM. The genetic linkage to ben gene and SCAR markers was identified by a pair of near isogenic lines H121 and Hben121. Southern blotting analysis and segregation ratio of F-2 progeny revealed that OPG18/943 and OPG18/972 were single-copy in genome, and locus of OPG18/943 and OPG18/972 were allelic and sequence tagged sites. It is the first report on molecular markers linked to ben or Ben genes. The markers are useful to marker-assisted selection for the breeding and tag ben gene with map-based cloning.

Analysis of the Germplasm Resources and Genetic Relationships Among Hybrid Cymbidium Cultivars and Native Species with RAPD Markers

The random amplified polymorphic DNA （RAPD） marker was assessed to detect the genetic relationships among 48 hybrid Cymbidium cultivars from Japan, Korea, China, and USA, and 2 species of native Cymbidium. Twenty primers were screened from 100 random decamer primers, and a total of 258 DNA bands were amplified, 253 of which （98.1%） were polymorphic. The average number of polymorphic DNA bands amplified by each primer was 12.6. All cultivars were distinguishable when a number of primers were considered. Genetic similarities among the cultivars and species were estimated based on the amount of band sharing ranging from 0.364-0.817 with an average of 0.581. According to the data, a dendrogram of genetic relationship, which was constructed using the UPGMA method, showed that all the tested cultivars and native species were classified into five cluster groups with the similarity coefficient of 0.592. It revealed that the genetic relationships among tested accessions were to some extent related with their origin, flower colour, branch type, and genealogy. It further indicated that the RAPD technique is a useful tool for studying the genetic relationships among hybrid Cymbidium cultivars.

Molecular identification of sex in Hippophae rhamnoides L. using isozyme and RAPD markers

In many dioecious plants, gender affects economic value, breeding schemes and opportunities for commercial harvests. Hippophae rhamnoides L. is a dioecious plant species in which female genotypes are commercially preferred over male genotypes. Its berries have rich medicinal, nutritional and pharmaceutical properties because of their large amounts of vitamins, essential oils, proteins, fatty acids, free amino acids and flavanoids. Primary limitation for breeding H. rhamnoides L. is its dioecious nature, since gender cannot be identified by traditional methods. Therefore, some reliable and quick methods need to be developed. This commu- nication deals with the development of isozyme and RAPD markers for early sex identification in this dioecious tree. The isozyme analysis was conducted with four enzyme systems, viz. peroxidase, esterase, malate dehydrogenase and catalase. The peroxidase enzyme system produced a female specific sex marker, which successfully differentiated between the staminate and pistillate geno- types ofH. rhamnoides L. Thirty five random decamer primers were used in our study and one male sex linked marker was identified. OPD-20 （5＇-ACTTCGCCAC-3＇） displayed a band at 911 bp that expressed polymorphism between male and female genotypes. The staminate and pistillate genotypes could be distinguished using RAPD marker OPD-209n. These results revealed the immense poten- tial of peroxidase isozyme patterns and RAPD as genetic markers for sex identification in H. rhamnoides L.

DNA polymorphisms and genetic relationship among populations of Acacia leucophloea using RAPD markers

RAPD （randomly amplified polymorphic DNA） markers were employed to characterize polymorphisms among 5 provenances of Acacia leucophloea and to detect genetic relatedness of the species with 6 other acacias （A. holosericea, A. auriculiformis, A. mangium, A. dealbata, A. ferruginea, and A. nilotica） widely grown in India. Of 194 markers scored for the provenances, 29.38% exhibited polymorphism. Also, 326 markers were generated among 7 species of Acacia, accounting for 55.82% of the polymorphisms. The fifteen 10-mer primers employed were capable of producing 1-8 polymorphic bands for the provenances, and 6-17 for all seven species of Acacia. The genetic similarity coefficient based on Jaccard＇ s coefficient revealed that provenances Thirumangalam and Dharmapuri were closely related. The dendrogram based on a sequential agglomerative hierarchical non-overlapping （SAHN） clustering analysis grouped 4 provenances of A. leucophloea （Dharapuram, Thirumangalam, Pudukottai and Dharmapuri） into one cluster and the other provenance, Sendurai, into a separate cluster. The genetic similarity matrix for 7 Acacia species showed that A. nilotica and A. dealbata were distantly related, while A. holosericea and A. ferruginea were very closely related. Cluster analysis grouped the species of Acacias into 3 major groups of which A. dealbata alone formed a separate group. The RAPD markers generated 36 provenance-specific markers and 162 species-specific markers that could have strong applications for species identification and tree breeding programs for A. leucophloea and for other Acacia species included in this study.

Inheritance of Resistance to SMV3 and Identification of RAPD Marker Linked to the Resistant Gene in Soybean

One SMV resistant soybean line (95-5383) was crossed with four susceptible soybean varieties/ line (HB1, Tiefeng21, Amsoy, Williams) and one resistant introduced line PI486355. Their F, and F2 individuals were identified for SMV resistance by inoculation with SMV3. The results showed that in the four crosses of resistant X susceptible, F1 were susceptible and the ratio of F2 populations was 1 resistant : 3 susceptible (mosaic and necrosis), indicating that 95-5383 carries one recessive gene that confer resistance to SMV3. There is segregation of susceptibility in F2 progenies from the cross of 95-5383 X PI486355, indicating that the SMV3 resistant gene in 95-5383 is located at different locus from PI486355. By bulked segregating analysis (BSA) in F2 populations of 95-5383 X HB1, one codominant RAPD marker OPN11980/1070 closely linked to SMV3 resistance gene amplified with RAPD primer OPN11 was identified. The DNA fragment OPNll980 was amplified in resistant parent 95-5383 and resistant bulk, and OPN111070 was amplified in susceptible parent HB1 and susceptible bulk. OPNll980/1070 was amplified in F,. Identification of the markers in F2 plants showed that the codominant marker OPNll980/1070 is closely linked to the SMV resistance locus in 95-5383, with genetic distance of 2.1cM.

Genetic Diversity of Testa Pigments and RAPD Marker of Yellow-Seeded Rapeseed (Brassica napus L.)

14 yellow-seeded rapeseed lines (Brassia napus L.) from different genetic sources were used to analyze diversity of testa pigments content, oil and protein content, and RAPD markers. The results showed that the anthocyanin and melanin were the most important pigments in testa and their content were responsible for the variation in seed color ranging from orange to black yellow, 14 yellow-seeded lines could be classified into 3 groups: high anthocyanin content group with anthocyanin content over 2. 54 mg g-1 DW, the seed color was light yellow or orange; low pigments content group with low content of anthocyanin and melanin, the testa was transparent and the seed color was light yellow, greenish yellow or twany; high melanin content group with melanin content over 178. 4U(A290nm) , the testa was black, the seed color was black yellow. Oil content changed from 36.2% to 45. 5%, protein content from 21.1% to 27.7% , and the correlation analysis revealed that the oil content is highly significantly negatively correlated with the protein content. The cluster analysis showed that the extensive genetic variation existed among 14 yellow-seeded lines by using unweighted paired group method with arithmetic average (UPGMA) based on RAPD markers which were amplified with decamer primers, the genetic similarity among them ranged from 0. 25 to 0.909, and 14 yellow-seeded lines could put into 2 clusters corresponding to genome difference.

Detection of RAPD Markers Linked to Gene lx_1 in Soybeans

Near-isogenic lines of soybean lipoxygenase, which contain genes Lox, lx1, lx2, lx3, lx1.3, lx2.3,respectively, were used for polymorphic analysis by RAPD technique.520 10-mer-oligonucleotideprimers were screened, and thirteen primers showed polymorphism among near-isogenic lines.There were six primers showed special polymorphic bands among lines lx1 and lx1.3. Especially,primer S352 presented the stable results in which a 900 bp band was found in the lines lx1 andlx1.3, and primer S352900 was detected with F2 generation of cross 96P11×Century-1, indicatedprimer S352900 could be identified as a RAPD marker linked to gene lx1 in soybeans, the distanceof linkage was 7.6 cM.

Discrimination of Wild Tea Germplasm Resources (Camellia sp.) Using RAPD Markers

Discrimination of 24 wild tea germplasm resources (Camellia sp.) using RAPD markers was conducted. The result showed that RAPD markers were very effective tool and method in wild tea germplasm discrimination. There were 3 independent ways to discriminate tea germplasms, a) unique RAPD markers, b) specific band patterns and c) a combination of the band patterns or DNA fingerprinting provided by different primers. The presence of 16 unique RAPD markers and the absence of 3 unique markers obtained from 12 primers made it possible to discriminate 14 germplasms. Using the unique band patterns of primer OPO-13 could discriminate 10 tea germplasms. It was of much importance using minimum primers to obtain maximum discrimination capacity. All the 24 wild tea germplasms could be discriminated easily and entirely by the band patterns combination or DNA fingerprinting obtained from OPO-13, OPO-18, OPG-12 and OPA-13, including two wild tea trees of very similar morphological characteristics and chemical components.

Study on Identification of RAPD Marker of Phytophthora sojae Associated with Rps1-k

A near-isogenic lines(NILs) -Williams and Williams82 is used to identify molecular marker linked to the resistance gene Rps1-k by RAPD. Genomic DNAs extracted from soybean leaves of the NILs were analyzed by RAPD using 160 different 10-nt random primers. Some specific DNA fragments were amplified from Williams82 with 4 primer(OPF-16, OPB-05, OPD-06 and OPH-05) which contains Rps1-k. All these specific DNA fragments were not detected in Williams. The experiment with OPH-05 was repeated 3 times and the results were the same. Using primer- OPH-05 to detect other resistance cultivars with Rps1-k, almost everyone can amplify the specific DNA fragment. So it is inferred that the specific DNA fragment is probably linked to Rps1-k.

Detecting RAPD Markers Linked to Ripe Rot Resistance Genes in Chinese Wild Vitis

With F1 individuals of the cross combination 88-110 of 83-4-96 ( V. quinquangularis Rehd.) X Muscat Rose ( V. vinifera L.), the RAPD marker OPC15-1300 linked to ripe rot ( Gloeosporium fructi-genum Berk.) resistance genes in Chinese wild Vitis was gained using bulked segregation analysis (BS A). And it was found that OPC15-1300 could be hereditary from the resistant parent (83-4-96) after the marker was tested in 50 Fj plants of the cross combination 88-110, 32 accessions of 8 Chinese wild Vitis species and 14 cultivars of V. vinifera L. Also, it has provided a solid basis for molecular marker-assisted selection (MAS) and for possibly cloning disease resistance genes in the future.

Genetic Diversity of the Palestinian Fig (<i>Ficus carica</i>L.) Collection by Pomological Traits and RAPD Markers

Analysis of differentiation (genetic diversity and related relationships) among 22 landrace (Ficus carica L. sativa) and 2 wild form (F. carica L. caprificus) accessions of fig growing under the same environmental conditions in the Palestinian Fig Collection, Til, Nablus, Palestine, using PCR-based Random Amplified Polymorphic DNA (RAPD) and pomological markers, revealed considerable genetic diversity. The phenotypic analysis shows that pomological traits were permitted to evaluate morphological variability of fig landraces. The Jaccard similarity coefficient between landraces was determined by cluster analysis using the UPGMA method. Based on the genetic relationships among genotypes as illustrated by the dendrograms, generated from pomological and RAPD data by UPGMA clustering method, the following 12 genotypes: Qaisi, Mwazi, Barqawi, Inaqi, Swadi, Kharobi, Hmadibiadi, Sfari, Khdari, Biadi, Qrawi, and Slati, may be considered as distinct landraces. The remaining genotypes may be considered as synonymous (4) (Hmadi and Hmari, and Ajloni and Adloni), or closely related (6) landraces (Zraqi and Ghzali, Blati and Neami, and Qraee and Khurtmani). The wild fig forms clustered together and may be considered as distinct genotypes. Clustering patterns obtained from the combined (pomological and RAPD) markers had higher discriminatory power to discriminate fig landraces than using either pomological or RAPD markers alone. These results proved the importance of both pomological and RAPD markers to elucidate in part denomination problems and relationships among cultivars. Wide phenotypic and molecular diversity found in fig germplasm indicates a considerable potential for improving this crop.

Genetic Variation of Sago Palm (Metroxylonsagu Rottb.) Progenies with Natural Pollination by Using RAPD Markers

Sago palm is flowering and fruiting just once in their life cycle. Sago palms that grow naturally and semi cultivated were generally occurred natural pollination to form fruits and seeds, if not cut down to take the starch contained in their trunk. Sago palm pollination may occur as self-pollinated and cross-pollinated. If cross-pollinated was occurred in the pollination process, it will be varied of their progenies. This study aims to reveal the genetic variation of sago palms progenies with naturally pollinated process. The research method is to collect seeds from one parent trees that have produced ripe fruit. Fruit seeds germinated to be made and tested genetic variation using RAPD markers. Isolation of DNA is done by using the fresh young leaves. DNA amplification is done by using RAPD primers. The results showed that the progenies derived from naturally pollinated of sago palms were genetically varied based on RAPD markers and also varied based on morphological phenotypic. Variations occurred in the progenies of sago palm indicated that the sago palms were estimated cross-pollinated naturally, as a result fruits and seeds with genetically differences.

Assessment of Genetic Variation in Soybean (<i>Glycine max</i>) Accessions from International Gene Pools Using RAPD Markers: Comparison with the ISSR System

Soybean (Glycine max) is one of the most important crops in the world in terms of total production and usage. It is also among the least diverse species. The main objectives of the present study were to 1) assess the level of genetic variation among soybean (G. max) accessions from different countries using Random Amplified Polymorphic DNA (RAPD) markers and 2) compare Inter Simple Sequence Repeats (ISSR) and RAPD marker systems in detecting polymorphic loci in soybeans (G. max). Genomic DNAs from 108 soybeans (G. max) accessions from 11 different gene pools were analyzed using several ISSR and RAPD primers. The average level of polymorphic loci detected with the RAPD primers was 35%. The soybean accessions from the China, Netherlands, and Canada gene pools were the least genetically variable with 25%, 26%, and 30% of polymorphic loci, respectively. Accessions from Hungary (43%) and France (48%) showed the highest level of polymorphism based on the RAPD analysis. Overall, RAPD data revealed that the accessions from different countries are closely related with 64% genetic distance values below 0.40. The levels of polymorphic loci detected with the RAPD and ISSR marker systems were in general moderate and similar even if they target different regions of the genome. A combination of different marker systems that include RAPD/ISSR, microsatellites (SSR), and SNPs should provide the most accurate information on genetic variation of soybean (G. max) accessions.

Molecular characterization of Cuban endemism Carica cubensis Solms using random amplified polymorphic DNA (RAPD) markers

The objective of this work is to present an appropriate set of RAPD (random amplified polymorphic DNA) markers using single and multiplex PCR analysis suitable for the characterization of the endemic Cuban species Carica cubensis and the establishment of genetic relationships with the cultivated species Carica papaya. RAPD markers presented a high level of polymorphism. In addition, the incorporation of more than one RAPD primer in the PCR analysis increased the number of obtained bands and the polymorphism of these bands. A total of 73 RAPD bands were detected (45 of them polymorphic) with the nine RAPD markers assayed using single and multiplex PCR analysis. Results demonstrated a reduced genetic variability within the tested Carica cubensis accessions. The observed clustering in this species could be better explained according to geographic proximity and can indicate the similar precedence of the isolated studied populations. C. cubensis seem to be subspecies of C. papaya adapted to the environmental conditions of the mountains of Cuba or a endemic species close to C. papaya. The implications of these results in the creation of effective germplasm core collection in Carica species have been also discussed.

Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics of a NaCI-Tolerant Potato Cell Line

Salinity is a serious threat to agricultural production. Potato （Solanum tuberosum） is an important food crop characterised for having low to moderate salinity tolerance. Tissue cultures may be relevant to improve salt tolerance in potato through selection of salt-tolerant cell lines and subsequent regeneration of plants. In this work, the authors used the random amplified polymorphic DNA （RAPD） markers to investigate the occurrence of genetic polymorphism in a potato calli line tolerant to 150 mM NaCI. Out of 40 primers screened, eight generated polymorphic patterns that distinguished salt-tolerant line from the control. Although the macroscopic appearance was similar in both lines, ultrastructural study revealed alterations in salt-grown cells. These showed that plastids less differentiated with a lower number of grana had more and larger starch grains than control cells. In conclusion, RAPD analysis revealed that NaCl-adapted line is a somaclonal variant and the ultrastructural study showed changes essentially at the plastids.

Assessment of Genetic Diversity and Relationships among Maize （Zea mays L,） Varieties in Iraq Using Random Amplified Polymorphic DNA （RAPD） Markers

RAPD （random amplified polymorphic DNA） markers were performed to fingerprint 20 varieties of maize. Twenty operon primers generated informative RAPD patterns and selected for further RAPD analysis. These 20 primers yielded 291 of main bands, out of which 169 were polymorphic bands across tested varieties. Each selected primer produced between 59 bands （OPC-01） to 142 bands （OPX-04）. Amplification products （DNA amplicons） and fragments size ranged from 260 bp （OPT-06） to 2,365 bp （OPP-05）. The largest number of polymorphic bands （20 bands） was produced by primer OPX-04 while, the lowest number of polymorphic bands （1 band） was produced by primer OPA-03. The primers of the most interest of this purpose were those that produced more variety specific DNA profiles, such as OPD-03, OPE-18, OPF-05, OPL-11 and OPX-04. The primer efficiency ranged from 0.20 in primer OPA-12 to 0.01 in primer OPA-03. The highest polymorphism and value of discrimination in this study were obtained with primers OPX-04, while, the lowest polymorphism and value of discrimination were obtained with primers OPA-03. The lowest genetic distance was 0.1941 between varieties Manlcet and Biotech Bag, while, the highest genetic distance was 0.6433 between varieties Pio 3751 and Buhooth 106. Cluster analysis （phylogenetic tree） by UPGMA （unweighted pair-group method of arithmetic means） based dendrogram revealed that they were four main genetic groups. The overall analysis of the results reveals that the genetic relationships among the maize varieties were related to some of their morphological characters as well as to their geographical origins at the molecular genetics.

APPLICATION OF RAPD IN GENETIC DIVERSITY STUDY ON GRACILARIA LEMANEIFORMIS Ⅲ.PHASE AND SEX RELATED MARKERS

Random amplification of polymorphic DNA (RAPD) was conducted by using 10 ran-dom primers (P-1 to P-10) in Gracilaria lemaneiformis. Phase and sex specific bands were amplified byprimers P-2, P-6, P-7 and P-8: for P-2 a 1 .4 kb band was found in female gametophytes and tetrasporo-phytes, for P-6, a 0.6 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0. 76 kbband appeared in male gametophytes and tetrasporophytes; for P-7, a 0.72 kb band appeared in femalegametophytes and tetrasporophytes; for P-8, a 0. 73 kb band only appeared in male gametophytes.

Identification of a RAPD marker linked to a blast resistance gene in Oryza sativa L

Marker-aided selection has received more attention in recent years. This relies on the exploitation of dose linkage between molecular markers and target gene(s). We report here a randomly amplified polymorphic DNA (RAPD) marker tightly linked to the blast resistance gene Pi-ll(t) derived from Hongjiaozhan, which confers the resistance to race ZB1 of Pyricularia oryzae Cav.

Subspecies specific RAPD markers in rice

The cultivated rice(Oryza sativa L.)is known to contain two major subspecies,indica(O. stativa L. ssp. indica)and japonica(O. sativa L. ssp.japonica). The indica and japonica differentiation