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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

Research Progress on Genetic Diversity of Cultivated Soybean Germplasm Resources in China

In this paper,by analyzing the genetic diversity of cultivated soybean germplasm resources in China,the environmental and genotypic factors that affect the genetic diversity of cultivated soybean germplasm resources were explored to further expand the genetic basis of the existing germplasm resources of cultivated soybean in China. Moreover,research progress on genetic diversity of cultivated soybean in China was summarized,which not only revealed the geographical characteristics of genetic diversity of cultivated soybean in China,but also proposed direction for research of genetic diversity of soybean.

Genetic Differentiation and Conservation of 14 Surviving Individuals of Euryodendron excelsum Endemic to China

Random amplification polymorphicDNA(RAPD)markers were used to assess the genetic variations and the evolutionary relationships among all 14 individuals of a critically endangered Euryodendron excelsum(Theaceae)population distributed in Ba Jia Zhen,Yangchun,Guangdong,China.Twenty-three random primers detected 156 sites,out of which 95(60.26%)were polymorphic loci.The number of the observed alleles was 1.6090,and the number of the effective alleles was 1.3471.Nei’s gene diversity was 0.1993,and Shannon index was 0.1534.A relatively high level of genetic variation was identified in E.excelsum.An unweighted pair group method with arithmetic mean(UPGMA)tree established from Jaccard similarity coefficients suggested that 14 individuals were clustered into two subgroups and that the No.2 plant was genetically distant from the rest of the individuals.The UPGMA clustering was also supported by a principle components analysis of RAPD phenotypic data.The management and conservation strategy of E.excelsum was proposed based on our results.