Application of DNA fingerprints for identification and genetic analysis of Carica papaya and other Carica species

Summary Various DNA fingerprint probes were applied to Carica papaya and other Carica species for both identification and genetic analysis.Each of the Carica papaya cultivars is characterized by a specific DNA fingerprints pattern. Various Carica species also have specific patterns which distinguish them from one another. Band sharing levels were used to estimate the relatedness between the various Carica species.Genetic analysis of 11 progeny from a cross between the Carica papaya cultivars 17/82 and 112 suggests that application of DNA fingerprinting to Carica papaya breeding, could make the process more efficient. Genetic analysis of the DNA fingerprint bands revealed no linkage or allelic relationship among the bands analyzed, indicating that these loci are not clustered in the Carica genome.     

Molecular Marker based Genetic Diversity Analysis in Rice (Oryza sativa L.) using RAPD and SSR markers

The availability of an array of molecular marker systems allowed comparing the efficiency of two of these marker systems to estimate the relationships among various taxa. The objective of this study was to assess the genetic diversity among 40 cultivated varieties and five wild relatives of rice, Oryza sativa L. involving simple sequence repeat (SSR) randomly amplified polymorphic DNA (RAPD) markers. The accessions were evaluated for polymorphisms after amplification with 36 decamer primers and 38 SSR primer pairs. A total of 499 RAPD markers were produced among the 40 cultivated varieties and five wild relatives with a polymorphism percentage of 90.0. Out of 38 SSR primer pairs used, only one locus viz., RM115 was monomorphic. The average Polymorphism Information Content (PIC) value was 0.578 and it ranged from a low of zero (RM 115) to a high of 0.890 (RM 202). The Mantel matrix correspondence test was used to compare the similarity matrices and the correlation coefficient was 0. 582. The test indicated that clusters produced based on RAPD and SSR markers were not conserved since matrix correlation value was 0.582 as against the minimum required value of 0.800. The two marker systems contrasted most notably in pair-by-pair comparisons of relationships. SSR analysis resulted in a more definitive separation of clusters of genotypes indicating a higher level of efficiency of SSR markers for the accurate determination of relationships between accessions that are too close to be accurately differentiated by RAPD markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic mapping of a PRSV-P resistance gene in “highland papaya” based on inheritance of RAF markers

Papaya ringspot virus type P (PRSV-P) is a significant disease of Carica papaya. A major gene for PRSV-P resistance has been mapped in Vasconcellea cundinamarcensis, a distant relative of C. papaya. This was achieved by genetic mapping of the resistance phenotype and inherited, dominant, polymorphic randomly amplified DNA fingerprint (RAF) markers in F2 progenies of V. parviflora and V. cundinamarcensis. The parents of this cross confer resistance to several major diseases that affect C. papaya including PRSV-P in V. cundinamarcensis. Heredity of DNA markers and PRSV-P resistance was studied in the intrageneric population presented due to intergeneric fertility barriers between Carica and Vasconcellea. Genetic polymorphism between parents, based on RAF markers, was 75% with more than 70% of markers generated showing mendelian segregation for the expected ratios 1:3 or 1:1 (p < 0.05). Preferential inheritance of markers from either parent was not detected in the F2, indicating stable transfer of the genetic material. Discrete V. parviflora and V. cundinamarcensis linkage maps were compiled from 79 and 83 framework markers, delineating to 10 and 11 groups respectively. F1 and F2 progeny were screened for resistance to PRSV-P under controlled conditions. The resistant phenotype segregated 3:1 in the F2 and mapped to V. cundinamarcensis linkage group 7 with adjacent RAF markers within 4 cM. The framework maps of V. parviflora and V. cundinamarcensis presented cover 630.2 and 745.4 cM respectively, accounting for between 47–52 and 49–55 percent of the predicted genome lengths. These maps provide a platform for further genetic study of disease resistance characteristics identified in these species and the development of DNA markers tightly linked to these traits, which could be applied to the breeding of resistant C. papaya cultivars.     

RAPD and SCAR markers linked to sex determination in Eucommia ulmoides Oliv.

Eucommia ulmoides Oliv. is strictly a dioecious perennial tree native to China. The pistillate plants are economically more useful than the staminate plants. The random amplified polymorphic DNA (RAPD) technique was used to screen markers of sex determination in this species. A 569 bp RAPD marker, marker linked to sex determination in E. ulmoides (MSDE), was found in all the pistillate but not in the staminate plants; its exclusiveness to pistillate plants was confirmed by Southern blotting. MSDE was sequenced and specific primers were synthesized to generate a 569bp pistillate-specific SCAR marker, SCARmr. SCARmr could be useful for screening E. ulmoides plants for gender even before they reach reproductive maturity, resulting in considerable saving of time and economic resources.     

Genetic control of isozymes in Carica papaya L.

The genetic control of isozymes was investigated in papaya. Out of six buffer systems tested, Histidine-citrate pH 6.5 gave the most consistent and greatest number of well-resolved enzymes. Nine enzyme systems (ACO, IDH, MDH, PGM, PGI, SKD, TPI, UGP, 6-PGD) out of 28 were resolved. Based on the initial screening of 131 accessions of papaya from Central and South America for electromorph variants, parents were selected for various combinations of controlled crosses. These controlled crosses were then used to elucidate the inheritance of isozymes in papaya. Evidence of Mendelian inheritance was obtained for eight out of nine polymorphic loci: Aco-1, Aco-2, Pgi-2, Pgm-1, Skd, Tpi-1, Tpi-2, and Ugp. No controlled cross was available to study the genetic control of the polymorphic locus Pgm-2. Because they were not variable in all accessions included in this study, the loci Idh, Mdh-1, Mdh-2, and 6-Pgd, were also not examined by segregation analysis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic diversity and relationships among Lablab purpureus genotypes evaluated using RAPD as markers

Summary Genetic variation in 40 accessions of Lablab purpureus was evaluated using random amplified polymorphic DNA as markers. A high level of genetic variation in this species was detected but this was mainly restricted to the difference between cultivated and wild forms. Of the cultivated genotypes, genetic variation among Asian collections was significantly higher than that among African collections. The three most divergent cultivated genotypes were all from Asia. Four of the five wild accessions, two from Zimbabwe and the other two from Zambia, were closely related. The other one, CPI 31113 collected from Uganda, was highly divergent. The two commercial forage varieties used in Australia, Rongai from Kenya and Highworth from India, were not very different.     

Genetic relationship of Mediterranean mandarins (Citrus deliciosa Tenore) using RAPD markers

Summary Random amplified polymorphic DNA (RAPD) analysis was carried out to evaluate polymorphism and genetic similarity between 39 Mediterranean mandarin genotypes. One hundred eleven amplification products were identified using 21 random primers. An average of 2.2 RAPD markers was obtained for each primer, corresponding to 42% of the amplification products. Genotype-specific RAPD markers were also found, mainly in known hybrids. UPGMA cluster analysis revealed the low level of genetic variation between accessions of Mediterranean mandarins, whereas their hybrids with other Citrus species showed greater genetic dissimilarity. Twenty accessions yielded very similar patterns, suggesting either that they could be a single clone, or that the technique was not able to detect genomic variation. However, for the other specimens genetic polymorphism can easily be detected by RAPD, although the genetic variation between accessions was quite low. The large number of hybrids and the low polymorphism between accessions support the hypothesis that Mediterranean mandarins are all true hybrid of Common mandarins (Citrus reticulata Blanco).     

Assessment of genetic variability in grapefruits (Citrus paradisi Macf.) and pummelos (C. maxima (Burm.) Merr.) using RAPD and SSR markers

The genetic variability of 38 grapefruit (Citrus paradisi Macf.) and three pummelos (C. maxima (Burm.) Merr..) accessions was evaluated using RAPD, and single sequence repeat (SSR) analyses. Approximately49% of the 198 RAPD were polymorphic, and 4.6 alleles per SSR loci were identified. PIC values changed from 0.093 to 0.450. A UPGMA phenetic tree was constructed and two main grapefruit groups were identified. The grapefruit accessions `do Cabo' and `Siamesa-Filipinas'clustered very close to the pummelos in Group A. The Group B consisted of three sub-groups, which comprised all of the other grapefruit accessions. The majority of grapefruit accessions showed a narrow genetic base suggesting that the observed morphological polymorphism within the group must be associated with somatic mutations, which were not detected by these molecular markers. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic relationships between cultivated and wild olives of Corsica and Sardinia using RAPD markers

In order to ensure the genetic diversity of the Olea europaea complex,it is necessary to characterize the cultivated varieties and the wildpopulations. In the present study, we focused on the olives growing on twoMediterranean islands, Corsica and Sardinia. On these two islands, there areolives with many denominations, as well as forests of oleasters. Here, it wasproposed to determine the relationships among cultivated and wild olives.Some Italian denominations were studied in addition to assess the influenceof the mainland on the two islands in this respect.The 59 RAPD markers obtained showed the existence of manysynonymous, and homonymous. A dendrogram was constructed using theUPGMA method and a FCA was carried out. The results of these twoanalyses showed the existence of a genetic divergence between the oleastersand the cultivated varieties. They suggest that some of the Corsicanvarieties were probably selected from local wild forms, contrary to theSardinian varieties. They also show that there are feral forms growing onboth islands, which result from hybridization between oleasters andvarieties.     

RAPD markers linked to a rust resistance gene in cultivated groundnut (Arachis hypogaea L.)

Groundnut rust (Puccinia arachidis Speg.) is an important air borne pathogen, which causes substantial losses in groundnut yield and quality. Although large numbers of accessions were identified as rust resistant in wild, interspecific derivative and cultivated groundnut species, transfer of resistance to well-adapted cultivars is limited due to linkage drag, which worsens yield potential and market acceptance. A F₂ mapping population comprising 117 individuals was developed from a cross between the rust resistant parent VG 9514 and rust susceptible parent TAG 24. Rust resistance was governed by single dominant gene in this cross. We identified 11 (out of 160) RAPD primers that exhibited polymorphism between these two parents. Using a modified bulk segregant analysis, primer J7 (5′CCTCTCGACA3′) produced a single coupling phase marker (J7₁₃₅₀) and a repulsion phase marker (J7₁₃₀₀) linked to rust resistance. Screening of the entire F₂ population using primer J7 revealed that the coupling phase marker J7₁₃₅₀ was linked with the rust resistance gene at a distance of 18.5 cM. On the other hand, the repulsion phase marker J7₁₃₀₀ was completely linked with rust resistance. Additionally, both J7₁₃₀₀ (P = 0.00075) and J7₁₃₅₀ (P < 0.00001) were significantly associated with the rust resistance. Marker J7₁₃₀₀ identified all homozygous rust resistant genotypes in the F₂ population and was present in all the eight susceptible genotypes tested for validation. Thus, J7₁₃₀₀ should be applicable for marker-assisted selection (MAS) in the groundnut rust resistance breeding programme in India. To the best of our knowledge this is the first report on the identification of RAPD markers linked to rust resistance in groundnut.     

Assessment of genetic relatedness of the genera Ananas and Pseudananas confirmed by RAPD markers

Genetic relationships among 18 accessions, including 16 of Ananas and two of Pseudananas, were investigated using RAPD molecular markers. The procedure for DNA extraction was adapted from the method of Dellaporta et al. (1983) where an incubation in proteinase K and a purification step were included. From the total of 148 markers scored,132 (89.2%) were polymorphic. The similarity matrix was used for cluster analysis. The phenogram developed from the RAPD bands showed that for most of the cases, the accessions within a species grouped together. Nevertheless, a moderate infraspecific genetic variation was observed. For example, DNA data grouped all A. comosus accessions with a mean similarity coefficient of 0.85. Comparable results were obtained with all other species investigated. The highest genetic divergence was found withinA. lucidus where the mean similarity coefficient among accessions was0.75. A similar level of genetic polymorphism was observed among species,therefore, a definition about which species were involved in the constitution of A. comosus genotypes was not possible. These results agree with the breeders standpoint suggesting that all Ananas species belong to the primary gene pool of pineapple. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence of gene introgression in apple using RAPD markers

Summary A genomic remnant of Malus floribunda clone 821 introgressed into the cultivated apple M. x domestica Borkh. was identified using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). Using a set of 59 oligonucleotide decamer primers, polymorphic DNA markers were identified among three pooled DNA samples. Based on the presence or absence of bands among bulked apple scab-resistant selections and cultivars, bulked scab-susceptible cultivars, and a M. floribunda clone 821 sample, one primer, A 15, identified amplified fragments in the scab-resistant bulked sample that was also unique to the M. floribunda clone 821. The unique band from M. floribunda clone 821 was amplified in four out of 17 scab-resistant selections/cultivars. This RAPD, designated OA15₉₀₀, identifies an introgressed fragment that has as yet no known function.     

Identification and genetic diversity analysis of date palm (Phoenix dactylifera L.) varieties from Morocco using RAPD markers

Genetic variation among 43 date palm (Phoenix dactylifera L.) accessions, including 37 accessions from Morocco and 6 cultivars from Iraq and Tunisia, was studied using Random Amplified Polymorphic DNA (RAPD) markers. The pre-screening of 123 primers on four genotypes allowed selection of 19 primers which revealed polymorphism and gave reproducible results. All 43 analysed genotypes were distinguishable by their band patterns. RAPD technology therefore appears very effective for identifying accessions of date palm. RAPD-based genetic distance was used to determine the relationships between the accessions. The grouping-association identified by cluster analysis was rather weak. However, morphologically similar varieties clustered together. A relatively low polymorphism and a lack of evident organisation are observed among the date palm varieties grown in Morocco. This could be related to the mode of introduction and maintenance of the Moroccan date palm germplasm involving limited foundation germplasm, exchange of cultivars between plantations, and periodic development of new recombinant cultivars following sexual reproduction. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic diversity of wild coffee (Coffea arabica L.) using molecular markers

Genetic diversity was studied using RAPD markers among119 coffee (Coffea arabica L.) individuals representing 88 accessions derived from spontaneous and subspontaneous trees in Ethiopia, the primary centre of species diversity, six cultivars grown locally in Ethiopia, and two accessions derived from the genetic populations Typica and Bourbon, spread in the 18^th century, which gave rise to the most currently grown cultivars. Twenty-nine polymorphic fragments were used to calculate a similarity index and construct dendrograms. The Ethiopian material was separated from the Typica- and Bourbon-derived accessions and classified in four groups: one with most of the collected material from southwestern Ethiopia and three from southern and southeastern Ethiopia. Almost all detected diversity was found in the southwestern group while the southern and southeastern groups presented only 59% of identified markers. The genetic distances were low between the southwestern group and the southern and southeastern groups, and between the southwestern group and the Typica- and Bourbon-derived accessions. The cultivated coffee derived from the genetic populations Typica and Bourbon appeared little differentiated from wild coffee growing in the southwest. The results supported the hypothesis that southwestern Ethiopian coffee trees could have been introduced recently in the south and southeast. A separate analysis of the 80accessions classified in the southwestern group allowed identifying particular spontaneous- and subspontaneous-derived accessions and redundancies in the collected material from southwestern Ethiopia. RAPD markers did not detect any within-collection polymorphism except for two trees that were identified as off-types in the CATIE field genebank. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD markers linked to resistance to downy mildew disease in soybean

To determine and utilize RAPD markers linked to resistance to downymildew incited by Peronospora manshurica in soybean, a resistantcultivar `AGS129' was crossed to a susceptible cultivar `Nakhon Sawan 1'(NS1). F₂ and BC₁ populations were advanced from the F₁ and evaluatedfor resistance to the disease. ²-test demonstrated that the resistancewas controlled by a single dominant gene (Rpmx). Near-isogenic lines(NILs) and bulked segregant analysis (BSA) were used to identify RAPDmarkers linked to the gene. Six DNA bulks namely F₅(R), F₅(S),BC₆F₃(R), BC₆F₃(S), F₂(R) and F₂(S) were set up by pooling equalamount of DNA from 8 randomly selected plants of each disease responsetype. A total of 180 random sequence decamer oligonucleotide primerswere used for RAPD analysis. Primer OPH-02 (5 TCGGACGTGA 3 andOPP-10 (5 TCCCGCCTAC 3) generated OPH-02₁₂₅₀ and OPP-10₈₃₁fragments in donor parent and resistant bulks, but not in the recurrentparent and susceptible ones. Co-segregation analysis using 102 segregatingF₂ progenies confirmed that both markers were linked to the Rpmxgene controlling downy mildew disease resistance with a genetic distance of4.9 cm and 23.1 cm, respectively. Marker OPH-02₁₂₅₀ was presentin 13 of 16 resistant soybean cultivars and absent in susceptible cultivars,thus confirming a potential for MAS outside the mapping population.     

Genetic variation among cultivars of red clover (Trifolium pratense L.) detected by RAPD markers amplified from bulk genomic DNA

Summary The use of random amplified polymorphic DNA (RAPD) markers obtained from bulked samples was investigated for cultivar identification in red clover. Pooled samples were examined in order to minimize variation within cultivars. To determine the appropriate number of individuals to include in the bulked samples representing each cultivar, DNA samples from two, three, four, five, ten and twenty individuals were pooled. Twenty was found to be an appropriate number of red clover individuals per bulk in order to amplify only the DNA sequences shared among most individuals in each cultivar. Fourteen 10-mer primers were used to amplify genomic DNA from combined leaf samples of 15 red clover cultivars from European, Japanese and North American origins. A total of 79 amplified products, of which 55 were polymorphic, was obtained. Cultivar-specific bands were observed with 13 primers. The amplification patterns obtained from two primers could distinguish all 15 red clover cultivars. Rogers' genetic distances for all 105 pairwise comparisons were calculated to evaluate relationships among these cultivars. Cluster analysis based on these genetic distances separated these 15 cultivars into three groups, with two of the groups consisting of a single Japanese cultivar each, while the third group included cultivars from European, North American, and Japanese origins.     

Evaluation of the genetic diversity among elite tea (Camellia sinensis var. sinensis) accessions using RAPD markers

The diversity of 27 superior tea (Camellia sinensis var. sinensis) accessions from Korea, Japan and Taiwan was examined with RAPD-PCR (Random Amplified Polymorphic DNA Polymerase Chain Reaction) markers. Out of the 50 primers screened, 17 primers generated 58 polymorphic and reproducible bands. A minimum of 3 primers was sufficient to distinguish all the 27 accessions studied. The Shannon's index used to partition diversity into inter- and intra-group, revealed that 71 percent of variability resided within groups and 29 percent between groups. Diversity was greatest within the Korean group followed by Taiwan and Japan. The relatively high diversity observed in Korea might reflect the larger genetic base of its plantations while the low diversity in Japan could be explained by the long and intensive tea selection programme in this country. A dendrogram based on the UPGMA-link method using Jaccard's distances and multivariate Factorial correspondence analysis clustered the tea accessions into two main groups, regrouping the Taiwan cultivars on the one side and the Korean and Japanese accessions on the other side. This suggests that the Taiwan tea studied here may have a different origin from that of Korea and Japan. This revised version was published online in July 2006 with corrections to the Cover Date.     

Random amplified polymorphic DNA (RAPD) markers variability among cultivars and landraces of common beans (Phaseolus vulgaris L.) of south-Brazil

To evaluate the variability among cultivars and landraces of common bean(Phaseolus vulgaris L.), 15 cultivars and 18 landraces of common bean (Phaseolus vulgarisL.), a undefined species of Phaseolus,two landraces of Vigna angularis L., and a landrace of soybean (Glycine maxL.), were screened with fifteen oligonucleotide primers in PCR reactions. An average of 20.3 RAPD bands were scored per primer. A total of 304 amplification products were scored of which 88.8% were polymorphic among Phaseolus genotypes. Based on the RAPD markers, four major clusters were formed. Three clusters corresponded to the soybean, to the two Vigna angularis landraces, and to the Phaseolus sp. landrace, respectively. The fourth cluster include all the landraces and cultivars of Phaseolus vulgaris. This large group could be separated into three subgroups that were correlated with the phaseolin patterns and the average seed weight of the genotypes. The analysis shows that most of the landraces collected in South Brazil (17 out of 18) belong to the Andean gene pool, and most of the cultivars (13 out of 15) belong to the Middle American gene pool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fingerprinting of alfalfa meiotic mutants using RAPD markers

Summary A calendar of female sporogenesis and gametogenesis was made for both apomictic tetraploid (2n=4x=36) Brachiaria brizantha and Brachiaria decumbens and their apomictic F₁ hybrids with sexual tetraploid (2n=4x=36) Brachiaria ruziziensis. Microgametogenesis was used as a reference. Apospory was facultative in both species and hybrids. Environmental conditions had variable effects on the level of apomixis according to each genotype. Ratios of segregation into sexuals and apomicts in the interspecific hybrids suggest an oligogenic determinism with dominant apomixis in the genus Brachiaria. Highly apomictic and partially male fertile hybrids were identified and will be used in an improvement program to transfer genes for apomixis into the sexual species B. ruziziensis.