Evaluation of the extent of genetic variability among Theobroma cacao accessions using RAPD and RFLP markers

 Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to evaluate genetic relationships within the Theobroma cacao species and to assess the organization of its genetic diversity. Genetic variability was estimated with 18 primers and 43 RFLP probes on 155 cocoa trees belonging to different morphological groups and coming from various geographic origins. The majority of the RFLP probes issued from low-copy DNA sequences. On the basis of on the genetic distance matrices, the two molecular methods gave related estimates of the genetic relationship between genotypes. Although an influence of cocoa morphological groups and geographical origins of trees was observed, a lack of gene differentiation characterized the T. cacao accessions studied. The continuous RFLP variability observed within the species may reflect the hybridization and introgressions between trees of different origins. Nevertheless, the Nacional type was detected to be genetically specific and different from well-known types such as Forastero, Criollo and Trinitario. Some of those genotypes were characterized by a low heterozygosity rate and may constitute the original Nacional pool. These results also provide information for the constitution of a cocoa tree core collection. Received: 10 June 1996/Accepted: 11 October 1996     

Genetic variability of Triatoma rubrovaria (Reduviidae: Triatominae) from Brazil, Argentina and Uruguay as revealed by two different molecular markers

Randomly amplified polymorphic DNA (RAPD) and nuclear ribosomal DNA sequence analyses were used to assess the genetic population structure of the South American triatomine species Triatomo rubrovario throughout its geographical distribution. To investigate the genetic variability at both intraspecific and intrapopulational levels the RAPD profiles and the nucleotide sequences of the rDNA intergenic spacers, ITS-1 and ITS-2, were analysed and compared. The phenetic analysis based on RAPD profiles show three distinct clusters diverging by similarity coefficients ranging from 0.62 to 0.96. The ITS-1 and ITS-2 sequence variability detected may be considered very high, suggesting reproductive isolation between populations. A total of seven composite haplotypes (CH) were found, among which three are specific for Brazil, other three for Uruguay, and the last one common for the three countries studied. The population studied in Argentina does not represent an independent CH. Sequence analyses proved that the five populations studied are easily differentiable and that there is heterogeneity within each one. True mutations and indels are the responsible of sequence differences between haplotypes and populations, suggesting that divergence processes may presently go on within this species. The large intraspecific variability detected may underlie the known plasticity of T. rubrovaria, making it a potential intradomiciliary invader and consequently an appropriate vector for Chagas disease transmission. Therefore, this triatomine species must be continuously monitored throughout.     

Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers

Genetic variation within and between five populations of Oryza granulata from two regions of China was investigated using RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat amplification) markers. Twenty RAPD primers used in this study amplified 199 reproducible bands with 61 (30.65%) polymorphic; and 12 ISSR primers amplified 113 bands with 52 (46.02%) polymorphic. Both RAPD and ISSR analyses revealed a low level of genetic diversity in wild populations of O. granulata. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between regions. As the RAPD markers revealed, 73.85% of the total genetic diversity resided between the two regions, whereas only 19.45% and 6.70% were present between populations within regions and within a population respectively. Similarly, it was shown by ISSR markers that a great amount of variation (49.26%) occurred between the two regions, with only 38.07% and 12.66% between populations within regions and within a population respectively. Both the results of a UPGMA cluster, based on Jaccard coefficients, and pairwise distance analysis agree with that of the AMOVA partition. This is the first report of the partitioning of genetic variability within and among populations of O. granulata at the DNA level, which is in general agreement with a recent study on the same species in China using allozyme analysis. Our results also indicated that the percentage of polymorphic bands (PPB) detected by ISSR is higher than that detected by RAPD. It seems that ISSR is superior to RAPD in terms of the polymorphism detected and the amplification reproducibility. Received: 29 March 2000 / Accepted: 15 May 2000     

Detection and analysis of genetic variation in Hordeum spontaneum populations from Israel using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to analyse genetic diversity within and between Hordeum spontaneum populations sampled from Israel. Nei's index of genetic differentiation was used to partition diversity into within and between population components. Fifty-seven per cent of the variation detected was partitioned within 10 H. spontaneum populations. Using principal component and multiple regression analysis, part of the variation detected between populations was seen to be associated with certain ecogeographical factors. Fifty-eight per cent of the distribution of the phenotypic frequencies of three RAPD phenotypes detected using a single primer in 20 H. spontaneum populations could be accounted for by four ecogeographical variables, suggesting adaptive variation at certain RAPD loci.     

Characterization of Colletotrichum lindemuthianum Isolates from the State of Minas Gerais, Brazil

Anthracnose disease of common bean (Phaseolus vulgaris), caused by Colletotrichum lindemuthianum, is responsible for extensive yield losses worldwide. This pathogen is known to vary greatly in its pathogenicity. Control strategies include chemical control and, mainly, the development of resistant cultivars, taking into account the population structure of C. lindemuthianum. The objective of this study was to investigate the pathogenic and genetic diversity and population structure among C. lindemuthianum isolates collected in Minas Gerais state, Brazil. When these isolates were inoculated on 12 differential cultivars, a total of 10 races were identified within a series of 48 isolates collected in Minas Gerais, Brazil. Races 65, 81 and 73 were the most frequent races and occurred in most of the regions. This study also detected race 337, which had not been reported previously in the literature. Random amplified polymorphic DNA (RAPD) analysis performed on the same 48 isolates revealed great genetic diversity, clustering the series into five groups at a maximum similarity value of 89.6%. There was no clear relationship between the loci sampled by RAPD markers and the pathogenic characterization. Analysis of molecular variance showed that 96.06% of the variability was contained within regions and 3.94% among regions, indicating a high exchange of genetic material among the regions of the State. Most of the variability was detected within races (75.24%). The pathogenicity and RAPD assays corroborated the broad genetic diversity of the pathogen and the results have been useful in breeding for resistance to anthracnose.     

Fertilization and defoliation frequency affect genetic diversity of Festuca pratensis Huds. in permanent grasslands

Permanent pastures and meadows are species-rich vegetation systems that play an important role in the ecology and agriculture of temperate climates. Intensive management reduces species diversity and may also influence the genetic diversity within individual species and populations. The objective of this study was to assess genetic variability of meadow fescue, an important component of species-rich grasslands, and to determine whether fertilization and defoliation frequency influence genetic variability within natural populations. Genetic diversity of six natural populations and three cultivars of Festuca pratensis was investigated using randomly amplified polymorphic DNA (RAPD) markers and agronomic traits. Samples of natural populations were taken from two unrelated long-term experiments, where treatments had been applied for 11–38 years. RAPD analysis detected a clear genetic distinction of the cultivars from the natural populations. Genetic variability within cultivars was lower than within natural populations. Analysis of molecular variance ( AMOVA ) showed a significant effect of management on genetic variability. Fertilization and frequent defoliation led to a reduction in genetic variability within natural populations. Analysis of agronomic traits was only partially congruent with the results of RAPD analysis. This study shows that significant genetic variability exists within cultivars and natural populations of Festuca pratensis and can be reduced by intensive management.     

有性生殖对栗疫病菌群体结构的影响

采用RAPD方法对来源于栗疫病菌8个不同子囊壳的子囊孢子后代和无性生殖的对照群体各23个菌株进行了群体结构的比较。从RAPD随机引物中筛选出扩增多态性丰富的4条引物,共扩增出条带73条,多态性检测率为100%。研究结果表明,在8个子囊壳和无性生殖群体中的基因多样性,64.27%由群体内部引起,只有35.73%的多样性由群体之间的基因差异引起。各子囊壳群体间存在的基因流动很小(Nm=0.8994)。有性群体和无性群体之间的遗传距离为0.1389,基因流动值为3.4212,说明子囊壳群体和无性生殖群体之间存在一定的系统关系。分析表明栗疫病菌子囊孢子后代在自然界的传播对自然界的病菌的多样性起重要的作用。     

Vegetative compatibility and genetic analysis of Colletotrichum lindemuthianum isolates from Brazil

The causal agent of common bean anthracnose, Colletotrichum lindemuthianum, has considerable genetic and pathogenic variability, which makes the development of resistant cultivars difficult. We examined variability within and between Brazilian pathotypes of C. lindemuthianum through the identification of vegetative compatibility groups (VCGs) and by RAPD analysis. Two hundred and ninety-five nit mutants were obtained from 47 isolates of various pathotypes of the fungus collected from different regions, host cultivars and years. In complementation tests, 45 VCGs were identified. Eighteen RAPD primers were employed in the molecular analyses, producing 111 polymorphic bands. Estimates of genetic similarities, determined from the Sorence-Dice coefficient, ranged from 0.42 to 0.97; the dendrogram obtained by cluster analysis revealed 18 groups of isolates. RAPD and VCG markers presented high genotypic diversity. The number of significant associations (P=0.05) between RAPD, VCG and pathogenicity markers ranged from 0 (VCG) to 80% (pathogenicity). The test of multilocus association (rd) for RAPD markers was significantly different from zero (P<0.001), suggesting linkage disequilibrium. However, the results for VCG markers show the presence of recombination mechanisms. In conclusion, RAPD markers and VCGs were useful for detecting genetic variability among isolates of C. lindemuthianum. We found considerable diversity among isolates from the same geographic origin within a short interval; this suggests rapid evolution. There is a need for further studies to elucidate the population structure of this pathogen in agro-ecosystems.     

Preliminary study on genetic variability of Dicrocoelium dendriticum determined by random amplified polymorphic DNA

Genetic variability of adult specimens of Dicrocoelium dendriticum has been studied using random amplified polymorphic DNA (RAPD). The worms were collected from the infected livers of different sheep from several localities in León province (NW Spain). DNA fragments were amplified by means of decamer primer oligonucleotides of arbitrary sequence. Some primers produce complex and highly variable patterns of amplified DNA in D. dendriticum. Intra- and inter-population genetic variability of adult parasites were analyzed, scoring polymorphic and monomorphic reproducible bands by means of the Jaccard similarity, and dendrograms showing genetic relationships between individuals were obtained using the FITCH method. Genetic variability seems to be high in this parasite and genetic similarity within a population (worms infecting a single animal) is similar to the average similarity between worms from different sheep. These results suggest that each sheep is infected by numerous genetically different parasites from one or more populations of infected ants.     

Assessment of genetic variability through ISSR and RAPD markers in <Emphasis Type="Italic">Commiphora wightii</Emphasis> (Arn.) Bhandari

Commiphora wightii (Arn.) Bhandari is a commercially, medicinally and traditionally important tropical shrub widely used to treat various ailments and disorders. Demand of this plant is increasing in the pharmaceutical and perfumery industries due to the presence of guggulsterone E and Z, two important isomers conferring lipid- and cholesterol-lowering, and anti-cancerous properties. Ruthless and unscientific harvesting of oleo-gum resin by local populations from the wild, with negligible conservation efforts has made this species endangered and led to its inclusion in the Red Data Book of IUCN. It is imperative to have broad information regarding the extent of genetic variability available in the species to accelerate the breeding and conservation programs. Therefore, the present study was undertaken to analyze the extent of genetic variability existing among the C. wightii germplasm collected from Rajasthan and Haryana, the diversity rich Indian states, using ISSR and RAPD markers. A total of 100 (50 each) RAPD and ISSR markers were screened of which 37 RAPD and 43 ISSR primers were able to amplify DNA fragments. RAPD markers were more efficient, detecting 74.16 % polymorphism, compared to ISSR which detected 62.52 % polymorphism. Also, the values of average number of polymorphic bands per assay, polymorphism information content (PIC), diversity index (DI) and marker index (MI) were more for RAPD (7.76, 0.19, 0.38 and 2.53, respectively) than for ISSR (7.02, 0.13, 0.32 and 1.88) markers. The UPGMA dendrogram constructed using individual as well as combined data of the two marker systems separated the collected accessions into two major clusters containing 47 and 4 accessions, respectively, while one accession from Bikaner was not included in any cluster. Genetic similarity values obtained from Jaccard’s coefficient using combined data of both the marker systems were between 0.50 and 0.97. These results indicated the existence of wide genetic variability within this species and can be used for further research in the area of germplasm conservation, population genetics and plant breeding.     

Identification and Characterization of Malaysian River Catfish, Mystus nemurus (C&V): RAPD and AFLP Analysis

This work represents the first application of the amplified fragment length polymorphism (AFLP) technique and the random amplified polymorphic DNA (RAPD) technique in the study of genetic variation within and among five geographical populations of M. nemurus. Four AFLP primer combinations and nine RAPD primers detected a total of 158 and 42 polymorphic markers, respectively. The results of AFLP and RAPD analysis provide similar conclusions as far as the population clustering analysis is concerned. The Sarawak population, which is located on Borneo Island, clustered by itself and was thus isolated from the rest of the populations located in Peninsular Malaysia. Both marker systems revealed high genetic variability within the Universiti Putra Malaysia (UPM) and Sarawak populations. Three subgroups each from the Kedah, Perak, and Sarawak populations were detected by AFLP but not by RAPD. Unique AFLP fingerprints were also observed in some unusual genotypes sampled in Sarawak. This indicates that AFLP may be a more efficient marker system than RAPD for identifying genotypes within populations.     

Genetic variability of an endangered Bromeliaceae species (Pitcairnia albiflos) from the Brazilian Atlantic rainforest

Pitcairnia albiflos is a Bromeliaceae species endemic to Brazil that has been included as data-deficient in the extinction risk list of Brazilian flora. We analyzed genetic variability in P. albiflos populations using RAPD markers to investigate population structure and reproductive mechanisms and also to evaluate the actual extinction risk level of this species. Leaves of 56 individuals of P. albiflos from three populations were collected: Urca Hill (UH, 20 individuals), Chacrinha State Park (CSP, 24 individuals) and Tijuca National Park (TNP, 12 individuals). The RAPD technique was effective in characterizing the genetic diversity in the P. albiflos populations since it was possible to differentiate the populations and to identify exclusive bands for at least two of them. Even if there is low genetic diversity among them (CSP-UH = 0.463; CSP-TNP = 0.440; UH-TNP = 0.524), the populations seem to be isolated according to the low genetic diversity observed within them (H(pop) CSP = 0.060; H(pop) UH = 0.042; H(pop) TNP = 0.130). This fact might be the result of clonal and self-reproduction predominance and also from environmental degradation around the collection areas. Consequently, it would be important to protect all populations both in situ and ex situ to prevent the decrease of genetic variability. The low genetic variability among individuals of the same population confirms the inclusion of this species as critically endangered in the risk list for Brazilian flora.     

Population Structure of Puccinia recondita in Western Europe During 1995, as Assessed by Variability in Pathogenicity and Molecular Markers

The population structure of Puccinia recondita f. sp. tritici (Prt) in western Europe was examined by assessing variability in pathogenicity and in randomly amplified polymorphic DNA (RAPD) among 61 single uredinial isolates. The isolates were chosen to represent pathotypes detected in a previous survey of pathogenic variability in the fungus in western Europe in 1995. Thirty‐five pathotypes were identified by assessing infection types produced by the 61 isolates on 24 differential lines, each with a single gene for resistance to Prt. In contrast, only 18 RAPD phenotypes were identified by scoring 19 polymorphic RAPD bands generated with eight RAPD primers. When analysed by cluster and bootstrap analyses, the pathogenicity and RAPD results revealed little evidence for robust distinct clusters among the isolates. Multiple isolates of several pathotypes collected from widely separated locations such as Belgium, Germany, France, Italy and Switzerland had the same RAPD phenotype, providing evidence of clonal migration over considerable distances in western Europe. Some variability (one or two band differences) was observed in RAPD phenotype within several pathotypes, indicating the possible occurrence of genetic changes independent of pathogenicity, and/or the independent development of pathotypes with different genetic backgrounds. Two groups of isolates identified in the 1995 survey, differentiated by pathogenicity for genes Lr3a, Lr3bg, Lr3ka and Lr30, were not distinguished by RAPD phenotype, indicating that the groups probably do not constitute separate lineages within the pathogen population. Little correlation was apparent between the polymorphisms observed in pathogenicity and RAPD phenotypes. The similarity in the genetic backgrounds of the isolates, as assessed by RAPD markers, suggest that the observed differences in pathogenicity may have arisen by selection for specific virulences corresponding to genes for resistance in wheat cultivars grown in the region. Three isolates of pathotype 3, restricted in its distribution to southern France during 1995, were distinct from all other isolates in RAPD phenotype. Circumstantial evidence suggests that this pathotype originated from northern Africa, and that it belongs to a group of leaf rust pathogens specialized to durum wheats.     

Microsatellite markers provide evidence for sexual reproduction of Mycosphaerella graminicola in Saskatchewan.

This study examined the genetic structure of a Saskatchewan population of Mycosphaerella graminicola, cause of the foliar disease Septoria tritici blotch of wheat. Such knowledge is valuable for understanding the evolutionary potential of this pathogen and for developing control strategies based on host resistance. Nine pairs of single-locus microsatellite primers were used to analyze the genomic DNA of 90 isolates of M. graminicola that were collected using a hierarchical sampling procedure from different locations, leaves, and lesions within a wheat field near Saskatoon. Allelic series at eight different loci were detected. The number of alleles per locus ranged from one to five with an average of three alleles per locus. Genetic diversity values ranged from 0.04 to 0.67. Partitioning the total genetic variability into within- and among-location components revealed that 88% of the genetic variability occurred within locations, i.e., within areas of 1 m(2), but relatively little variability occurred among locations. Low variability among locations and a high degree of variability within locations would result if the primary source of inoculum was airborne ascospores, which would be dispersed uniformly within the field. This finding was confirmed by gametic disequilibrium analysis and suggests that the sexual reproduction of M. graminicola occurs in Saskatchewan.     

Population genetics in the conservation of the Azorean shrub Viburnum treleasei Gand.

We assess population genetic structure and variability in the endangered Azorean endemic Viburnum treleasei Gand., an evergreen shrub or small tree, occurring in eight out of nine islands of the archipelago. We combine RAPD and ISSR markers in eight populations and four islands covering the three subgroups of islands that compose the archipelago, and one population of V. tinus from the Portuguese mainland. Most of the genetic variability was found within populations, which is in accordance with the bi-parental reproductive strategy favored by the taxon. Gene flow estimations for the combined RAPD and ISSR markers suggest that the main cause for population variability between the studied populations is genetic drift. In accordance with the genetic structure indicators obtained, conservation measures should consider that translocation of individuals between islands must be avoided. In specific cases, the analysed populations may require the implementation of augmentation strategies due to their depleted state. With this study, a genetic background is now available to better define conservation measures for the taxon.     

Genetic variation among populations of the Hessian fly Mayetiola destructor (Diptera: Cecidomyiidae) in Morocco and Syria

The RAPD-PCR technique was used to study genetic variation within and among geographical populations of the Hessian fly, Mayetiola destructor (Say), from Morocco and Syria, associated with the fly's ability to overcome resistance in three wheat cultivars containing H5, H13 and H22 resistance genes. Variation was detected both for the level of susceptibility of the cultivars and RAPD profiles of M. destructor populations. By the use of RAPD-PCR, high genetic variability was detected among individuals and populations of M. destructor within and between areas separated geographically. The DNA fingerprints of populations of M. destructor were area-specific with Nei's measures of genetic distance ranging from 0.156 (between Abda and Beni Mellal, Morocco) to 1.977 (between Marchouch, Morocco and Lattakia, Syria). Cluster analysis of the genetic distances among the populations, identified the Syrian population as an outlier. A highly significant correlation (r = 0.81) observed between the genetic and geographic distances among the populations, provided genetic support for dispersal of the fly from its presumed origin in West Asia to Morocco.     

Comparative analysis of genetic diversity of sesame (Sesamum indicum L.) from Vietnam and Cambodia using agro-morphological and molecular markers

The purpose of this study was to comparatively analyze the genetic diversity of sesame (Sesamum indicum L.) using agro-morphological and molecular markers. Twelve sesame populations collected from three regions in Cambodia and Vietnam were used in this study. A high genetic variation was revealed both by agro-morphological and RAPD markers within and among the 12 sesame populations. The range of agro-morphological trait based average taxonomic distance among populations (0.02 to 0.47) was wider than that of RAPD based genetic distance (0.06 to 0.27). The mean distance revealed by agro-morphological markers (0.23) and RAPD markers (0.22) was similar. RAPD based analysis revealed a relatively higher genetic diversity in populations from South Vietnam as compared to the other two regions. Interestingly, populations from this region also had higher values for yield related traits such as number of capsules per plant, number of seeds per capsule, and seed yield per plant suggesting positive correlation between the extent of genetic variation within population and yield related traits in sesame. A highly significant positive correlation (r = 0.88, P < 0.001) was found between agro-morphological and RAPD markers in estimating the genetic distance between populations. Both methods suggested the existence of a substantial amount of genetic diversity both in the Vietnamese and Cambodian populations. Although both agro-morphological and RAPD markers were found to be useful in genetic diversity analysis in sesame, their combined use would give superior results.     

Detection of genetic variability in various isolates of cattle tick, <Emphasis Type="Italic">Boophilus microplus</Emphasis> from Tamil Nadu,India using PCR-RAPD analysis

The genomic DNA from ten isolates of the cattle tick, Boophilus microplus collected in and around Chennai, India, was analyzed by random amplified polymorphic DNA (RAPD) using PCR. Selected five random primers were used for the study of genetic variability among different isolates of B. microplus. A high degree of genetic polymorphism with a different pattern of RAPD profiles for each tick isolate was detected with all these random primers. This variability was also confirmed by similarity coefficient values and dendrogram which were performed using mean RAPD profiles for all the primers between various isolates of ticks. The findings suggest the existence of a complex genotypic diversity of the tick B. microplus in an endemic region such as Chennai.     

Genetic variation in Antarctic populations of the moss Sarconeurum glaciale

Sixty-six isolates of the moss Sarconeurum glaciale were collected from sites in continental Antarctica at Ross Island, southern Victoria Land and the Vestfold Hills. Genetic variation within and among the populations was estimated using isozymes and random amplified polymorphic DNA (RAPD) technology. Isozyme results only reproducibly showed variation between the populations with one enzyme; RAPDs indicated significantly higher levels of genetic variability within and among the Vestfold Hills samples than in the Ross Sea region samples. A dendrogram produced from the RAPD bands suggested that the Ross Island and southern Victoria Land samples form one population, and those from the Vestfold Hills form a separate and more variable population, possibly resulting from separate colonisation events on the continent. Received: 15 March 1996 / Accepted: 1 May 1997     

Genetic structure of wild bean populations in their South-Andean centre of origin

 The genetic structure of wild common bean populations was studied in the South-Andean centre of origin of the species. Plants were collected from 21 populations in Argentina and genetic variability was assessed for molecular and resistance markers. Polymorphism was weak for phaseolin, the major seed-storage protein, and for RAPD markers, while a high level of polymorphism was observed for resistance to anthracnose, one of the most important diseases of common bean. For the three traits, within-population variability was important and represented between 43.6% and 67.5% of the total variation. Although among-population differentiation was significant for all the traits, no correlation was found between the population distances calculated from RAPDs and resistance. These results indicate that pathogen selection pressure may be an important factor influencing the distribution of variability within and among host plant populations. Received: 28 October 1997 / Accepted: 25 November 1997