Karyotype and genetic relationship based on RAPD markers of six wild buckwheat species (<Emphasis Type="Italic">Fagopyrum</Emphasis> spp.) from southwest of China

Karyotypes of six wild buckwheat species from Sichuan Pan’xi Regions of China were investigated in present study. This study will help in understanding evolutionary mechanisms and relationships among the taxes in this section. The karyotypes of three species, F. densovillosum, F. gracilipes var. odontopterum and F. gracilipes are being reported for the first time. F. esculentum, F. cymosum, F. tataricum and F. densovillosum are diploid while F. gracilipes var. odontopterum and F. gracilipes are tetraploid, 2n = 4x = 32. At diploid level, the variation of the buckwheat chromosome karyotypes is bigger; however, on the tetraploid level, it shows a higher similarity. The karyotype evolutionary trend of buckwheat indicated that F. densovillosum seemed to be more advanced than F. gracilipes var. odontopterum and F. gracilipes. F. densovillosum, F. gracilipes var. odontopterum. and F. gracilipes were found more closely related genetically whereas F. tataricum clustered into a separate group. F. esculentum and F. cymosum clustered together in one separate group.     

Relationships among different geographical groups,agro-morphology,fatty acid composition and RAPD marker diversity in Safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis>)

Patterns of geographical diversity, and the relationship between agro-morphological traits and fatty acid composition were assessed for 193 safflower (Carthamus tinctorius) accessions representing forty countries. Accessions were assigned to eight groups based on geographical proximity. Cluster and Principal Component analyses were performed to assess patterns of diversity among the accessions and to select the most distant accessions from each of eight groups for analysis of randomly amplified polymorphic DNA (RAPD) markers. There was a large amount of diversity for agro-morphological traits, fatty acid composition, and RAPD markers. Most correlations among different traits were rather low. Plant height showed a positive correlation with days to flowering (r = 0.63**). Palmitic acid was positively correlated with stearic acid and oleic acid values, and negatively correlated with linoleic acid (P < 0.01). Oleic acid and linoleic acid showed a strong negative correlation (r = −0.89**). The first three principal components together explained 59% of the variation, however, neither principal component analysis (PCA) nor marker analysis revealed a clear relationship between diversity pattern and geographical origin. Accessions from some geographical regions tended to group together, such as accessions from South Western Asia, Central Western Europe, and the Mediterranean region. The correlation between the morphological matrix and the genetic matrix based on RAPD markers was not significant (r = 0.027). Wide diversity in safflower germplasm indicates a considerable potential for improving this crop for both agronomic and quality traits.     

Genetic diversity analysis in Boerhavia diffusa L. of different geographic locations in India using RAPD markers

Boerhavia diffusa is extensively used in herbal medicines as well as in the Ayurvedic system, because it contains a set of clinically important compounds. In the present study, the genetic variability in Boerhavia diffusa between accessions of different geographical origin within the Indian territory is assessed through random amplified polymorphic DNA (RAPD) markers. Twenty-eight accessions of Boerhavia were screened with eighteen primers of which nine were found to be the most informative. The degree of polymorphism was found to be high in accessions collected from different places of Uttar Pradesh (Set II) in comparison to other states of India (Set I). A relatively lower level of polymorphism was recorded in accessions collected from diverse locations around Lucknow (Set III). Accessions from neighbouring geographical regions exhibited more similarity than those from distant regions (as revealed by the set I analysis). Certain diagnostic markers may be correlated with morphological character(s) such as plant type. BDL appeared most distinct and divergent from the rest of the accessions and the BDJ plant in set II also showed least similarity estimate. Fragments of 5.62 Kb and 4.47 Kb with primer GN59 was found to be unique for BDP and BD2 having ovate leaf character, whereas ovoid leaf genotype exhibited 0.79 Kb (GN34 primer) fragment. Similarly a unique band type (0.35 Kb) with primer GN83 was present in BDL and BD1 that share light pink flower. Jaccard's, and Nei and Li similarity coefficient values amongst the accessions were in the range of 0.22 to 0.89 and 0.33 to 0.93, respectively. Association of RAPD markers with the leaf characteristics, flower colour as well as with geographical locations has been made. This shows that RAPD markers are also useful for the study of genetic structure of Boerhavia populations.     

The genetic characterization of Turkish watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis>) accessions using RAPD markers

Genetic diversity of the Turkish watermelon genetic resources was evaluated using different Citrullus species, wild relatives, foreign landraces, open pollinated (OP) and commercial hybrid cultivars by RAPD markers. The germplasm was consisted of 303 accessions collected from various geographical regions. Twenty-two of 35 RAPD primers generated a total of 241 reproducible bands, 146 (60.6%) of which were polymorphic. Based on the RAPD data the genetic similarity coefficients were calculated and the dendrogram was constructed using UPGMA (Unweighted pair-group method with arithmetic average). Cluster analysis of the 303 accessions employing RAPD data resulted in a multi-branched dendrogram indicating that most of the Turkish accessions belonging to var. lanatus of Citrullus lanatus (Thunb.) Matsum et Nakai were grouped together. Accessions of different Citrullus species and Praecitrullus fistulosus (Stocks) Pangalo formed distant clusters from C. lanatus var. lanatus. Among 303 accessions, a subset of 56 accessions was selected representing different groups and a second dendrogram was constructed. The genetic similarity coefficients (GS) within the Turkish accessions were ranged from 0.76 to 1.00 with 0.94 average indicating that they are closely related. Taken together, our results indicated that low genetic variability exist among the watermelon genetic resources collected from Turkey contrary to their remarkable phenotypic diversity.     

Genetic diversity and relationships among cultivated and wild accessions of tartary buckwheat (Fagopyrum tataricum Gaertn.) as revealed by RAPD markers

The methodology of sampling and the selection of a proper marker systemfor the analysis of accessions are major concerns in the evaluation of gene bank material. In our study the RAPD analysis of bulked DNA samples and single seedsDNA was successfully employed to evaluate intra- and inter-population geneticvariability of cultivated and wild tartary buckwheat accessions. The bulkingapproach enabled the distinction of all 40 analysed accessions and theirseparation into geographically well defined clusters. Three wild populations,two from Sichuan and one from Qinghai, formed a group that was geneticallyrelatively distant from wild populations from Tibet and all cultivatedlandraces which, on the other hand, exhibited very close relationships. Thesingle seed study that was used after bulked DNA analysis provided detailedinformation of the genetic variation present within some accessions of specialinterest. A moderate level of genetic variability was detected betweenaccessions and the variability was partitioned into between- andwithin-population components. On average, most of the detected variation ispresent between F. tataricumpopulations. The genetic and geographic distribution of variability is furtherdiscussed. We demonstrated the usefulness of combining bulking and single seedstudy approaches for the effective evaluation of genetic variability inF. tataricum accessions that couldalso have wider applicability in the management of plant genetic resources andphylogenetic studies.     

Genetic variability in Turkmen populations of Pistacia vera L.

Seedlings of Pistacia vera L. developed from seeds of two separate populations in Turkmenistan, Kepele and Agachli, were evaluated for their growth potential and genetic polymorphism. Plant growth rate as well as random amplified polymorphic DNA (RAPD) analysis showed distinct differences between the two populations. In plant height growth rate, 17 Agachli accessions were 1.3 times higher on average than that of 10 accessions of Kepele (significant at p = 0.046) and 1.2 times higher for trunk diameter growth rate (p = 0.062). Cluster analysis divided most accessions into two main genetic groups according to their geographic origin. The Agachli group was further divided into two subgroups. One Kepele accession (K4), was genetically different from the rest and clustered on a separate outgroup. Two Agachli accessions (A12 and A17) were outside the two main populations clusters. Accessions K9 and K10 from Kepele were exceptional and were clustered in each of the two Agachli's subgroups, indicating a close genetic relationship between the two populations. In addition, high similarity values (0.58–1.00) and small genetic distances reflect plausible gene flow between Kepele and Agachli, which are 100 Km apart. Mantel test revealed significant relationship between the RAPD and the morphological traits matrices, pointing to the genetic basis for the measured differences in the growth rate. Growing the accessions on the same plot, under similar conditions enabled the evaluation of genotypic differences. The combination of morphological traits and molecular markers will further assist in preservation of genetic variability and cultivation of useful genotypes of P. vera L.     

Geographic Pattern of Genetic Diversity Among 43 Ethiopian Mustard(Brassica carinata A. Braun) Accessions as Revealed by RAPD Analysis

As an oilseed crop, the cultivation of Ethiopian mustard (Brassica carinata) is restricted only to Ethiopia. Even though geographic diversity is a potent source of allelic diversity, the extent of genetic diversity among germplasm material of Ethiopian mustard from different countries has not been assessed. Forty-three accessions, comprising 29 accessions from eight different geographic regions of Ethiopia and 14 exotic accessions from Australia, Pakistan, Spain, and Zambia were analysed for their genetic diversity using random amplified polymorphic DNA (RAPD) technique. A set of 50 primers yielded a total of 275 polymorphic bands allowing an unequivocal separation of every Ethiopian mustard accession. The usefulness of the 50 RAPD primers in measuring heterozygousity and distinguishing accessions was variable such that polymorphic information content (PIC) varied from 0.05 to 0.40, band informativeness (BI) from 0.05 to 0.65 and primer resolving power (RP) from 0.15 to 6.83. Jaccard's similarity coefficients ranged from 0.44 to 0.87 indicating the presence of a high level of genetic diversity. On the average, Australian and Ethiopian accessions were the most similar while, Spanish and Zambian accessions were the most distant ones. Cluster analysis grouped the 43 accessions into four groups, which has quite a high fit (r = 0.80) to the original similarity matrix. With no prior molecular information, the RAPD technique detected large genetic diversity among the 43 accessions from five different countries and their grouping by dendrogram and principal coordinate analysis (PCoA) was inclined towards geographic differentiation of RAPD markers. Conversely, RAPD differentiation along geographic origin was not apparent within the Ethiopian accessions.     

Genetic Diversity Among Brazilian Cultivars and Landraces of Tomato Lycopersicon Esculentum Mill. Revealed by RAPD Markers

Random amplified polymorphic DNA markers (RAPD) were used to estimate the variability of 35 tomato accessions (Lycopersicon esculentum Mill.). A total of 257 reproducibly scorable bands were obtained from 20 primers, 78.6% of which were polymorphic. The percentage distribution of RAPD markers shows a bimodal distribution, and the frequency of rare alleles is similar in commercial and landrace accessions. Genetic distances among accessions were calculated and a dendrogram showing the genetic relationships among them was constructed allowing for the separation of four groups. Twenty out of 23 Brazilian landraces fell within one group, whereas commercial cultivars were distributed in the four groups. AMOVA analysis of RAPD data showed that, despite the high within Brazilian landraces and commercial cultivars variation, these two groups are significantly different, indicating that landraces can be a source of variation for breeding programs.     

Analyses of the seed protein contents on the cultivated and wild buckwheat <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> resources

The seed protein contents of 179 accessions belonging to ten species of genus Fagopyrum were determined by means of the Kjeldahl method. The results indicated that there are significant differences of seed protein content among different accessions within the same species. The average protein content of F. esculentum is 12.94%, ranging from 8.81–18.71%, and the protein content of Sibano is the highest, up to 18.71%, Gantian 1 the lowest with 8.81%. The average protein content of tartary buckwheat is 12.17%, with the range from 7.82–18.94%, and the protein content of cultivated tartary buckwheat Qianku 2 is the highest (18.59%), and that of Ganku 1 (7.82%) the lowest. Moreover, there are significant differences of seed protein content among different buckwheat species and between the different buckwheat types (between common buckwheat and tartary buckwheat, between the diploid and the tetraploid, between the cultivated and the wild, and between the big-achene group and the small-achene group). The average seed protein content of F. giganteum is the highest in all tested species, up to 17.81% with the range of 13.91–21.27%, and that of F. callianthum the lowest, only 8.31%. This study was financed by the Natural Science Foundation of China (30270852, 30471116), Program for New Century Excellent Talents in University (NCET-2004-0913), Guizhou Key International Cooperation Project (Qianke Hewai G Zi 2005#400108), Guizhou Oversea Talent Project (Qianren Xiangmu Zizhu Hetong 2004#02), and Key Project of Students in Guizhou Normal University (GNU-SP-2005-SJXY-A01)     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.     

Interrelationships among Coconut (Cocos Nucifera L.) Accessions Using RAPD Technique

The genetic relationships among 33 coconut germplasm accessions were analyzed using RAPD markers. The germplasm accessions were collected from various coconut growing regions viz. South Asia (SA), South East Asia (SEA), South Pacific (SP), Atantic and America, and Africa. Forty-five random primers produced a total of 399 polymorphic markers. The Polymorphism Information Content (PIC) ranged from 0.031 to 0.392 and the Marker Index (MI) ranged from 6.28 to 0.031 among the primers. Based on the MI a set of 5, 10 and 15 informative and reproducible primers were identified. The mantel matrix correlation was calculated to compare the similarity matrices of a set of reproducible informative primers and global primers. There was significant correlation among the similarity matrices (r ≥ 0.50). The similarity matrix based on 399 polymorphic markers was used to construct the dendrogram to show the genetic relationship among the accessions. Similarity values ranged between 0.573 and 0.846. There was less genetic similarity (based on Jaccard's coefficient) among South Pacific and South East Asian accessions. The clustering pattern obtained in the present study was in agreement with the earlier reports based on RFLP, SSRs and AFLPs.     

RAPD polymorphisms in Aegilops geniculata Roth (Ae. ovata auct. non L.)

Genetic diversity of eighteen accessions of Ae. geniculata (2n=28; UUMM) was assessed using the random amplified polymorphic DNA (RAPD) technique. We optimized RAPD conditions including the template DNA, the concentration of AmpliTaq DNA polymerase Stoffel fragment, and MgCl₂ concentration for revealing polymorphisms. Thirty-eight decamer oligonucleotides were individually used as primers under optimized conditions. Seventeen of these primers produced polymorphic RAPDs among the 18 accessions of Ae. geniculata. Polymorphisms were recorded by noting presence or absence of an amplification product from the total genomic DNA. Comparisons of unique and shared amplification products of each pair of accessions were used to generate genetic similarity coefficients (GSCs). These GSCs were used to construct a phenogram using an unweighted pair-group method with arithmetical averages (UPGMA). The phenogram shows that RAPD data is useful in the measurement of genetic variation or similarity within a species. It also indicates that we can select eight or nine accessions of the eighteen accessions to maintain at least 80% genetic variability of the Chinese collection of Ae. geniculata. Address for correspondence: Dr X-Y. Zhang, USDA-ARS-FRRL, Utah State University, Logan, UT 84322-6300, who is visiting the USA under an agreement between USDA-ARS and CMA-CAAS on germplasm resources.     

Genetic variation among highly endangered <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) Pennell from Southern India as detected using RAPD analysis

Randomly amplified polymorphic DNA (RAPD) fingerprinting approach was applied to assess genetic diversity in different accessions of rejuvenating and intellect-promoting ancient ayurvedic medicinal herb Bacopa monnieri (L.) Pennell collected from four Southern Indian states, along with in vitro micropropagated samples maintained in the laboratory for 5 years. With the 10 analyzed primers, 110 distinct bands, in the size range of 250–870 bp were observed, and among which 14 (12.72%) were polymorphic. Among the random primers used, only OPD 02 generated highest percentage of polymorphism with 30.77. Whereas OPD 08 generated a maximum of 18 amplified bands, but with only 1% polymorphism. Cluster analysis done on the basis of similarity co-efficient generated from RAPD profiles indicated all the accessions were divided into two sub-groups based on genetic distances under one major cluster. Major cluster is only the lone loose group of the Bm.2 collected from Kerala (KER) and in vitro micropropagated plant (IVMP) maintained in the lab. The other sub-group consisted of Bm.1 and Bm.3 collected from Tamil Nadu (TN) and Andhra Pradesh (AP) respectively, which are genetically similar and showed similarity with accessions from Karnataka Bm.4 (KK). These two sub-groups were joined together at 0.66 genetic distance level. Overall, the levels of genetic similarity within the accessions varied from 0.24 to 0.80, the matrix ranged from 0.36 to 0.80, with a mean value of 0.68 indicating genetic similarity at low level.     

Characterization of genetic variation and relationships among Choix germplasm accessions using RAPD markers

Choix, a plant in the tribe Maydeae of the grass family, has been cultivated in Asia for several thousand years. It is a potential gene resource for improvement of other cereal crops because of its nutritional value and tolerance to stress. Genetic variation and relationships among 21 Choix lachryma-jobi L. accessions were characterized by random amplified polymorphic DNA (RAPD) markers. A total of 205 DNA fragments across all materials were amplified with 31 random primers, averaging 6.61 per primer. Among amplified fragments, 115 showed polymorphism averaging 3.71 per primer. Of amplified markers, 56.1% were polymorphic, indicating considerable variation at the DNA level among these accessions. Some fragments were accession-specific. Pair-wise genetic similarity (GS) among 21 accessions ranged from 0.809 to 0.301. The 21 accessions clustered into two major groups. Three exotic Choix accessions clustered together. Three other Choix accessions, collected from Guangxi, China, clustered into a cohesive subgroup. Four wild types of Choix clustered into the same subgroup. These results indicated that the classification by RAPD data reflected the differences in geographic origins and evolution in Choix.     

Low levels of genetic diversity detected by RAPD analysis in geographically distinct accessions of Bacopa monnieri

Brahmi (Bacopa monnieri) is atraditional Indian medicinal plant known for its natural nootropicaction of saponins present in large amount in its shoots. Acollection of 24 B.monnieri accessions from differentagro-climatic zones of India and an introduction from Malaysiamaintained in the field genebank at CIMAP was analysed for RAPDvariation. Among the 40 random primers tested, 29 primers generatedone or more polymorphic bands. The number of polymorphic bandsgenerated was primer dependent, ranging from 2 to maximum of 8.Similarity matrices were generated from the RAPD data on the basis ofNei's estimates of similarity indices and dendrograms wereconstructed based on UPGMA clustering. All the accessions were foundto be in the range of 0.8–1.0 of similarity, which isindicative of a narrow genetic base among the various accessions witha medium level of polymorphism. It was possible to differentiateindividual accessions, showing differences in morphological andgrowth properties at DNA level. The observed low levels of geneticvariation were attributed to interplay of sexual and vegetative modesof reproduction and similarity of local environments in habitats ofB. monnieri.     

A study of genetic diversity of sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) in Vietnam and Cambodia estimated by RAPD markers

Sesame (Sesamum indicum L.) is a traditional oil crop cultivated throughout South East Asia. To estimate the genetic diversity of this crop in parts at the region, 22 sesame accessions collected in Vietnam and Cambodia were analyzed using 10 RAPD markers. The 10 primers generated 107 amplification products of which 88 were polymorphic fragments (83%). Genetic diversity of all populations was H_t = 0.34 when estimated by Nei’s genetic diversity and species diversity was H′_sp = 0.513 when estimated by Shannon diversity index. Genetic distance ranged from 0.03 to 0.43, with a mean genetic distance of 0.23. The unweighted pair group method with arithmetic averages (UPGMA) cluster analysis for the 22 accessions divided the material in four groups. The dendrogram revealed a clear division among the sesame accessions based on their geographical region. Interestingly, some geographically distant accessions clustered in the same group, which might indicate the human factor involved in the spreading of sesame varieties. The high level of polymorphism shown suggests that RAPD techniques can also be useful for the selection of parents in sesame (Sesamum indicum L.) breeding program and for cultivar differentiation.     

Diversity among landraces of Indian snapmelon (<Emphasis Type="Italic">Cucumis melo</Emphasis> var. <Emphasis Type="Italic">momordica</Emphasis>)

Diversity among 36 snapmelon landraces, collected from 2 agro-ecological regions of India (9 agro-climatic sub-regions), was assayed using RAPD primers, morphological traits of plant habit and fruit, 2 yield-associated traits, pest and disease resistance and biochemical composition (TSS, ascorbic acid, titrable acidity). Typical differences among accessions were observed in plant and fruit characteristics and snapmelon germplasm with high titrable acidity and possessing resistance to downy mildew, Cucumber mosaic virus, Zucchini yellow mosaic virus, Papaya ringspot virus, Aphis gossypii and Meloidogyne incognita was noticed in the collection. RAPD based grouping analysis revealed that Indian snapmelon was rich in genetic variation and region and sub-region approach should be followed across India for acquisition of additional melon landraces. Accessions of var. agrestis and var. momordica clustered together and there was a separate cluster of the accessions of var. reticulatus. Comparative analysis of the genetic variability among Indian snapmelons and an array of previously characterized reference accessions of melon from Spain, Israel, Korea, Japan, Maldives, Iraq, Pakistan and India using SSRs showed that Indian snapmelon germplasm contained a high degree of unique genetic variability which was needed to be preserved to broaden the genetic base of melon germplasm available with the scientific community. N. P. S. Dhillon and Ranjana contributed equally to this work and are considered the first authors.     

Genetic diversity of <Emphasis Type="Italic">Nelumbo</Emphasis> accessions revealed by RAPD

Total 65 lotus accessions in genus Nelumbo mainly collected from China, were subjected to random amplified polymorphic DNA (RAPD) markers to estimate the genetic diversity and to test the genetic basis of the relationships between morphotypes and molecular markers. Seventeen primers generated a total of 195 highly reproducible and discernible loci, among which 173 were polymorphic. Percent polymorphism varied from 66.7 to 100 with an average of 88.72, and five primers out of them, OPC05, OPG10, OPN20, OPP09 and OPS17, showed 100% polymorphism. A relatively high genetic diversity was detected among all the samples with the similarity coefficient values ranging from 0.45 to 0.85, and Nei’s gene diversity (h) 0.30, and Shannon index (I) 0.46. The UPGMA dendrogram clustered 65 accessions in four clusters and the clustering pattern showed two groups, N. nucifera ssp. nucifera and those accessions related to the American lotus, and some special cultivars, landraces, hybrids and the American lotus. Principal Coordinate Analysis (PCA) further indicated that the genetic diversity of Nelumbo accessions was not evenly distributed, instead, was presented by a clustered distribution pattern. Similar to the results revealed by the dendrogram, two main groups representing the two subspecies of N. nucifera, as well as some special landraces, cultivars of Chinese lotus, the Japanese lotus and hybrids out of the two groups were obtained. Neither the UPGMA dendrogram nor the PCA analysis exhibited strict relationship with geographic distribution and morphotypes among the accessions.