Genetic variability of Coffea arabica L. accessions from Ethiopia evaluated with RAPDs

The genetic diversity of 50 wild and semi-wild accessions of the Coffea arabica L. germplasm collection, gathered by the FAO and ORSTOM missions to Ethiopia, and maintained in Colombia by CENICAFE, was evaluated with RAPD markers. The evaluation was carried out in two phases: In phase one, the polymorphism of 8 Ethiopian accessions of different geographic origin, plus the cultivated variety 'Caturra' was assessed with the RAPD technique with forty-two 10-mer oligonucleotides. In phase two, 51 accessions were assessed with a set of 5 polymorphic primers that reproduced, with a correlation of 95%, the groups generated by the 24 polymorphic primers found in phase one. Principal Coordinate Analysis of molecular data revealed that a closely related group consisting of 86% of the Ethiopian C. arabica accessions evaluated are significantly different from the Caturra variety and could be used in a genetic breeding initiative to increase the variability of cultivated varieties. The results also indicate that a larger polymorphism is present in the Colombian replica of FAO Ethiopian coffee germplasm collection than previously reported.     

基于简化基因组测序技术的甘薯HRM分子标记开发及其应用

目前甘薯中针对SNP位点的分子标记开发及应用的报道较少。为开发甘薯特异SNP分子标记,本研究利用简化基因组测序技术对甘薯种质材料进行测序,筛选稳定的SNP位点,将其开发为基于HRM技术的分子标记,并在甘薯种质材料中进行验证。通过对23个甘薯种质材料简化基因组测序数据的分析,共发现835 756个SNP多态性位点,筛选其中的3 650个含有SNP多态性位点的高质量测序片段,成功设计了134对引物;初步验证发现,22对(16.42%)引物没有扩增产物,15对(11.19%)引物扩增产物2个以上,36对(26.87%)引物的扩增产物没有差异。61对(45.52%)引物在8个样本中的扩增特异性好,而且样本之间有差异。最后筛选34对扩增多态性丰富的引物对52份甘薯种质材料进行扫描,发现材料间多态性介于27.27%~90.91%之间,平均多态性达到59.35%。聚类分析表明,参试52份甘薯种质材料之间的差异较小,多数材料与骨干亲本南瑞苕、徐薯18之间关系较近,国外引进甘薯材料和地方品种差异较大。建议在今后甘薯育种中尽量选用地方品种和国外甘薯品种,从而更好地拓宽甘薯遗传背景。本研究初步建立了基于简化基因组技术和HRM技术开发甘薯SNP分子标记的新思路,为今后甘薯SNP分子标记开发提供了参考。同时本研究开发的甘薯分子标记,丰富了甘薯分子标记类型,为甘薯研究者开展快速的分子标记研究提供了支持。     

Analysis of genetic diversity in a sweet potato (Ipomoea batatas L.) germplasm collection from Tanzania as revealed by AFLP

Sweet potato (Ipomoea batatas L.) is the fifth most important crop in the developing countries after rice, wheat, maize and cassava. The amplified fragment length polymorphism (AFLP) method was used to study the genetic diversity and relationships of sweet potato accessions in the germplasm collection of Sokoine University of Agriculture, Morogoro and Sugarcane Research Institute, Kibaha, Tanzania. AFLP analysis of 97 sweet potato accessions using ten primer combinations gave a total of 202 clear polymorphic bands. Each one of the 97 sweet potato accessions could be distinguished based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the un-weight pair-group method using arithmetic average. AFLP-based genetic similarity varied from 0.388 to 0.941, with a mean of 0.709. Cluster analysis using genetic similarity divided the accessions into two main groups suggesting that there are genetic relationships among the accessions. Principal Coordinate analysis confirmed the pattern of the cluster analysis. Analysis of molecular variance revealed greater variation within regions (96.19%) than among regions (3.81%). The results from the AFLP analysis revealed a relatively low genetic diversity among the germplasm accessions and the genetic distances between regions were low. A maximally diverse subset of 13 accessions capturing 97% of the molecular markers diversity was identified. We were able to detect duplicates accessions in the germplasm collection using the highly polymorphic markers obtained by AFLP, which were found to be an efficient tool to characterize the genetic diversity and relationships of sweet potato accessions in the germplasm collection in Tanzania.     

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.     

Determination of genetic variability by RAPD markers in cauliflower,cabbage and kale local cultivars from France

RAPD markers were used to analyse the genetic variability among a cole crop cultivar collection from France and to identify possible duplicate accessions for facilitating the germplasm multiplication. We surveyed 24 cauliflower, 24 cabbage and 48 kale populations using 62, 100 and 89 RAPD markers respectively on 40 seed bulk samples per accession. Genetic distances were calculated on the basis of these markers. Using the phylogenetic package PAUP we compared the genetic clusters obtained for the RAPD markers with the groups based on morphologic and agronomic data. For cauliflowers and cabbages a drastic selection by growers led to an extensive differentiation of morphologic and precocity traits related to geographic origin, which matches the grouping based on RAPD markers. Furthermore, RAPDs detected in some cases misclassification of accessions in the collection. Kales formed an homogeneous group probably due to extensive genetic exchanges leading to a continuous diversity. For the other crops, different horticultural types were separated in several subgroups by the RAPD markers suggesting different genetic origins. RAPD markers provide a rapid and informative approach to analyze the genetic variability in a collection. It can be applied to autogamous (spring cauliflower) or allogamous (winter cauliflower) crops, even in cases where the intra-population diversity is extensive (cabbages).     

Development and use of microsatellite markers for genetic diversity analysis of cañahua (<Emphasis Type="Italic">Chenopodium pallidicaule</Emphasis> Aellen)

Cañahua (Chenopodium pallidicaule Aellen) is a poorly studied, annual subsistence crop of the high Andes of South America. Its nutritional value (high in protein and mineral content) and ability to thrive in harsh climates make it an important regional food crop throughout the Andean region. The objectives of this study were to develop genetic markers and to quantify genetic diversity within cañahua. A set of 43 wild and cultivated cañahua genotypes and two related species (Chenopodium quinoa Willd. and Chenopodium petiolare Kunth) were evaluated for polymorphism using 192 microsatellite markers derived from random genomic cañahua sequences produced by 454 pyrosequencing of cañahua genomic DNA. Another 424 microsatellite markers from C. quinoa were also evaluated for cross-species amplification and polymorphism in cañahua. A total of 34 polymorphic microsatellite marker loci were identified which detected a total of 154 alleles with an average of 4.5 alleles per marker locus and an average heterozygosity value of 0.49. A cluster analysis, based on Nei genetic distance, clearly separated from wild cañahua genotypes from the cultivated genotypes. Within the cultivated genotypes, subclades were partitioned by AMOVA analysis into six model-based clusters, including a subclade consisting sole of erect morphotypes. The isolation by distance test displayed no significant correlation between geographic collection origin and genotypic data, suggesting that cañahua populations have moved extensively, presumably via ancient food exchange strategies among native peoples of the Andean region. The molecular markers reported here are a significant resource for ongoing efforts to characterize the extensive Bolivian and Peruvian cañahua germplasm banks, including the development of core germplasm collections needed to support emerging breeding programs.     

Genetic variation in radish (Raphanus sativus L.) germplasm from Pakistan using morphological traits and RAPDs

Genetic diversity of 30 radish (Raphanus sativus L.) accessions was investigated at the phenotypic level with morphological characters and at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Thirty-six morpho-physiological traits were recorded from seedling stage to harvest. The 31 primers used generated 202 RAPD bands, of which 158 (78.2%) were polymorphic. Multivariate procedures were used to classify the germplasm on the basis of phenotypic traits and RAPD fragments. Dendrograms were generated for the Euclidean distance from the morphological data and the Nei's genetic distance from the RAPD markers. Phenotypically, all the accessions were classified into four major groups corresponding to the different forms of cultivated radish. The morphological diversity existing within each of these groups suggested that they should be discriminated into the three botanical convarieties, sativusT (large-rooted), caudatus (pod-type) and oleifer (oilseed-type). Clustering of the accessions did not show any pattern of association between the morphological characters and the collection sites. Instead, landrace groups were associated with their morphological similarities and horticultural uses. On the other hand, the intra-specific genetic relationships of several accessions based on RAPD analysis were related primarily to their collection sites rather than to their phenotypic affinities. The level of polymorphism exhibited by the various convarieties could be exploited in genetic mapping populations to tag economically important traits. These genotypes also could serve as a useful germplasm source for root, leaf, pod and seed. This preliminary study of traditional radish landraces from Pakistan provides useful information regarding their horticultural potential.     

Evaluation of the discriminance capacity of RAPD, isoenzymes and morphologic markers in apple (Malus x domestica Borkh.) and the congruence among classifications

Twenty-one apple accessions were characterised and classified taxonomically with randomly amplified polymorphic DNA (RAPD). The results were compared with morphological and isoenzymatic classifications of the same material by calculating the congruence between similarity matrices. The results confirmed the greater discrimination capacity of RAPD compared with isoenzymes, although the taxonomic clusters obtained with both types of markers were congruent. The genetic relationships calculated using RAPD and isoenzymes showed little congruence with the morphological relationships among accessions. Isoenzymatic markers are useful in germplasm banks to evaluate the diversity of newly introduced accessions. The use of RAPD markers are especially beneficial to discriminate between material that is genetically similar, to evaluate genetic variability within a collection and to choose the components of the core collection.     

Genetic diversity of potato landraces from northwestern Argentina assessed with simple sequence repeats (SSRs)

Andean potato varieties are cultivated in the northwest of Argentina and constitute the most important staple food for the local farmers. The genetic diversity of 155 accessions conserved at the Genebank of Balcarce (INTA) was tested using four microsatellites. Three commercial potato varieties of Tuberosum group and one accession of Curtilobum group were used as outgroups. The presence of bands was scored for each microsatellite and for each accession and the data were analysed by principal coordinate analysis. The polymorphism information content was obtained for each molecular marker from banding patterns. Analysis of molecular variance was carried out with a variable number of accessions for each landrace, from different departments and sites within departments. More than one genotype was detected in the majority of the potato landraces. Some accessions within each landrace did not differentiate. AMOVA revealed that most of the genetic variation occurred among sites within departments and among local varieties. These findings are discussed considering the agricultural practices carried out in the Andean farming system.     

Application of random amplified polymorphic DNA to study genetic diversity in Paspalum scrobiculatum L. (Kodo millet, Poaceae)

Summary Genetic diversity and patterns of geographic variation among collections of Paspalum scrobiculatum (kodo millet) and P. polystachyum were studied using molecular markers generated through the random amplified polymorphic DNA (RAPD) method. A high level of polymorphism in RAPD markers was observed among the individual accessions, demonstrating the high genetic diversity of the crop. The markers obtained from the RAPD method were analyzed with the cluster analysis, principal coordinates and minimum spanning tree methods. Three major groups were resolved, one representing the African accessions, and two for the Indian accessions. The accessions of the north African kodo millet and P. polystachyum (considered conspecific with P. scrobiculatum) were quite distinct. The Australian kodo millet showed higher affinity to the African types. The study demonstrated that the RAPD technique can be applied to resolving degrees and patterns of genetic variation at the population and species levels, identifying cultivars, and defining gene pools of this crop.     

Analysis of the contribution of Mesoamerican and Andean gene pools to European common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) germplasm and strategies to establish a core collection

Common bean (Phaseolus vulgaris L.) was introduced in Europe from both Mesoamerican and Andean centres of origin. In this study, a collection including 544 accessions from all European regions showed that the Andean phaseolin types ‘T’ (45.6%) and ‘C’ (30.7%) prevailed over the Mesoamerican ones ‘S’ (23.7%), and accessions with cuboid seed shape (34.9%), maroon coat darker colour seed (44.3%), uniform seed colour (69.6%) were the most frequent. European accessions with phaseolin ‘S’ showed a significantly larger average seed size compared to those from America in the same phaseolin class while those presenting ‘T’ and ‘C’ phaseolin did not. This suggests that, during crop expansion in Europe, sampling or selection favoured the large-seeded races within the Mesoamerican ‘S’ gene pool or, possibly, introgression from Andean germplasm did occur. A core collection was developed using sampling approaches based on the information available in the genebank databases and on phaseolin patterns. Four sampling strategies were used: simple random sampling, and three random-stratified samplings, by logarithm of frequency of accessions by country, by European region, and by phaseolin pattern, respectively. Two sampling strategies resulted in core collections significantly different for phaseolin electrophoretic patterns from the whole collection. Stratification by phaseolin patterns increased the frequency of ‘S’ types (‘C’ type = 33%, ‘T’ type = 5.7% and ‘S’ type = 31.3%). The core collections were validated using seven seed characters, and no significant difference was observed in all strategies. This first developed European bean core collection will help to assess the contribution of the two American gene pools to the European germplasm and their relative importance for breeding purposes.     

Analysis of genetic diversity in Indian taro [Colocasia esculenta (L.) Schott] using random amplified polymorphic DNA (RAPD) markers

Taro [Colocasia esculenta (L.) Schott] germplasm accessions collected from different parts of India were subjected to RAPD (Random Amplified Polymorphic DNA) analysis to assess the genetic diversity prevalent and also to test the genetic basis of morphotypic classification. Thirteen random decamer primers out of the 22 tested were used to analyse 32 taro accessions belonging to 28 morphotypes. Three out of these thirteen primers analysed showed 100 per cent polymorphism. Per cent polymorphism varied from 60 to 100 among the polymorphic primers. High genetic diversity was revealed as the similarity coefficient values ranged from 0.50 to 0.98. No two accessions analysed in the present study showed a similarity coefficient value of one thereby indicating their distinctness and presence of high genetic diversity in Indian taro germplasm. Dendrogram obtained from UPGMA analysis grouped 32 accessions in four clusters and three accessions were placed as outliers. Clustering pattern did not show any strict relationship with geographical distribution, morphotype classification and genotypic diversity. Further, accessions classified, as belonging to the same morphotypic group did not always cluster together. Presence of a very close genepool of the wild, weedy and cultivated forms with extreme levels of phenotypic and genotypic variation is suggested as the reason for high genetic diversity reported. Usefulness of DNA markers such as RAPD in characterising and assessing the genetic diversity in Indian taro germplasm is hereby demonstrated.     

RAPD markers in the analysis of genetic diversity among common bean germplasm from Central Himalaya

A set of 99 common bean germplasm collected from central Himalaya was investigated for their genetic variability using random amplified polymorphic DNA (RAPD) markers. Ten oligonucleotide primers, selected from 60 initially screened, generated 123 amplicon products. Of these, two amplicons were shared by all the accessions whereas 112 were polymorphic at least in two pair wise comparison. Nine unique bands identified were as low as 0.32 kb M.W. to as high as 3.5 kb and were confined to eight collections. All primers produced polymorphic amplicons though the extent of polymorphism varied with each primer. The primer OPF-17 was found to be most powerful and efficient as it generated a total of 17 bands of which 15 were polymorphic. RAPD markers data were analysed statistically using NTSYSpc.2.02e software and a dendrogram was generated using Jaccard’s similarity coefficient. The similarity coefficient values varied from 0.19 to 0.91. Grouping analysis revealed the categorization of 99 germplasm into 12 major branches with different level of similarity. Three branches namely branch 2, 3 and 5 out of 12 had only one accession. Branch 1 which consisted of three accessions was the most divergent as revealed by Jaccard’s similarity coefficient. Branching pattern of the accessions did not show any correlation with morphological data or altitudinal alignment of the accessions.     

Assessment of genetic diversity in Solanum trilobatum L., an important medicinal plant from South India using RAPD and ISSR markers

Solanum trilobatum L. is an Indian medicinal plant containing rich amount of steroidal glyco-alkoloids that can be used as precursor for commercial steroid production. Two efficient marker systems such as Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were used for the first time to assess the genetic diversity across 14 S. trilobatum accessions obtained from five South Indian states. Twenty out of 60 RAPD primers generated 189 distinct bands of which 160 were polymorphic with an average of 8 polymorphic bands per primer. A maximum of up to 15 fragments were amplified with an average of 9.45 bands per primer and the amplicons varied in size between 100 and 3,000 bp. The percentage of polymorphism ranged from 55.5 to 100, with an average of 84.6. ISSR profiling using 7 out of 20 primers amplified 83 bands and the number of amplified fragments varied from 2 to 16 with a size range of 200–1,800 bp. Totally 72 polymorphic bands were obtained using 7 ISSR primers at an average of 10.28 polymorphic bands per primer. Polymorphism percentage varied from 50 to 100 among the selected accessions resulting in an average percentage of polymorphism of 86.7. The PIC values ranged from 0.49 to 0.93 for RAPD and 0.16 to 0.90 for ISSR primers. The study pointed out that ISSR markers were more efficient than RAPD markers in evaluating the degree of genetic variation in S. trilobatum. The UPGMA cluster analysis grouped all Tamil Nadu accessions in one cluster and other state accessions in another cluster. The Principal component analysis also substantiates this clustering pattern. Thus the phylogenetic relationship and a high genetic variation revealed in the present study could provide a baseline data for conservation and improvement of this plant in future. Also the molecular markers identified in this study will be helpful in authentication of this species to prevent adulteration in herbal medicine.     

Genetic diversity of 38 spinach (<Emphasis Type="Italic">Spinacia oleracea</Emphasis> L.) germplasm accessions and 10 commercial hybrids assessed by TRAP markers

Target region amplification polymorphism (TRAP) markers were used to assess genetic variability among 38 germplasm accessions and 10 commercial hybrids of spinach (Spinacia oleracea L.), an economically important leafy vegetable crop in many countries. Germplasm accessions with different geographic origins and 10 commercial hybrids were examined. For assessing genetic diversity within accessions, DNA was extracted from 12 individual seedlings from six germplasm accessions and two hybrids. A relatively high level of polymorphism was found within accessions based on 59 polymorphic TRAP markers generated from one fixed primer derived from the Arabidopsis-like telomere repeat sequence and two arbitrary primers. For evaluating interaccession variability, DNA was extracted from a bulk of six to 13 seedlings of each accession. Of the 492 fragments amplified by 12 primer combinations, 96 (19.5%) were polymorphic and discriminated the 48 accessions from each other. The average pair-wise genetic similarity coefficient (Dice) was 57.5% with a range from 23.2 to 85.3%. A dendrogram indicated that the genetic relationships among the accessions were not highly associated with the geographic locations in which the germplasms were collected. The seven commercial hybrids were grouped in three separate clusters, suggesting that the phenotype-based breeding activities tended to reduce the genetic variability. This preliminary study demonstrated that TRAP markers are effective for fingerprinting and evaluating genetic variability among spinach germplasms. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Characterization of <Emphasis Type="Italic">Prosopis cineraria</Emphasis> (L.) Druce germplasm with suitable horticultural traits from the hot arid region of Rajasthan,India

Sixteen germplasm accessions of Prosopis cineraria with suitable horticultural traits were identified from north-western Rajasthan, India, propagated clonally by budding on seedling rootstock and maintained in the field gene bank. Morphological characterization of seven-year-old trees of these accessions by 21 traits indicated a lot of variation among the accessions tested. Higher number of flowers per raceme was found in accession CIAH/K2, higher width of ripened pod in CIAH/K5, higher number of seeds per pod in CIAH/K12 and a higher weight of seed per pod in CIAH/K6. Overall, CIAH/K16 was found to be a superior genotype for most of the useful traits. High significant positive correlation was obtained with traits useful for horticultural values. Out of 62 random decamer primers for random amplification (RAPD) reaction, and four minisatellite core sequence for direct amplification of minisatellite DNA (DAMD) screened with these accessions, 12 RAPD and 2 DAMD primers were found polymorphic. Average polymorphism resolved by these markers among the accessions was 93.2%. Genetic diversity revealed by Jaccard’s co-efficient was between 0.11 and 0.77, and four major clusters were identified among these accessions by phylogenetic analysis using NTSYSpc-2.02e software. This study shows the existence of high genetic diversity within these accessions.     

Microsatellites and RAPD Markers to Study Genetic Relationships Among Cowpea Breeding Lines and Local Varieties in Senegal

Genetic diversity in local cowpea varieties and breeding lines from Senegal were studied using random amplified polymorphic DNA (RAPD) and microsatellite (SSR) techniques. Among the 61 RAPD primers used, twelve show polymorphism. Fifteen of the 30 microsatellite primer pairs were polymorphic, detecting one to nine alleles per locus. The RAPD and SSR data were analyzed both separately and in combination to assess relationships among genetic lines. Although RAPD provided information on levels of genetic diversity, microsatellite markers are most effective in determining the relationship among cowpea accessions and varieties. The SSR results support the genetic diversification of cowpea in Senegal and underscore their potential in elucidating patterns of germplasm diversity of cowpea in Senegal. An erratum to this article is available at .     

Development of Core Collection in Pigeonpea [<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millspaugh] using Geographic and Qualitative Morphological Descriptors

Pigeonpea is an important pulse crop grown by smallholder farmers in the semi-arid tropics. Most of the pigeonpea cultivars grown to date are selections from the landraces, with a narrow genetic base. With the expansion of the crop to newer areas, problems of local importance are to be addressed. Hence, an economically feasible and faster germplasm evaluation mechanism, such as a core collection, is required. This article describes the development of core collection from 12,153 pigeonpea accessions collected from 56 countries and maintained at ICRISAT, Patancheru, India. The germplasm accessions from 56 countries were placed under 14 clusters based primarily on geographic origin. Data on 14 qualitative morphological traits were used for cluster formation by Ward’s method. From each cluster ≈10% accessions were randomly selected to constitute a core collection comprising 1290 accessions. Mean comparisons using Newman–Keuls test, variances’ comparisons by Levene’s test, and comparison of frequency distribution by χ²-test indicated that the core collection was similar to that of the entire collection for various traits and the genetic variability available in the entire collection is preserved in the core collection. The Shannon–Weaver diversity index for different traits was also similar for both entire and core collection. All the important phenotypic associations between different traits available in the entire collection were preserved in the core collection. The core collection constituted in the present study facilitates identification of useful traits economically and expeditiously for use in pigeonpea improvement.