Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship

Microsatellite amplification was performed on cassava (Manihot esculenta) and six other different species (all wild) of the Manihot genus. We used ten pairs of microsatellite primers previously developed from cassava, detecting 124 alleles in a sample of 121 accessions of the seven species. The number of alleles per locus ranged from four to 21 alleles, and allelic diversity was greater in the wild species than in cassava. Seventy-nine alleles, including unique ones, were detected in the wild species but were not found in the crop. The lower level of heterozygosity in some wild species probably resulted from a combination of fine-scale differentiation within the species and the presence of null alleles. Overall, microsatellite primers worked across the genus, but, with increasing genetic distance, success in amplifying loci tended to decrease. No accession of M. aesculifolia, M. carthaginensis, and M. brachyloba presented a banding pattern at locus Ga-140; neither did one appear for M. aesculifolia at locus Ga-13. Previous work with amplified fragment length polymorphism (AFLP) markers and this microsatellite analysis show that these three wild taxa are the most distant relatives of the crop, whereas the wild forms M. esculenta subsp. flabellifolia and M. esculenta subsp. peruviana appear to be the closest.     

AFLP analysis of relationships among cassava and other Manihot species

 Despite the worldwide importance of cultivated cassava (M. esculenta Crantz) its origin and taxonomic relationships with other species in the genus have not been clearly established. We evaluated a representative sample of the crop’s diversity and six wild taxa with AFLPs to estimate genetic relationships within the genus. Groupings of accessions of each species by data analysis corresponded largely with their previous taxonomic classifications. A mixed group, consisting of Manihot esculenta subsp. flabellifolia and M. esculenta subsp. peruviana, was most similar to cassava, while M. aesculifolia, M. brachyloba, and M. carthaginensis were more distant. Species-specific markers, which may be useful in germ-plasm classification or introgression studies, were suggested by the unique presence of AFLP products in samples of each of the three wild species. Heterogeneity of similarities among individuals of certain species suggested the existence of intraspecific gene pools, a hypothesis that was supported by morphological or ecogeographic evidence with varying degrees of success. Quantitative assessment of genetic diversity revealed greater homogeneity among cassava accessions than among itsclosest wild relatives. The demonstration of unique genetic diversity in the two M. esculenta subspecies and their genetic similarity to the crop supports the hypothesis that these materials may be the ancestors of cassava. Received: 4 November 1996 / Accepted: 20 December 1996     

AFLP analysis of African cassava (Manihot esculenta Crantz) germplasm resistant to the cassava mosaic disease (CMD)

Amplified fragment length polymophism was assessed in 20 land races and nine elite lines of cassava from Africa, resistant and susceptible to the cassava mosaic disease (CMD). Eleven accessions from a representative core collection from Latin America, previously studied by AFLPs, were included as a reference. AFLP data from all accessions was analyzed by both the unweighted pair group mean average (UPGMA) and multiple cluster analysis (MCA) methods of analysis. Genetic differentiation between clusters and the coefficient of genetic differentiation was also calculated. Results reveal a genetic divergence between African and Latin American accessions, although some overlap was found between them. African land races resistant to CMD, were also found to be genetically differentiated from susceptible land races and from resistant elite lines. AFLP analysis identified a considerable number of duplicates in the African accessions, suggesting a sizeable percentage of redundancy. A unique AFLP fragment, found in a relatively high frequency in African accessions, but absent in the Latin American accessions, was found to be associated with branching pattern by QTL mapping in an F1 progeny derived from African and Latin American parents. The likely source and the utility of the unique AFLP fragment in understanding the processes of genetic divergence in Africa is discussed. Received: 15 May 1999 / Accepted: 28 July 1999     

感病与抗病圭亚那柱花草遗传多样性的AFLP分析

圭亚那柱花草(Stylosanthes guianensts Swartz)原产中南美洲及非洲,是一种重要的热带豆科牧草,已在我国华南热带、南亚热带地区种植并利用.由胶孢炭疽菌(Colletotrichum gloeosporioides(Penz.)Sacc.)引起的炭疽病是柱花草的主要病害.采用扩增片段长度多态性(AFLP)技术分析了42个圭亚那柱花草品系的遗传多样性,同时对其抗病性进行了接种鉴定.从96个选择性引物对中筛选出较好的4个,分别对42个圭亚耶柱花草品系进行扩增,共获得出225条带,其中多态性带215条,平均多态性水平为95.5%,表现出高度的多态性.采用NTSYS-pc软件计算了品系间的遗传相似系数,其变化范围为0.31～0.95.根据非加权成对平均数法(uPGMA)进行聚类分析,建立了42个品系的聚类树系图,以所有品系的平均遗传相似系数0.48为阈值,共分为5类.主成分分析表明:第一主成分和第二主成分对全部品系间遗传变异的贡献率分别为56.04%和6.40%,并建立了品系间相互关系的二维图,各品系在二维图中的分布与UPGMA分类相吻合.抗病性鉴定结果表明:各品系对两种典型的病原菌的抗性有差异,其中抗病品系对两种病原菌的抗病相关系数达到0.904,表明抗病品系对两种病原菌有共同抗性.此外,抗病品系在UPGMA聚类中呈随机分布.这些结果表明,AFLP技术是分析圭亚那柱花草遗传多样性的有效方法.     

Genetic differentiation analysis of African cassava (Manihot esculenta) landraces and elite germplasm using amplified fragment length polymorphism and simple sequence repeat markers

Molecular‐marker‐aided evaluation of germplasm plays an important role in defining the genetic diversity of plant genotypes for genetic and population improvement studies. A collection of African cassava landraces and elite cultivars was analysed for genetic diversity using 20 amplified fragment length polymorphic (AFLP) DNA primer combinations and 50 simple sequence repeat (SSR) markers. Within‐population diversity estimates obtained with both markers were correlated, showing little variation in their fixation index. The amount of within‐population variation was higher for landraces as illustrated by both markers, allowing discrimination among accessions along their geographical origins, with some overlap indicating the pattern of germplasm movement between countries. Elite cultivars were grouped in most cases in agreement with their pedigree and showed a narrow genetic variation. Both SSR and AFLP markers showed some similarity in results for the landraces, although SSR provided better genetic differentiation estimates. Genetic differentiation (Fst) in the landrace population was 0.746 for SSR and 0.656 for AFLP. The molecular variance among cultivars in both populations accounted for up to 83% of the overall variation, while 17% was found within populations. Gene diversity (H_e) estimated within each population varied with an average value of 0.607 for the landraces and 0.594 for the elite lines. Analyses of SSR data using ordination techniques identified additional cluster groups not detected by AFLP and also captured maximum variation within and between both populations. Our results indicate the importance of SSR and AFLP as efficient markers for the analysis of genetic diversity and population structure in cassava. Genetic differentiation analysis of the evaluated populations provides high prospects for identifying diverse parental combinations for the development of segregating populations for genetic studies and the introgression of desirable genes from diverse sources into the existing genetic base.     

Genetic mapping of resistance to bacterial blight disease in cassava (Manihot esculenta Crantz)

Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is a major disease of cassava (Manihot esculenta Crantz) in Africa and South America. Planting resistant varieties is the preferred method of disease control. Recent genetic mapping of an F₁ cross (TMS 30572 × CM 2177–2) led to the construction of the first molecular genetic map of cassava. To better understand the genetics of resistance to CBB, we evaluated individuals of the F₁ cross for CBB resistance by controlled greenhouse inoculations and visually assessed symptoms on days 7, 15, and 30 days after inoculation, using a scale where 0 = no disease and 5 = maximum susceptibility. Five Xam strains were used: CIO-84, CIO-1, CIO-136, CIO-295, and ORST X-27. Area under the disease progress curve (AUDPC) was used as a quantitative measure of resistance in QTL analysis by single-marker regression. Based on the AUDPC values, eight QTLs (quantitative trait loci), located on linkage groups B, D, L, N, and X of the female-derived framework map, were found to explain 9–20% of the phenotypic variance of the crop’s response to the five Xam strains. With the male-derived framework map, four QTLs on linkage groups G and C explained 10.7–27.1% of the variance. A scheme to confirm the usefulness of these markers in evaluating segregating populations for resistance to CBB is proposed. Received: 20 September 1999 / Accepted: 30 December 1999     

Microsatellite variation in cassava (Manihot esculenta, Euphorbiaceae) and its wild relatives: further evidence for a southern Amazonian origin of domestication

Genetic variation at five microsatellite loci was used to investigate the evolutionary and geographical origins of cassava (Manihot esculenta subsp. esculenta) and the population structure of cassava's wild relatives. Two hundred and twelve individuals were sampled, representing 20 crop accessions, 27 populations of cassava's closest wild relative (M. esculenta subsp. flabellifolia), and six populations of a potentially hybridizing species (M. pruinosa). Seventy-three alleles were observed across all loci and populations. These data indicate the following on cassava's origin: (1) genetic variation in the crop is a subset of that found in the wild M. esculenta subspecies, suggesting that cassava is derived solely from its conspecific wild relative. (2) Phenetic analyses group cassava with wild populations from the southern border of the Amazon basin, indicating this region as the likely site of domestication. (3) Manihot pruinosa, while closely related to M. esculenta (and possibly hybridizing with it where sympatric), is probably not a progenitor of the crop. Genetic differentiation among the wild populations is moderately high (F:(ST) = 0.42, rho(ST) = 0.54). This differentiation has probably arisen primarily through random genetic drift (rather than mutation) following recent population divergence.     

Characterization of microsatellite markers in cassava based on microsatellite-AFLP technique

We developed molecular markers for cassava based on the microsatellite-amplified fragment length polymorphism (M-AFLP) technique. Twenty primer pairs were developed and used for the analysis of 48 samples of Manihot species, consisting of M. esculenta (33), M. esculenta ssp flabellifolia (3), M. chlorosticta (3), M. carthaginensis (3), M. filamentosa (3), and M. tristis (3). Nine microsatellite loci that were polymorphic among these Manihot species were identified, giving 32 polymorphic alleles and from two to seven alleles per locus. Unbiased and direct count heterozygosity varied from 0.0233 to 0.7924 and 0.0000 to 0.7083, respectively. There was significant deviation (P < 0.05) from Hardy-Weinberg equilibrium at five loci. Genotypic data from the Manihot species were subjected to genetic diversity analysis. We found that M. chlorosticta and M. esculenta ssp flabellifolia were the closest populations, while M. filamentosa and M. esculenta ssp flabellifolia were the most divergent. Considering within M. esculenta, the samples from Nigeria and Fiji were the most closely related, while those from Venezuela and of unknown origin were the most divergent. We conclude that the M-AFLP technique is an effective method for generating microsatellite markers that are useful for genetic diversity analysis in Manihot species.     

Genetic diversity of traditional South American Landraces of Cassava (Manihot esculenta Crantz): an analysis using microsatellites

The extent and structure of the genetic variability of traditional varieties of cassava (Manihot esculenta Crantz) have been little documented, despite considerable evidence for this crop? great varietal diversity in traditional agroecosystems. We used microsatellite markers to assess the genetic structure of traditional landraces of sweet and bitter cassava collected from five South American sites. As reference, we used a sample of 38 accessions from a world collection of cultivated cassava. For a total of 10 loci examined, we found 15 alleles that were not represented in this sample. Ten of these had been previously detected in wild Manihot species. The geographical structure of genetic variability was weak, but the genetic differentiation between bitter and sweet landraces was significant, suggesting that each form had evolved separately after domestication. Our results showed that traditional landraces form an important source of genetic diversity and merit more attention from managers of crop genetic resources.     

Comparative analysis of genetic variation among <Emphasis Type="Italic">Xanthomonas axonopodis pv manihotis</Emphasis> isolated from the western states of Nigeria using RAPD and AFLP

Xanthomonas axonopodis pv manihotis is the causal agent of cassava bacterial blight (CBB) worldwide. CBB disease is a major constraint to cassava cultivation, and losses can be extremely severe in regions where highly susceptible cultivars are grown. To develop an efficient disease management policy, the genetic diversity of the pathogens population must be known. There is dearth of information on the genetic diversity of X. axonopodis pv manihotis population in Nigeria. We used RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), a PCR-based technique, to characterize the X. axonopodis pv manihotis isolates from the western States of Nigeria. Thirteen strains Xam and 2 reference strains were tested with eight primers combination of AFLP and 4 RAPD primers. RAPD amplified DNA fragment data showed four major clusters at 80 % similarity coefficient level and two strains were not clustered by this analysis. Strains Kwa76A and Ond48A were also separated in the principal component analysis of the same data. Numerical analysis differentiated the AFLP patterns into four distinct clusters and grouped two strains separately at 66 % similarity. PCA assembly grouped the bacterial strains into 4 and one of the strains was singled out from the others. The two DNA analyses techniques seem to be complimentary to one another and informative on the genomic structure of Xam population in Western Nigeria. The genetic analysis presented here contributes to understanding of the Xam population structure in Western Nigeria.     

Using microsatellites,isozymes and AFLPs to evaluate genetic diversity and redundancy in the cassava core collection and to assess the usefulness of DNA-based markers to maintain germplasm collections

The cassava core collection was selected to represent, with minimum repetitiveness, the potential genetic diversity of the crop. The core (630 accessions) was chosen from the base collection (over 5500 accessions) on the basis of diversity of origin (country and geographic), morphology, isozyme patterns and specific agronomic criteria. To asses the genetic diversity of the core, 521 accessions were typed with four microsatellite loci. Allele diversity and frequency, and size variance of dinucleotide repeats (Rst statistic) were estimated. Microsatellite allele numbers and frequencies varied among countries: Colombia and Brazil had the largest number of different alleles across all loci. Mexico also had a high number, ranking fifth after Peru, Costa Rica and Venezuela (which tied). Unique alleles were present in accessions from Brazil, Colombia, Guatemala, Venezuela and Paraguay. A small number (1.34%) of potential duplicates were identified through isozyme and AFLP profiles. Thus, the present results indicated that traditional markers have been highly effective at selecting unique genotypes for the core. Future selections of cassava germplasm sets can be aided by DNA-based markers to ensure genetically representative, non-redundant samples. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hybridization in the section Mentha (Lamiaceae) inferred from AFLP markers

The amplified fragment length polymorphism (AFLP) method was used to evaluate genetic diversity and to assess genetic relationships within the section Mentha in order to clarify the taxonomy of several interspecific mint hybrids with molecular markers. To this end, genetic diversity of 62 Mentha accessions from different geographic origins, representing five species and three hybrids, was assessed. Three EcoRI/MseI AFLP primer combinations generated an average of 40 AFLP markers per primer combination, ranging in size from 50 to 500 base pairs (bp). The percentage of markers polymorphic ranged from 50% to 60% across all accessions studied. According to phenetic and cladistic analysis, the 62 mint accessions were grouped into two major clusters. Principal coordinates analysis separated species into well-defined groups, and clear relationships between species and hybrids could be described. Our AFLP analysis supports taxonomic classification established among Mentha species by conventional (morphological, cytological, and chemical) methods. It allows the assessment of phenetic relationships between species and the hybrids M. spicata and M. × piperita, largely cultivated all over the world for their menthol source, and provides new insights into the subdivision of M. spicata, based for the first time on molecular markers.     

Unmanaged sexual reproduction and the dynamics of genetic diversity of a vegetatively propagated crop plant, cassava (Manihot esculenta Crantz), in a traditional farming system

Occurrence of intervarietal or interspecific natural crosses has been reported for many crop plants in traditional farming systems, underlining the potential importance of this source of genetic exchange for the dynamics of genetic diversity of crop plants. In this study, we use microsatellite loci to investigate the role of volunteer seedlings (plants originating from unmanaged sexual reproduction) in the dynamics of genetic diversity of cassava (Manihot esculenta Crantz), a vegetatively propagated crop, in a traditional farming system in Guyana. A previous field study showed that farmers incorporate such plants into the germplasm for vegetative propagation, and that many of them are likely to be assigned by farmers to recognized varieties. Under strict vegetative propagation clonality of varieties is expected. The high proportion of polyclonal varieties observed suggests that incorporation of seedlings into the germplasm for propagation is a frequent event. The molecular variability assessed with microsatellite markers shows that there is high differentiation among heterozygous varieties, whereas populations of seedlings do not depart from the proportions expected under Hardy-Weinberg assumptions. Assignment of seedlings to a recognized variety on the basis of morphological similarity greatly increases genetic diversity within the variety. We argue that recombination and gene flow play a major role in the dynamics of genetic diversity of cassava in traditional farming systems. Documenting unmanaged sexual reproduction and its genetic consequences is a prerequisite for defining strategies of in situ conservation of crop plant genetic resources.     

Thai elite cassava genetic diversity was fortuitously conserved through farming with different sets of varieties

Many in situ conservation programs have been developed to preserve plant landrace diversity and to promote its sustainable utilization, but little is known about the effectiveness of the developed programs in conserving plant genetic diversity. We investigated the effectiveness of an unregulated (i.e., unplanned or open) conservation system maintained by Thai farmers in conserving Thai elite cassava (Manihot esculenta Crantz) varieties. Specifically, we compared genetic diversity of 266 cassava clones that were collected from 80 farms in eight provinces with 16 cassava landraces and varieties released since the 1970s through genotyping with 35 informative simple sequence repeat (SSR) markers. The SSR analysis revealed a large regional heterogeneity in cassava diversity, with a strong genetic differentiation of the assayed clones among the 80 farms (19.8 %) and across the eight provinces (11.8 %). Significant associations were also found between SSR variation and farm agro-ecological factors or some farming practices. However, there was no significant genetic differentiation (0.9 %) between the 266 farm clones and 16 reference varieties. These findings suggest that the Thai elite cassava genetic diversity was fortuitously conserved by the farmers through farming with different sets of varieties. Implications of these findings are discussed with respect to on-farm conservation of plant genetic resources.     

Screening of cassava (Manihot esculenta Crantz) accessions against Phytophthora palmivora induced tuber rot of cassava

Cassava (Manihot esculenta Crantz), is an important tropical tuber crop with global importance and plays a significant role in the food, nutritional and livelihood security of around 500 million people. In India, the low productivity of cassava attributes to the soil borne disease, particularly tuber rot caused by Phytophthora palmivora (Butl.) which is destructive and the attack is spreading in alarming rate in all the cassava growing regions causing heavy yield loss of more than 50%. Introduction of disease resistant varieties may alleviate the problem to a certain extent. This paper describes the screening procedures and findings on the disease resistant variety of cassava accession against tuber rot. Variety Sree Padmanabha imparted high resistance against tuber rot, while Sree Sahya was moderately resistant and all other accessions studied were found to be susceptible in in vitro and in field trials. In screening studies, a reproducible positive correlation was obtained between attached tubers in live plant with detached tubers which showed that detached tuber part can be used for the prediction of resistance in attached live plants of cassava for cultivar resistance. The procedure described here could be used as a simple, rapid and efficient method for screening of cassava accessions against tuber rot of cassava.     

Characterization of pathogenic and nonpathogenic strains of Xanthomonas axonopodis pv. manihotis by PCR-based DNA fingerprinting techniques

Strains of Xanthomonas axonopodis pv. manihotis (Xam) were characterized for pathogenicity and for DNA polymorphism using different PCR-based techniques. Using amplified restriction fragment length polymorphism (AFLP), strains were distinguished from each other and also from other Xanthomonas strains. Cluster analysis showed a high correlation between DNA polymorphism and pathogenicity. Four Xam strains were further analyzed using three PCR-based techniques, AFLP, AFLP-pthB and RAPD-pthB. Various primer combinations were used including primers specific to a Xam pathogenicity gene (pthB) along with RAPD or AFLP primers. The AFLP primer combinations EcoRI+T/MseI+A and EcoRI+T/MseI+T were the most efficient to discriminate among pathogenic and nonpathogenic Xam strains. Polymorphic bands were excised from the gel, amplified and cloned. Sequences analysis showed significant homology with bacterial pathogenicity island, genes involved in pathogenic fitness and regulators of virulence. Three cloned AFLP fragments were used as probes in DNA blot experiments and two of them showed significant polymorphism.     

Biological parameters of Bemisia tabaci (Gennadius) biotype B (Hemiptera: Aleyrodidae) on Jatropha gossypiifolia, commercial (Manihot esculenta) and wild cassava (Manihot flabellifolia and M. carthaginensis) (Euphorbiaceae)

Bemisia tabaci (Gennadius) is one of the most important pests of cassava in Africa and several countries of Asia due to the damage caused by direct feeding, the excretion of honeydew, and its capacity as a vector of cassava mosaic geminivirus. There is a general consensus that B. tabaci is a complex of morphologically indistinguishable populations with different biotypes. In the Americas, the polyphagous biotype B does not appear to feed on cassava. Recent studies indicate that it is possible, however, for biotype B to gradually adapt to cassava using phylogenetically related hosts. Therefore, the possibility that some wild species of cassava constitute intermediate hosts in the adaptation process may lead to the establishment of biotype B on commercial varieties of Manihot esculenta. In here, we evaluated Jatropha gossypiifolia, two wild species of cassava (Manihot flabellifolia and M. carthaginensis) and a commercial cassava variety (MCol 2063) as hosts of biotype B. The highest oviposition rate (2.7 eggs /two days) occurred on M. esculenta, although the development time (44 d) was the longest when compared to M. carthaginensis and J. gossypiifolia. About 60% of the population could reproduce on the wild cassava species vs. 55% on J. gossypiifolia and 27.5% on the commercial variety. Our data suggest that J. gossypiifolia is a suitable host and the wild species M. carthaginensis can constitute a potential intermediate host in the adaptation of biotype B to commercial varieties of cassava.     

Customisation of AFLP analysis for cassava varietal identification.

Amplified fragment length polymorphism (AFLP) markers were used in the characterization of eight cassava varieties. This nonradioactive AFLP system was customized in terms of the choice of restriction enzymes used and the selection of nucleotides added to the 3' end of primers. EcoRI/MseI and HindIII/MseI fragments generally gave monomorphic profiles while ApaI/TaqI fragments produced polymorphic profiles suggesting a genome with high G + C content. It was possible to identify the eight cassava varieties used in this study using CTG as selective bases at the TaqI primer. For cassava, the AFLP system provided a higher number of loci detected per run when compared to RAPD. The reliability accompanying AFLP analysis would thus make it suitable for the characterization of cassava varieties.     

Genetic diversity in colonial bentgrass (Agrostis capillaris L.) revealed by EcoRI-MseI and PstI-MseI AFLP markers.

Colonial bentgrass (Agrostis capillaris L.) is a potential source for genetic improvement of resistance to environmental stress and disease for other bentgrass species (Agrostis spp.). To conserve and study the existing genetic resources of colonial bentgrass for use in breeding, genetic diversity was investigated using amplified fragment length polymorphism (AFLP) markers. Included in this study were 22 accessions from US Department of Agriculture germplasm collected from 11 countries, in conjunction with 14 accessions from northern Spain and 3 commercial cultivars. Ten EcoRI-MseI and 6 PstI-MseI AFLP primer combinations produced 181 and 128 informative polymorphic bands, respectively. Cluster analysis of genetic similarity estimates revealed a high level of diversity in colonial bentgrass species with averages of 0.51 (EcoRI-MseI) and 0.63 (PstI-MseI). Greater genetic diversity was detected by the EcoRI-MseI AFLP primer combinations. A low but significant positive correlation (r = 0.44, p = 0.0099) between the 2 Jaccard similarity matrices was obtained by the Mantel test. Commercial cultivars of bentgrass showed a narrow genetic background. The assessment of genetic diversity among colonial bentgrass accessions suggested the potential value of the colonial bentgrass germplasm in turfgrass cultivar improvement.