Assessment of genetic diversity in Azadirachta indica using AFLP markers

 Genetic diversity was estimated in 37 neem accessions from different eco-geographic regions of India and four exotic lines from Thailand using AFLP markers. Seven AFLP selective primer combinations generated a total of 422 amplification products. The average number of scorable fragments was 60 per experiment, and a high degree (69.8%) of polymorphism was obtained per assay with values ranging from 58% to 83.8%. Several rare and accession-specific bands were identified which could be effectively used to distinguish the different genotypes. Genetic relationships within the accessions were evaluated by generating a similarity matrix based on the Jaccard index. The phenetic dendrogram generated by UPGMA as well as principal correspondence analysis separated the 37 Indian genotypes from the four Thai lines. The cluster analysis indicated that neem germplasm within India constitutes a broad genetic base with the values of genetic similarity coefficient ranging from 0.74 to 0.93. Also, the Indian genotypes were more dispersed on the principal correspondence plot, indicating a wide genetic base. The four lines from Thailand, on the other hand, formed a narrow genetic base with similarity coefficients ranging from 0.88 to 0.92. The lowest genetic similarity coefficient value (0.47) was observed between an Indian and an exotic genotype. The level of genetic variation detected within the neem accessions with AFLP analysis suggests that it is an efficient marker technology for delineating genetic relationships amongst genotypes and estimating genetic diversity, thereby enabling the formulation of appropriate strategies for conservation and tree improvement programs. Received: 20 October 1998 / Accepted: 28 November 1998     

Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers

To meet various breeding objectives and to conserve the existing genetic resources of mulberry for future use, the present study was undertaken to investigate the amount of genetic diversity and to establish the relationships between mulberry genotypes using fluorescence-based AFLP markers. Genetic diversity was estimated in 45 mulberry accessions from different eco-geographic regions of Japan and other parts of the world. Five primer combinations amplified an average of 110 AFLP markers per primer combination, ranging in size from 35 to 500 bp. A high degree of polymorphism was revealed by these combinations that ranged from 69.7 to 82.3% across all the genotypes studied. Several rare genotype-specific bands were also identified which could be effectively utilized to distinguish different genotypes. The wide range in genetic similarity coefficients (0.58–0.99) indicated that the mulberry germplasm collection represents a genetically diverse popu-lation. The phenetic dendrogram generated by the UPGMA method grouped 45 accessions into four major clusters, which was in agreement with the results from conventional methods. Clustering of some genotypes into strictly separate groups was not readily apparent and no clear interrelationships could be depicted, in spite of their different geographic origin. In addition, AFLP analysis provided sufficient polymorphism for DNA typing and contributed additional insights into the genetic structure of the mulberry germplasm. These results will help in the formulation of appropriate strategies for conservation and variety improvement in mulberry, for which little or no knowledge of genetic diversity is currently available. Received: 30 December 1999 / Accepted: 14 March 2000     

AFLP analysis of the phenetic organization and genetic diversity of Vigna unguiculataL. Walp. reveals extensive gene flow between wild and domesticated types

Amplified fragment length polymorphisms (AFLPs) were used to evaluate genetic relationships within cowpea [Vigna unguiculata (L.) Walp.] and to assess the organization of its genetic diversity. Nei’s genetic distances were estimated for a total of 117 accessions including 47 domesticated cowpea (ssp. unguiculata var. unguiculata), 52 wild and weedy annuals (ssp. unguiculata var. spontanea), as well as 18 perennial accessions of the wild subspecies pubescens, tenuis and alba. AFLP variation was also used to study genetic variation among and within domesticated and wild accessions based on their geographical origin (western, eastern and southern Africa). Wild annual cowpea (var. spontanea) (H _T =0.175) was more diverse than domesticated cowpea (H _T =0.108). Wild cowpea was more diverse in eastern (H _S =0.168) than in western Africa (H _S =0.129), suggesting an eastern African origin for the wild taxon. The AFLP data were consistent with earlier findings of a unique domestication event in cowpea in the northern part of the continent and suggested that domestication in eastern or southern Africa was unlikely. It did not allow a more precise localization of domestication due to extensive gene flow between wild and domesticated forms that has led to a large crop-weed complex distributed over the entire African continent. In addition, wild materials from northeastern Africa are still lacking. Overall, the superiority of the AFLP technique over isozymes resided in its ability to uncover variation both within domesticated and wild cowpea, and should be a powerful tool once additional wild material becomes available. Received: 11 September 2000 / Accepted: 14 June 2001     

Genetic structure of a Lima bean base collection using allozyme markers

 Genetic diversity and structure within a Lima bean (Phaseolus lunatus L.) base collection have been evaluated using allozyme markers. The results obtained from the analysis of wild and cultivated accessions confirm the existence of Andean and Mesoamerican gene pools characterised by specific alleles. Wild and cultivated accessions of the same gene pool are grouped. The Andean natural populations have a very limited geographic distribution between Ecuador and northern Peru. The Mesoamerican wild form extends from Mexico up to Argentina through the eastern side of the Andes. Andean and Mesoamerican cultivated accessions of pantropical distribution contribute substantially to the genetic diversity of the Lima bean base collection. Population genetic parameters, estimated from allozymes, confirmed the predominant selfing mating system of the Lima bean. The selfing mating system, the occurrence of small populations, and low gene flow lead to an interpopulation gene diversity (D_ST=0.235) higher than the intrapopulation gene diversity (H_S=0.032). On the basis of the results, guidelines are given to preserve and exploit the genetic diversity of this threatened species. The results also confirm the independent domestication of the Lima bean in at least two centres, one of which is located at medium elevation in the western valleys of Ecuador and northern Peru. Received: 3 June 1997 / Accepted: 17 June 1997     

Evaluation of genetic variation in the daylily (Hemerocallis spp.) using AFLP markers

The daylily (Hemerocallis spp.) is one of the most economically important ornamental plant species in commerce. Interestingly, it is also one of the most heavily bred crops during the past 60 years. Since the American Hemerocallis Society began acting as the official registry of daylily cultivars in 1947, more than 40 000 registrations have been processed. In order to determine the effects of intensive breeding on cultivar development, and to study relationships among different species, genetic variation in the daylily was estimated using AFLP markers. Nineteen primary genotypes (species and early cultivars) and 100 modern cultivars from different time periods were evaluated using 152 unambiguous bands (average 79% polymorphism rate) derived from three AFLP primer combinations. Overall, pairwise similarity estimates between entries ranged between 0.618 and 0.926 (average=0.800). When comparing cultivar groups from different time periods (1940–1998), genetic similarity was initially increased, compared to the primary diploid genotypes, remained constant from 1940 to 1980, and then steadily increased as breeding efforts intensified and hybridizers began focusing on a limited tetraploid germplasm pool derived by colchicine conversion. Among modern (1991–1998) daylily cultivars, genetic similarity has increased by approximately 10% compared to the primary genotypes. These data were also used to evaluate recent taxonomic classifications among daylily species which, with a few minor exceptions, were generally supported by the AFLP data. Received: 15 March 2000 / Accepted: 13 June 2000     

Pollen flow and the intrapopulation genetic structure of Heloniopsis orientalis on the forest floor as determined using microsatellite markers

Pollenflow, seedling dynamics, and the genetic structure of Heloniopsis orientalis (Thunb.) C. Tanaka (Liliaceae) were analyzed as part of an effort to understand the reproduction system of perennial herbs foundon the forest floor by means of microsatellite markers. It was possible to assign paternity to all offspring within this population by paternity analysis. Although no ordinary pollinator visits were observed during the flowering period, effective pollen flow occurred throughout the population. It appears that low frequencies of pollinator visits nonetheless permitted adequate pollen flow. Studies of seedling dynamics suggested restricted recruitment by means of seed. Gene flow in the present population seemed to depend mainly on pollen. Local genetic structuring caused by seeds falling near the mother plant and by vegetative reproduction was detected by means of spatial autocorrelation. Three main modes of development of genetic structure were found for this species: (1) asexual reproduction by adventitious buds, which was dominant (up to 20 cm from the parent plants); (2) sexual reproduction by seeds that fell from flower stalks (up to 60 cm from the parent plants); and (3) pollen that dispersed over a wide area (throughout the population). Received: 13 January 2000 / Accepted: 21 March 2000     

Assessment of genetic diversity within and among germplasm accessions in cultivated sorghum using microsatellite markers

Microsatellite markers are increasingly being used in crop plants to discriminate among genotypes and as tools in marker-assisted selection. Here we evaluated the use of microsatellite markers to quantify the genetic diversity within as well as among accessions sampled from the world germplasm collection of sorghum. Considerable variation was found at the five microsatellite loci analysed, with an average number of alleles per locus equal to 2.4 within accessions and 19.2 in the overall sample of 25 accessions. The collection of sorghum appeared highly structured genetically with about 70% of the total genetic diversity occurring among accessions. However, differentiation among morphologically defined races of sorghum, or among geographic origins, accounted for less than 15% of the total genetic diversity. Our results are in global agreement with those obtained previously with allozyme markers. We were also able to show that microsatellite data are useful in identifying individual accessions with a high relative contribution to the overall allelic diversity of the collection. Received: 10 August 1999 / Accepted: 27 August 1999     

Construction of a genetic linkage map for roses using RAPD and AFLP markers

A segregating population of diploid rose hybrids (2n = 2x = 14) was used to construct the first linkage maps of the rose genome. A total of 305 RAPD and AFLP markers were analysed in a population of 60 F₁ plants based on a so-called ”double-pseudotestcross” design. Of these markers 278 could be located on the 14 linkage groups of the two maps, covering total map lengths of 326 and 370 cM, respectively. The average distances between markers in the maps for 93/1–117 and 93/1–119 is 2.4 and 2.6 cM, respectively. In addition to the molecular markers, genes controlling two phenotypic characters, petal number (double versus single flowers) and flower colour (pink versus white), were mapped on linkage groups 3 and 2, respectively. The markers closest to the gene for double flowers, Blfo, and to the gene for pink flower colour, Blfa, cosegregated without recombinants. The maps provide a tool for further genetic analyses of horticulturally important genes as, for example, resistance genes and a starting point for marker-assisted breeding in roses. Received: 22 September 1998 / Accepted: 12 March 1999     

Assessment of the genetic diversity and genetic relationships of Lilium in China using ISSR markers

China is a center of natural distribution and diversity of genus Lilium around the world. In the study, the genetic diversity and genetic relationships of Lilium in China were analyzed by inter-simple sequence Repeat (ISSR) markers. The 6 highly polymorphic ISSR primers were selected to amplify the 20 Lilium species. The results showed that a total of 114 DNA bands were amplified, all of which were polymorphic loci (P = 100%), the effective number of alleles (Ne) was 1.2753, and the difference value between observed number of alleles (Na) and effective number of alleles (Ne) was 0.7247, and the Nei's genetic diversity (H) was 0.2048, and the Shannon's information index (I) was 0.3503. These results indicated that there is significant genetic difference among Lilium species in China. Taking the average genetic similarity coefficient (Gs) 0.5313 as the threshold, the 20 tested Lilium species were clustered into 5 groups, which was not entirely consistent with traditional clustering by morphological traits. The results obtained from this study can provide a reference for the molecular study of Lilium germplasm resources.     

The use of microsatellite markers for detection of genetic diversity in barley populations

 A barley lambda-phage library was screened with (GA)n and (GT)n probes for developing microsatellite markers. The number of repeats ranged from 2 to 58 for GA and from 2 to 24 for GT. Fifteen selected microsatellite markers were highly polymorphic for barley. These microsatellite markers were used to estimate the genetic diversity among 163 barley genotypes chosen from the collection of the IPK Genebank, Germany. A total of 130 alleles were detected by 15 barley microsatellite markers. The number of alleles per microsatellite marker varied from 5 to 15. On average 8.6 alleles per locus were observed. Except for GMS004 all other barley microsatellite markers showed on average a high value of gene diversity ranging from 0.64 to 0.88. The mean value of gene diversity in the wild forms and landraces was 0.74, and even among the cultivars the gene diversity ranged from 0.30 to 0.86 with a mean of 0.72. No significant differences in polymorphism were detected by the GA and GT microsatellite markers. The estimated genetic distances revealed by the microsatellite markers were, on average , 0.75 for the wild forms, 0.72 for landraces and 0.70 among cultivars. The microsatellite markers were able to distinguish between different barley genotypes. The high degree of polymorphisms of microsatellite markers allows a rapid and efficient identification of barley genotypes. Received: 26 November 1997 / Accepted: 19 January 1998     

Towards a saturated sorghum map using RFLP and AFLP markers

 A near-saturated sorghum genetic linkage map was produced using RFLP, AFLP and morphological markers. First a composite, essentially RFLP-based genetic linkage map was obtained from analyses of two recombinant inbred populations. This map includes 343 loci for 11 linkage groups spanning 1352 cM. Since this map was constructed with many previously mapped heterologous probes, it offers a good basis for synteny studies. Separately, an AFLP map was obtained from the analysis of 168 bands revealed from 12 primer pair combinations. It includes 137 loci for 11 linkage groups spanning 849 cM. Taking into account the different data sets, we constructed a combined genetic linkage map including 443 loci spanning 1899 cM. Two main features are to be noted: (1) the distribution of AFLPs along the genome is not uniform; (2) an important stretching of the former core map is induced after adding the AFLPs. Received: 10 May 1998 / Accepted: 13 July 1998     

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     

Evaluating genetic relationships between indigenous coconut (Cocos nucifera L.) accessions from Sri Lanka by means of AFLP profiling

 PCR-based DNA profiling of coconut palms indigenous to Sri Lanka was conducted using amplified fragment length polymorphism (AFLPs). A total of 322 amplification products were generated from the 42 genotypes with eight pairs of primers (EcoRI and MseI). Overall most variation was detected in the tall (Typica) rather than the intermediate (Aurantiaca) and dwarf (Nana) forms. A hierarchical analysis of molecular variance (AMOVA) was used to quantify and partition levels of variability into between- and within-form components. This revealed that for the inbreeding dwarf and intermediate forms most variation was observed between, rather than within, forms. In contrast, the outbreeding tall forms exhibited as much variation within as between forms. These observations have important implications for the maintenance and collection of coconut germplasm. This study also provided insights into the genetic (as opposed to phenotypic) relatedness of coconut accessions. Morphologically the Aurantiaca group of accessions are considered to be intermediate between the tall and dwarf accessions. Estimation of genetic relatedness based on AFLP analysis identified the Aurantiaca group as being more similar to the dwarf rather than the tall group. In addition, putative duplicate accessions were identified in the Aurantiaca group. Information emerging from this study will facilitate the management of coconut germplasm and optimise the choice of genetically divergent parents for crossing. Received: 16 June 1997 / Accepted: 14 October 1997     

A new aspect of genetic diversity of Indonesian oil palm (Elaeis guineensis Jacq.) revealed by isoenzyme and AFLP markers and its consequences for breeding

Oil palm (Elaeis guineensis Jacq.) plays an important economic role in some countries of Southeast Asia like Indonesia, which is the world’s second producer of palm and palm kernel oil. The quality improvement of planting material needs a better understanding of the genetic relationships between genotypes from different populations used in the breeding programmes. In this study, 48 parents, representative of four populations used in Indonesian Oil Palm Research Institute (IOPRI) breeding programmes, were analysed with five selected AFLP primer pairs and four isoenzymatic systems. One hundred and fifty eight scorable band levels were generated of which 96 (61%) were polymorphic. AFLP allowed us to identify off–type descendants which were excluded from analysis. The use of unbiased Rogers distance clearly separated the four studied populations. The Neighbor-Joining method re-groups two African populations which are known as originating from different regions. Nevertheless, the variability revealed is in accordance with oil palm breeders’ knowledge. The results obtained with AFLP showed that the crosses among the African sub-population, which is excluded in oil palm reciprocal recurrent selection (RRS) breeding programmes, may be more interesting than the crosses between the African and the Deli populations. Received: 9 August 1999 / Accepted: 28 February 2000     

Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon

Three different types of molecular markers, RAPD, AFLP and RFLP were used to measure genetic diversity among six genotypes of Cucumis melo L. Each line represented a different melon genotype: Piel de Sapo, Ogen, PI161375, PI414723, Agrestis and C105. A number of polymorphic RAPD, AFLP and RFLP bands were scored on all materials and the genetic similarity measured. Clustering analysis performed with the three types of markers separated the genotypes into two main groups: (1) the sweet type, cultivated melons and (2) the exotic type, not cultivated melons. While the data obtained suggest that all three types of markers are equally informative, AFLPs showed the highest efficiency in detecting polymorphism. Received: 30 December 1999 / Accepted: 24 January 2000     

Comparison of the genetic diversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O. sativa L.) using RFLP markers

Forty fourth single-copy RFLP markers were used to evaluate the genetic diversity of 122 accessions of common wild rice (CWR, Oryza rufipogon Griff.) and 75 entries of cultivated rice (Oryza sativa L. ) from more than ten Asian countries. A comparison of the parameters showing genetic diversity, including the percentage of polymorphic loci (P), the average number of alleles per locus (A), the number of genotypes (Ng), the average heterozygosity (Ho) and the average genetic multiplicity (Hs) of CWR and indica and japonica subspecies of cultivated rice from different countries and regions, indicated that CWR from China possesses the highest genetic diversity, followed by CWR from South Asia and Southeast Asia. The genetic diversity of CWR from India is the second highest. Although the average gene diversity (Hs)of the South Asian CWR is higher than that of the Southeast Asian CWR, its percentage of polymorphic loci (P), number of alleles (Na) and number of genotypes (Ng) are all smaller. It was also found that the genetic diversity of cultivated rice is obviously lower than that of CWR. At the 44 loci investigated, the number of polymorphic loci of cultivated rice is only 3/4 that of CWR, while the number of alleles, 60%, and the number of genotypes is about 1/2 that of CWR. Of the two subspecies studied, the genetic diversity of indica is higher than that of japonica. The average heterozygosity of the Chinese CWR is the highest among all the entries studied. The average heterozygosity of CWR is about two-times that of cultivated rice. It is suggested that during the course of evolution from wild rice to cultivated rice, many alleles were lost through natural and human selection, leading to the lower heterozygosity and genetic diversity of the cultivated rice. Received: 19 May 1999 / Accepted: 26 April 2000     

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 genetic maps of Eucalyptus globulus and E. tereticornis

 Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient technique for detecting large numbers of DNA markers in eucalypts. We have used AFLP markers in a two-way pseudo-testcross strategy to generate genetic maps of two clones of different Eucalyptus species (E. tereticornis and E. globulus). Of 606 polymorphic fragments scored, 487 segregated in a 1 : 1 ratio, corresponding to DNA polymorphisms heterozygous in one parent and null in the other. In the maternal E. tereticornis map, 268 markers were ordered in 14 linkage groups (919 cM); the paternal E. globulus map had 200 markers in 16 linkage groups (967 cM). Results from PGRI software were compared with MAPMAKER. The average density of markers was approximately 1 per 3.9 cM. Framework markers were ordered with an average confidence level of 90%, covering 80–100% of the estimated Eucalyptus genome size. In order to investigate the homologies between the E. tereticornis and the E. globulus genetic linkage maps, we included 19 markers segregating 3 : 1 in the analysis. Some homeologous linkage groups were recognized. The linkage data developed in these maps will be used to detect loci controlling commercially important traits. Received: 17 July 1997 / Accepted: 13 October 1997     

Polymorphism, distribution, and segregation of AFLP markers in a doubled haploid rice population

We exploited the newly developed amplified fragment length polymorphism (AFLP) technique to study the polymorphism, distribution and inheritance of AFLP markers with a doubled haploid rice population derived from ‘IR64’/‘Azucena’. Using only 20 pairs of primer combinations, we detected 945 AFLP bands of which 208 were polymorphic. All 208 AFLP markers were mapped and distributed over all 12 chromosomes. When these were compared with RFLP markers already mapped in the population, we found the AFLP markers to be highly polymorphic in rice and to follow Mendelian segregation. As linkage map of rice can be generated rapidly with AFLP markers they will be very useful for marker-assisted backcrossing. Received: 11 April 1996 / Accepted: 14 June 1996     

Allozyme variation and population genetic structure of common wild rice Oryza rufipogon Griff. in China

In order to determine the genetic diversity and genetic structure of populations in common wild rice Oryza rufipogon, an endangered species, allozyme diversity was analyzed using 22 loci in 607 individuals of 21 natural populations from the Guangxi, Guangdong, Hainan, Yunnan, Hunan, Jiangxi and Fujian provinces in China. The populations studied showed a moderate allozyme variability (A=1.33, P=22.7%, Ho=0.033 and He=0.068), which was relatively high for the genus Oryza. The levels of genetic diversity for Guangxi and Guangdong were significantly higher than those for the other regions, and thus South China appeared to be the center of genetic diversity of O. rufipogon in China. A moderate genetic differentiation (F_ST=0.310, I=0.964) was found among the populations studied. Interestingly, the pattern of population differentiation does not correspond to geographic distance. An estimate of the outcrossing rate (t=0.324) suggests that the species has a typical mixed-mating system. The deficit of heterozygotes (F=0.511) indicates that some inbreeding may have taken place in outcrossing asexual populations because of intra-clone outcrossing events and ”isolation by distance” as a result of human disturbance. In order to predict the long-term genetic survival of fragmented populations, further studies on gene flow among the remaining populations and the genetic effects of fragmentation are proposed. Finally, some implications for the conservation of endangered species are suggested. Received: 22 June 1999 / Accepted: 20 December 1999