AFLP™ markers for DNA fingerprinting in cattle

This work reports on use of the recently described amplified fragment length polymorphism (AFLP) technology for DNA fingerprinting in cattle. The AFLP technology produces molecular markers through the high-stringency polymerase chain reaction (PCR)-amplification of restriction fragments that are ligated to synthetic adapters and amplified using primers, complementary to the adapters, which carry selective nucleotides at their 3' ends. While, for plants, the double digestion of genomic DNA with Eco RI and Mse I is suggested, in mammals the enzyme combination Eco RI/ Taq I produces clearer and more polymorphic AFLP patterns. In a sample of 47 Italian Holstein genotypes, 16 Eco RI/ Taq I primer combinations identified 248 polymorphic bands in a species known for its low level of restriction polymorphism. In spite of the low information content carried by each AFLP polymorphism (average polymorphism information content = 0·31), the number of fragments revealed by each primer combination increased significantly the level of genetic information gained in each experiment. AFLP patterns are reproducible in independent experiments and polymorphic fragments segregate in cattle families according to Mendelian rules.     

AFLP studies on downy-mildew-resistant and downy-mildew-susceptible genotypes of opium poppy

Downy mildew (DM) caused by Peronospora arborescens, is a serious disease in opium poppy (Papaver somniferum), which has a world-wide spread. The establishment of DM-resistant cultivars appears to be a sustainable way to control the disease. In this paper, we present the results of a study aimed at the identification of amplified fragment length polymorphism (AFLP) markers for DM-resistance in opium poppy. Three opium poppy genotypes (inbred over about 10 years): Pps-1 (DM-resistant), Jawahar-16 (DM-susceptible) and H-9 (DM-susceptible) were crossed in a diallel manner and the F1 progeny along with the parents were subjected to AFLP analysis of chloroplast (cp) and nuclear DNA with seven and nine EcoRI / MseI primer combinations, respectively. cpDNA AFLP analysis identified 24 Pps-1 (DM-resistant)-specific unique fragments that were found to be maternally inherited in both the crosses, Pps-1 × Jawahar-16 and Pps-1 × H-9. In the case of nuclear DNA AFLP analysis, it was found that 17 fragments inherited from Pps-1 were common to the reciprocal crosses of both (i) Pps-1 and Jawahar-16 as well as (ii) Pps-1 and H-9. This is the first molecular investigation on the identification of polymorphism between DM-resistant and DM-susceptible opium poppy genotypes and development of DM-resistant opium poppy genotype-specific AFLP markers. These AFLP markers could be used in future genetic studies for analysis of linkage to the downy mildew resistance trait.     

PCR-based molecular markers for assessment of somaclonal variation in <Emphasis Type="Italic">Pinus pinea</Emphasis> clones micropropagated <Emphasis Type="Italic">in vitro</Emphasis>

Four different markers [random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and selective amplified microsatellite polymorphism length (SAMPL)] were applied for evaluating somaclonal variation of micropropagated genotypes of stone pine (Pinus pinea L.). The total number of primers tested was 130, with 223 combinations assayed. A high number of them amplified successfully (178), representing 79.82 % of the total, and the average number of amplified fragments ranged from 2.47 (ISSR) to 65.76 (SAMPL). Based on internal controls, no problem of reproducibility was detected. Almost no somaclonal variation was detected within the clones. Of the tested markers, ISSR, AFLP, and SAMPL showed monomorphic amplification profiles, with only RAPD markers showing some interclonal variation.     

羊草种质基因组DNA的AFLP多态性研究

羊草是禾本科牧草之王 ,在当前我国西部生态建设和草原畜牧业发展中发挥着重要作用。用AFLP方法对2 7份我国不同地区分布的羊草 (Leymuschinensis (Trin .)Tzvel)材料进行了基因组DNA多态性分析 ,8对AFLP引物组合在 2 7个不同羊草基因型中共扩增出 5 37条带 ,产生出的DNA片段大小分布在 75bp - 5 30bp之间。其中单态性带 89条 ,占 16 .6 % ,多态性带 32 9条 ,占 6 1.3%。平均每对引物组合扩增的DNA带数为 6 6 .13,总的多态性比率为 78.84%。AFLP多态信息含量PIC值分布于 0 .0 - 0 .5之间 ,平均PIC值为 0 .2 16 ,出现的PIC最大值 (0 .5 )约占AFLP标记的 8.5 % ,说明羊草基因组DNA的多态性比较丰富。以 5 37个AFLP标记为原始数据 ,根据Nei和Li的方法对 2 7份羊草材料进行遗传变异和聚类分析的结果表明 :羊草种内有高频率的遗传变异发生 ,且与地理分布和生态环境密切相关 ;2 7份羊草不同基因型被划分为四大类群 ,不同类群相互间的遗传距离相对较大 ,在树状图中表现为较远的亲缘关系。对羊草种内遗传变异发生的原因和品种的形成进行了初步讨论。     

Diversity analysis in Indian genotypes of linseed (Linum usitatissimum L.) using AFLP markers

AFLP fingerprinting of 45 Indian genotypes of linseed was carried out to determine the genetic relationship among them. Sixteen primer combinations produced 1142 fragments with 1129 as polymorphic and 13 as monomorphic fragments. Polymorphic fragments varied from 44 (E-ACA/M-CTA) to 94 (E-AGC/M-CAC) with an average of 70.6 fragments per primer combination. The frequency of polymorphism varied from 93.7% to 100% with an average of 98.8% across all the genotypes. The PIC value ranged from 0.19 to 0.31 with an average of 0.23 per primer combination. The primer pair E-AGC/M-CAC showed the maximum PIC value (0.31) followed by E-AGC/M-CAG (0.29), E-AAC/M-CAG (0.26) and E-AGC/M-CTA (0.25). Resolving power (RP) and marker index (MI) varied from 13.73 to 43.50 and 8.81 to 28.91 respectively. The Jaccard's similarity coefficient varied from 0.16 to 0.57 with an average of 0.26 ± 0.05. The maximum genetic similarities (57%) were detected between genotypes Him Alsi-1 and Him Alsi-2, followed by Him Alsi-1 and GS41 and GS41 and LC-54. The genotypes R-552, Himani, RKY-14, Meera, Indira Alsi-32 and Suyog were found to be more divergent genotypes. The NJ clustering grouped all the 45 genotypes into three major clusters. In general the genotypes of cluster III had high oil content and those of cluster I had low oil content. At the population level, within population variance was much higher than between populations variance.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Feeding behavior of aphids on narrow‐leafed lupin (Lupinus angustifolius) genotypes varying in the content of quinolizidine alkaloids

Since the beginning of breeding narrow‐leafed lupins [Lupinus angustifolius L. (Fabaceae)] with a low alkaloid content, susceptibility to several aphid species has increased. Therefore, the probing and feeding behavior of Aphis fabae Scopoli, Aphis craccivora Koch, Acyrthosiphon pisum (Harris), Myzus persicae (Sulzer), and the well‐adapted Macrosiphum albifrons Essig (all Hemiptera: Aphididae) was studied over 12 h on narrow‐leafed lupin genotypes containing varying amounts and compositions of alkaloids. We used the electrical penetration graph (EPG) technique to obtain information on the influence of alkaloid content and composition on the susceptibility to various aphid species. Results indicated that the total time of probing of A. fabae, A. craccivora, A. pisum, and M. persicae increased with a reduced alkaloid content, whereas the alkaloid content had no influence on M. albifrons. Almost all of the individuals (>93%) conducted sieve element phases on the highly susceptible genotype Bo083521AR (low alkaloid content). A reduced occurrence of phloem phases was observed during the 12‐h recording on the alkaloid‐rich cultivar Azuro, especially for A. pisum (37.5%) and A. fabae (55.0%). Furthermore, aphids feeding on genotypes with low alkaloid content had in most cases significantly longer sieve element phases than when feeding on resistant genotypes (Kalya: low alkaloid content, yet resistant; Azuro: high alkaloid content, resistant), whereas M. albifrons showed the longest phloem phase on the alkaloid‐rich cultivar Azuro. As most significant differences were found in phloem‐related parameters, it is likely that the most important plant factors influencing aphid probing and feeding behavior are localized in the sieve elements. The aphids’ feeding behavior on the cultivar Kalya, with a low alkaloid content but reduced susceptibility, indicates that not only the total alkaloid content influences the feeding behavior but additional plant factors have an impact.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

Genetic diversity of durum wheat as determined by AFLP in fluorescence

The DNA of fifteen Italian cultivars of durum wheat (Triticum turgidum L. ssp. durum) were analyzed by in fluorescence amplified fragment length polymorphism (fAFLP) in order to obtain the characteristic fingerprintings of genotypes and assess their genetic relatedness. Among 64 combinations of fluorescence labelled primers, three different combinations were chosen as producing a total of 6630 AFLP fragments, 2277 (34.3 %) of them being polymorphic. By using this fAFLP methodology a DNA fingerprinting of each durum wheat cultivar was generated for genotype identification. Analysis of the genetic relationships show the low variability among durum wheat cultivars.     

Multicolour fluorescent detection and mapping of AFLP markers in chicken (Gallus domesticus)

We describe the mapping of amplified restriction fragment polymorphism (AFLP) markers in chicken (Gallus domesticus) using a multi-colour fluorescent detection system. DNA was used from a population consisting of four families with a total of 183 F2 individuals. The enzyme combination EcoRI/TaqI was used for double digestion, and fluorescently labelled fragments were analysed on an ABI PRISM 377 DNA sequencer. Polymorphic signals in the range of 50-500 bp were genotyped with the ABI PRISM Genotyper 2.0 software, which enabled the analysis of both dominant and incomplete dominant markers (with respect to AFLP, often referred to as codominant). In 19 sets consisting of 3 EcoRI/TaqI primer pair combinations each, a total of 475 polymorphic markers was detected. From these polymorphisms 344 markers could be mapped on the Wageningen linkage map. Fourteen markers were length polymorphisms of the same fragment and 28 markers Z-linked and uniformative; 64 AFLP markers appeared to be unlinked and 25 AFLP markers could not be accurately mapped on the basis of the genotyping results. The resulting AFLP/microsatellite linkage map is comprised of 33 linkage groups with a total of 835 loci.     

Association analysis,genetic diversity and structure analysis of tobacco based on AFLP markers

Knowledge in the area of genetic diversity could aid in providing useful information in the selection of material for breeding such as hybridization programs and quantitative trait loci mapping. To this end, 50 Nicotiana tabacum genotypes were genotyped with 21 primer combination of amplified fragment length polymorphism (AFLP). A total of 480 unambiguous DNA fragments and 373 polymorphic bands were produced with an average of 17.76 per primer combination. Also, the results revealed high polymorphic rate varing from 52.63 to 92.59 %, demonstrating that AFLP technique utilized in this research can be a powerful and valuable tool in the breeding program of N. tabacum. Cluster analysis based on complete linkage method using Jaccard’s genetic distance, grouped the 50 tobacco genotypes into eight clusters including three relatively big clusters, one cluster including Golden gift, Burly 7022 and Burly Kreuzung, one cluster consisting of two individuals (Pereg234, R9) and three single-member clusters (Pennbel69, Coker176 and Budisher Burley E), Recent genotypes showed high genetic distance from other genotypes belonging to cluster I and II. Association analysis between seven important traits and AFLP markers were performed using four statistical models. The results revealed the model containing both the factors, population structure (Q) and general similarity in genetic background arising from shared kinship (K), reduces false positive associations between markers and phenotype. According to the results nine markers were determined that could be considered to be the most interesting candidates for further studies.     

Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a polymerase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in order to estimate population genetic diversity and genetic structure parameters, one must assume that dominant (amplified) alleles are identical in state, recessive (unamplified) alleles are identical in state, AFLP fragments segregate according to Mendelian expectations and that the genotypes of an AFLP locus are in Hardy-Weinberg equilibrium (HWE). The HWE assumption is untestable for natural populations using dominant markers. Restriction fragment length polymorphism (RFLP) markers segregate as codominant alleles, and can therefore be used to test the HWE assumption that is critical for analysing AFLP data. This study examined whether the dominant AFLP markers could provide accurate estimates of genetic variability for the Aedes aegypti mosquito populations of Trinidad, West Indies, by comparing genetic structure parameters using AFLP and RFLP markers. For AFLP markers, we tested a total of five primer combinations and scored 137 putative loci. For RFLP, we examined a total of eight mapped markers that provide a broad coverage of mosquito genome. The estimated average heterozygosity with AFLP markers was similar among the populations (0.39), and the observed average heterozygosity with RFLP markers varied from 0.44 to 0.58. The average FST (standardized among-population genetic variance) estimates were 0.033 for AFLP and 0.063 for RFLP markers. The genotypes at several RFLP loci were not in HWE, suggesting that the assumption critical for analysing AFLP data was invalid for some loci of the mosquito populations in Trinidad. Therefore, the results suggest that, compared with dominant molecular markers, codominant DNA markers provide better estimates of population genetic variability, and offer more statistical power for detecting population genetic structure.     

Comparison of RAPD,ISSR, and AFLP Molecular Markers to Reveal and Classify Orchardgrass (Dactylis glomerata L.) Germplasm Variations

Three different DNA-based techniques, Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) markers, were used for fingerprinting Dactylis glomerata genotypes and for detecting genetic variation between the three different subspecies. In this study, RAPD assays produced 97 bands, of which 40 were polymorphic (41.2%). The ISSR primers amplified 91 bands, and 54 showed polymorphism (59.3%). Finally, the AFLP showed 100 bands, of which 92 were polymorphic (92%). The fragments were scored as present (1) or absent (0), and those readings were entered in a computer file as a binary matrix (one for each marker). Three cluster analyses were performed to express–in the form of dendrograms–the relationships among the genotypes and the genetic variability detected. All DNA-based techniques used were able to amplify all of the genotypes. There were highly significant correlation coefficients between cophenetic matrices based on the genetic distance for the RAPD, ISSR, AFLP, and combined RAPD-ISSR-AFLP data (0.68, 0.78, 0.70, and 0.70, respectively). Two hypotheses were formulated to explain these results; both of them are in agreement with the results obtained using these three types of molecular markers. We conclude that when we study genotypes close related, the analysis of variability could require more than one DNA-based technique; in fact, the genetic variation present in different sources could interfere or combine with the more or less polymorphic ability, as our results showed for RAPD, ISSR and AFLP markers. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationship among genotypes of Dactylis glomerata.     

Molecular polymorphism and linkage analysis in sweet passion fruit,an outcrossing species

One of the current challenges of tropical fruit crop improvement is to incorporate molecular marker‐based approaches into conventional breeding programmes. This study was designed to build an integrated genetic map of the sweet passion fruit (Passiflora alata), a diploid (2n = 18) outcrossing species which is greatly appreciated for in natura consumption, and reported to inspire cosmetic and pharmaceutical companies to create plant‐derived compounds. With this in mind, a full‐sib family of 180 individuals was genotyped using different molecular marker types, such as amplified fragment length polymorphisms (AFLP), microsatellite‐AFLP (M‐AFLP), simple sequence repeats (SSR), resistance gene analogues (RGA) and target region amplification polymorphism (TRAP). On average, the rate of polymorphism between the parental genotypes was 20.3%. We also searched for single nucleotide polymorphisms (SNPs) in some AFLP bands and in seven gene fragments, and found one SNP every 87 bp. All SNPs were biallelic and occurred most frequently in putative gene fragments (81.5%) rather than in AFLP bands (60.0%) analyzed. Excellent gel profiles were obtained allowing the recognition of all types of segregation expected for a progeny of an outcrossing species. Multipoint linkage analysis was performed using OneMap software, with logarithm of the odds (LOD) score ≥ 5.6 and recombination fraction <0.5. The resulting integrated map consists of 549 markers, 2.0% of which fit a segregation ratio of 1:1:1:1, 1.3% a ratio of 1:2:1, 27.3% a ratio of 3:1 and 69.4% a ratio of 1:1. The map spanned a total of 2073.0 cM, with an average distance between adjacent markers of 3.8 cM. This is the first linkage study on sweet passion fruit and should prove useful for quantitative trait loci mapping.     

Cleaved AFLP (cAFLP), a modified amplified fragment length polymorphism analysis for cotton

In certain plant species including cotton (Gossypium hirsutum L. or Gossypium barbadense L.), the level of amplified fragment length polymorphism (AFLP) is relatively low, limiting its utilization in the development of genome-wide linkage maps. We propose the use of frequent restriction enzymes in combination with AFLP to cleave the AFLP fragments, called cleaved AFLP analysis (cAFLP). Using four Upland cotton genotypes (G. hirsutum) and three Pima cotton (G. barbadense), we demonstrated that cAFLP generated 67% and 132% more polymorphic markers than AFLP in Upland and Pima cotton, respectively. This resulted in 15.5 and 25.5 polymorphic cAFLP markers per AFLP primer combination, as compared to 9.1 and 11.0 polymorphic AFLP. The cAFLP-based genetic similarity (GS) is generally lower than the AFLP-based GS, even though both marker systems are overall congruent. In some cases, cAFLP can better resolve genetic relationships between genotypes, rendering a higher discriminatory power. Given the high-resolution power of capillary-based DNA sequencing system, we further propose that AFLP and cAFLP amplicons from the same primer combination can be pooled as one sample before electrophoresis. The combination produced an average of 18.5 and 31.0 polymorphic markers per primer pair in Upland and Pima cotton, respectively. Using several restriction enzyme combinations before pre-selective amplification in combination with various frequent 4 bp-cutters or 6 bp-cutters after selective amplification, the pooled AFLP and cAFLP will provide unlimited number of polymorphic markers for genome-wide mapping and fingerprinting.     

Differentiation of bermudagrass (Cynodon spp.) genotypes by AFLP analyses

 Bermudagrasses (Cynodon spp.) are major turfgrasses for home lawns, public parks, golf courses and sport fields, and are widely adapted to tropical and warmer temperate climates. Morphological and physiological characteristics are not sufficient to differentiate some bermudagrass genotypes because the differences between them are often subtle and subject to environmental influence. In this study, a DNA-typing technique, amplified fragment length polymorphism (AFLP), was used to differentiate bermudagrass genotypes and to explore their genetic relationships. Twenty seven bermudagrass cultivars and introductions, mostly from the Coastal Plain Experiment Station in Tifton, Ga., were assayed by the radioactive (³²P) and the fluorescence-labeled AFLP methods. The AFLP technique produced enough polymorphism to differentiate all 27 bermudagrass genotypes, even the closely related ones. An average of 48–74 bands in the 30–600-bp size range was detected by the ³²P-labeled AFLP method. The results indicated that most of the 14 primer combinations tested in this study could be used to distinguish bermudagrass genotypes, and that some single primer-pairs could differentiate all 27 of them. To test the reliability and reproducibility of the AFLP procedure, three DNA isolations (replications) of the 27 bermudagrass genotypes were assayed using five primer pairs. Only 0.6% of the bands were evaluated differently among the three replications. One replication of one genotype (which was most likely a planting contaminant) was grouped in an unexpected cluster using the Unweighted Pair Group Mean Average (UPGMA) method. A one- or two-band difference in scoring did not change the clustering of genotypes or the replications within genotypes. The 27 genotypes were grouped into three major clusters, many of which were in agreement with known pedigrees. Trees constructed with different primer combinations using ³²P- and fluorescence-labelling formed similar major groupings. The semi-automated fluorescence-based AFLP technique offered significant improvements on fragment sizing and data handling. It was also more accurate for detection and more efficient than the radioactive labelling method. This study shows that the AFLP technique is a reliable tool for differentiating bermudagrass genotypes and for determining genetic relationships among them. Received: 28 July 1998 / Accepted: 3 November 1998     

A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the DNA polymorphism in elite blackgram genotypes. A total of 25 random and 16 ISSR primers were used. Amplification of genomic DNA of the 18 genotypes, using RAPD analysis, yielded 104 fragments that could be scored, of which 44 were polymorphic, with an average of 1.8 polymorphic fragments per primer. Number of amplified fragments with random primers ranged from two (OPA-13) to nine (OPK-4) and varied in size from 200 bp to 2,500 bp. Percentage polymorphism ranged from 16.6% (OPK-7) to a maximum of 66.6% (OPE-5, OPH-2, and OPK-8), with an average of 42.7%. The 16 ISSR primers used in the study produced 101 bands across 18 genotypes, of which 55 were polymorphic. The number of amplified bands varied from two (ISSR 858) to ten (ISSR 810), with a size range of 200–2,200 bp. The average numbers of bands per primer and polymorphic bands per primer were 6.3 and 3.4, respectively. Percentage polymorphism ranged from 25% (ISSR 885) to 100% (ISSR 858), with an average percentage polymorphism of 57.5% across all the genotypes. The 3-anchored primers based on poly(GA) and poly(AG) motifs produced high average polymorphisms of 54.98% and 58.32%, respectively. ISSR markers were more efficient than the RAPD assay, as they detected 57.4% polymorphic DNA markers in Vigna mungo as compared to 42.7% for RAPD markers. The Mantel test between the two Jaccards similarity matrices gave r =0.32, showing low correlation between RAPD- and ISSR-based similarities. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in ISSR and combined data of RAPD and ISSR.     

Optimization of AFLP for extremely large genomes over 70 Gb

Here, we present an improved amplified fragment length polymorphism (AFLP) protocol using restriction enzymes (AscI and SbfI) that recognize 8‐base pair sequences to provide alternative optimization suitable for species with a genome size over 70 Gb. This cost‐effective optimization massively reduces the number of amplified fragments using only +3 selective bases per primer during selective amplification. We demonstrate the effects of the number of fragments and genome size on the appearance of nonidentical comigrating fragments (size homoplasy), which has a negative impact on the informative value of AFLP genotypes. We also present various reaction conditions and their effects on reproducibility and the band intensity of the extremely large genome of Viscum album. The reproducibility of this octo‐cutter protocol was calculated using several species with genome sizes ranging from 1 Gb (Carex panicea) to 76 Gb (V. album). The improved protocol also succeeded in detecting high intraspecific variability in species with large genomes (V. album, Galanthus nivalis and Pinus pumila).     

Genetic diversity of Dioscorea dumetorum (Kunth) Pax using Amplified Fragment Length Polymorphisms (AFLP) and cpDNA

We have utilized Amplified Fragment Length Polymorphisms (AFLP) in conjunction with chloroplast DNA (cpDNA) sequence data to study the genetic diversity in 53 accessions of Dioscorea dumetorum from six countries in West and Central Africa. Our results provide a comparison of the two marker systems with regards to their applicability to differentiate intraspecific genotypes and the grouping of the accessions based on localities of collection. A total of 1052 AFLP fragments (of which 94.1% were polymorphic) produced from twelve primer combinations indicate a relatively high level of polymorphism among the accessions. Three major genetic groups that do not strictly follow a geographic distribution pattern were identified using Neighbour-joining and the principal coordinate (PCo) analyses. Accessions from Togo showed higher numbers of private fragments and the highest percentage polymorphism (59.4%). The detection of highest genetic diversity in accessions from Nigeria and Togo and their relationship to other accessions suggest that these countries are the centre of origin and diversity of D. dumetorum. The moderately high genetic diversity (average of 61%) is suggesting great influence on the D. dumetorum germplasms through exchange and transfer of cultivars among local farmers in the sub-region. In contrast, DNA sequence data from the psbA-trnH and the rpoB-trnC chloroplast regions revealed no variation among accessions from the different localities and clearly differentiated by AFLP patterns. The results demonstrate the usefulness of the AFLP marker in generating high polymorphism in the D. dumetorum accessions from West and Central Africa and hence may be used for agronomic purposes.