Molecular AFLP Analysis of the Genotypes of Pepper Capsicum annuum Cultivars

The results of AFLP study of 14 Capsicum annuum cultivars are presented. In spite of the known low genomic variation of large-fruited sweet pepper, AFLP analysis proved to be suitable for detecting polymorphism and genotyping pepper cultivars. Nine primer pairs were selected to allow identification of the cultivars under study. Among-cultivar polymorphism detectable with these primers was estimated at 16.5%. A characteristic AFLP pattern was obtained for each cultivar. Several cultivar-specific fragments were revealed for seven cultivars. On the basis of the AFLP data, genetic distances between cultivars were determined and a tree was constructed by means of hierarchic cluster analysis (UPGMA) with the Jacquard coefficient. It was assumed that this information is useful in breeding programs involving the cultivars examined.     

Genetic Variability in Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) Estimated by Morphological Data and Amplified Fragment Length Polymorphism Markers

Data on genetic similarity among crop cultivars is of vital importance for the plant breeder. The objectives of this study were to group pepper (Capsicum annuum L.) genotypes into clusters according to their distances as estimated by morphological traits and amplified fragment length polymorphism (AFLP) markers and to assess the relationships between the two. Thirty-nine pepper genotypes obtained from different countries were grown in the greenhouse at University of the Free State, South Africa, during 2001 and 2002 in a randomized complete block design with three replications. A total of 20 different morphological traits were measured and six AFLP primer pairs were used to estimate pairwise genetic distances. Both datasets showed high genetic distances among the different genotypes, indicating high genetic diversity among them. The mean genetic distance among Ethiopian pungent elongated-fruit genotypes, was lower than that between them and the introduced ones. Morphological and AFLP distance estimations generally clustered together genotypes with similar fruit sizes. Significant, positive correlation was observed between morphological and AFLP diversity estimations. The narrow genetic basis among the Ethiopian pungent elongated-fruit cultivars suggests that the pepper breeding program of Ethiopia should focus on enriching its germplasm through local collection and introductions from other parts of the world.     

A Comparison of AFLP and RAPD Markers for Genetic Diversity and Cultivar Identification in Cotton

AFLP and RAPDmarkers were employed in sixteen diploid cotton (Gossypium sp) cultivars for genetic diversity estimation and cultivar identification. Polymorphism information content (PIC) and percent polymorphism were found to be more for AFLP markers as compared to RAPD markers. Average Jaccard’s genetic similarity index was found to be almost similar using either AFLP or RAPD markers. All the cultivars could be distinguished from one another using AFLP markers and also by the combined RAPD profiles. Cultivar identification indicators like resolving power, marker index and probability of chance identity of two cultivars suggested the usefulness of AFLP markers over the RAPD markers. AFLP and RAPD analyses revealed limited genetic diversity in the studied cultivars. Cluster analysis of both RAPD and AFLP data produced two clusters, one containing cultivars of G. herbaceum and another containing cultivars of G. arboreum species. Highly positive correlation between cophenetic matrices using RAPD and AFLP markers was observed. AFLP markers were found to be more efficient for genetic diversity estimation, polymorphism detection and cultivar identification.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Development of a CAPS marker for the Pvr4 locus: a tool for pyramiding potyvirus resistance genes in pepper.

The Pvr4 resistance gene in pepper confers a complete resistance to the three pathotypes of potato virus Y (PVY) and to pepper mottle virus (PepMoV). In order to use this gene in a marker-assisted selection (MAS) program and to permit the pyramiding of several potyvirus resistance genes in the same cultivar, tightly linked amplified fragment length polymorphism (AFLP) markers were obtained by the bulked segregant analysis method. Eight linked AFLP markers were mapped in an interval from 2.1 +/- 0.8 to 13.8 +/- 2.9 cM around this locus. The closest codominant AFLP marker was converted into a codominant CAPS (cleaved amplified polymorphic sequence) marker using data from the alignment of the two allele sequences. We have further characterized the relevance of the CAPS marker for MAS programs in different pepper breeding lines.     

Monitoring of cultivar identity in tissue culture-derived date palms using RAPD and AFLP analysis

In the present study, two polymerase chain reaction (PCR)-based methods namely, randomly amplified polymophic DNA (RAPD) and amplification fragment length polymorphism (AFLP) were employed to assess genetic variations, which may appeared, in tissue culture-derived date palm (Phoenix dactylifera) offshoots. Analysis of RAPD banding patterns generated by PCR amplification using 37 random primers gave no evidences for somaclonal variations and the percentage of polymorphic bands in a total of 259 scored bands was zero. Meanwhile, analysis of AFLP banding patterns generated using 13 primer combinations pointed to minor genetic variations in the AFLP banding patterns. The percentage of genetic variations (polymorphism) in tissue culture-derived date palm offshoots belonging to cultivars Sakkoty, Gandila and Bertamoda was 2.6, 0.79 and 1 %, respectively, as revealed by AFLP analysis. The low percentage of genetic variations confirms the genetic stability of tissue culture-derived dry date palm cultivars.     

Establishment of AFLP technique and assessment of primer combinations for mei flower

Mei flower is one of the most famous ornamental flowers in eastern Asia for its blossoming in early spring. Amplified fragment length polymorphism (AFLP) is one of the most frequently used techniques for analysis of genetic variation and is used herein for the first time inPrunus mume. This research provides a detailed and modified AFLP protocol for Mei genomic DNA digested withEcoRI/PstI restriction endonuclease combinations. The 10 best primer pairs of high polymorphism were screened from 256 primer combinations that could reliably and repetitively distinguish 14 Mei samples and would be suitable for genetic analysis of more cultivars. Ten primer pairs produced up to a total of 524 AFLP bands and up to 233 polymorphic bands. The ratio of polymorphic bands scoped from 35.71% to 59.67%, and the average ratio was 44.46% in the 10 primers. AFLP is an effective, inexpensive, and timesaving technique for the genetic differentiation of the Mei cultivars, as evidenced in this study.     

Comparative analyses of genetic diversities within tomato and pepper collections detected by retrotransposon-based SSAP, AFLP and SSR

The retrotransposon-based sequence-specific amplification polymorphism (SSAP) marker system was used to assess the genetic diversities of collections of tomato and pepper industrial lines. The utility of SSAP markers was compared to that of amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. On the basis of our results, SSAP is most informative of the three systems for studying genetic diversity in tomato and pepper, with a significant correlation of genetic relationships between different SSAP datasets and between SSAP, AFLP and SSR markers. SSAP showed about four- to ninefold more diversity than AFLP and had the highest number of polymorphic bands per assay ratio and the highest marker index. For tomato, SSAP is more suitable for inferring overall genetic variation and relationships, while SSR has the ability to detect specific genetic relationships. All three marker results for pepper showed general agreement with pepper types. Additionally, retrotransposon sequences isolated from one species can be used in related Solanaceae genera. These results suggest that different marker systems are suited for studying genetic diversity in different contexts depending on the group studied, where discordance between different marker systems can be very informative for understanding genetic relationships within the study group.     

Evaluation of AFLPs for germplasm fingerprinting and assessment of genetic diversity in cultivars of tomato (Lycopersicon esculentum L.).

Cultivated tomato (L. esculentum L.) germplasm exhibits limited genetic variation compared with wild Lycopersicon species. Amplified fragment length polymorphism (AFLP) markers were used to evaluate genetic variation among 74 cultivars, primarily from California, and to fingerprint germplasm to determine if cultivar-specific patterns could be obtained. All 74 cultivars were genotyped using 26 AFLP primer combinations; of the 1092 bands scored, 102 AFLP bands (9.3%) were polymorphic. Pair-wise genetic similarity coefficients (Jaccard and Nei-Li) were calculated. Jaccard coefficients varied from 0.16 to 0.98 among cultivar pairs, and 72% of pair-wise comparisons exceeded 0.5. UPGMA (unweighted pair-group method with arithmetic averaging) clustering and principle component analysis revealed four main clusters, I-IV; most modern hybrid cultivars grouped in II, whereas most vintage cultivars grouped in I. Clusters III and IV contained three and two cultivars, respectively. Some groups of cultivars closely related by pedigree exhibited high bootstrap values, but lower values (<50%) were obtained for cluster II and its four subgroups. Unique fingerprints for all 74 cultivars were obtained by a minimum of seven AFLP primer pairs, despite inclusion of some closely related cultivars. This study demonstrated that AFLP markers are effective for obtaining unique fingerprints of, and assessing genetic diversity among, tomato cultivars.     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

AFLP analysis of genetic variability in New Guinea impatiens

New Guinea impatiens ( Impatiens hawkeri) is an economically important floral crop, however, little work has been conducted to further our understanding of the genetics of this crop. In this study, we used amplified fragment length polymorphism (AFLP) technology to investigate the level of polymorphism present among 41 commercial cultivars of New Guinea impatiens, study their genetic relatedness, and assess the genetic diversity in this material. An efficient DNA extraction protocol was developed, and a total of 48 EcoRI and MseI primer combinations were used for PCR amplification. Amplification products were then subjected to polyacrylamide gel electrophoresis. The AFLP analysis showed that all 41 cultivars generated between 73 and 130 scoreable polymorphic bands per primer combination. Gower's Genetic Dissimilarity estimates for the entire set of cultivars ranged between 0.940 and 0.488. A dendogram was generated from these dissimilarity data that revealed four groupings among these 41 cultivars. The implications of these results on genotypic variation, genetic relationships, and genetic diversity in New Guinea impatiens will be discussed.     

Amplified fragment length polymorphism fingerprinting of 16 banana cultivars (Musa cvs.)

Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits.     

Genetic diversity of Phytophthora capsici isolates from pepper and pumpkin in Argentina

Phytophthora capsici is a soilborne oomycete plant pathogen that limits pepper production worldwide. The population structure varies significantly depending on the location (e.g. Peru vs. USA) and little is known about the diversity of P. capsici in Argentina. Our objective was to assess the diversity of P. capsici in Argentina at key pepper production areas. Forty isolates were recovered 2006-2009 from pepper and one isolate from pumpkin at 11 locations. Isolates were assessed for mating type, mefenoxam sensitivity and multilocus single nucleotide polymorphism (SNP) genotype profiles. Ten isolates with identical SNP profiles also were genotyped with amplified fragment length polymorphism (AFLP) markers. All 41 isolates had the A1 mating type and were sensitive to mefenoxam. Genotypic analysis using eight polymorphic SNP markers indicated 87% of the isolates had the same multilocus genotype, which is fixed for heterozygosity at seven of the eight SNP sites. AFLP analyses confirmed these findings, and overall it appears that clonal reproduction drives the population structure of P. capsici in Argentina. The implications for breeding resistant peppers and overall disease management are discussed.     

漆树品种的AFLP分析及评价(简报)

漆树(Toxicodendron vernicifluum(stokes)F.A.Barkley)隶属于漆树科(Anacardiaceae)漆树属(Tox- icodendron)的落叶乔木,是我国重要的特用经济林木。漆树栽培与生漆使用在我国已有几千年的历史,在长期栽培过程中形成了许多农家品种,它们具有一定的形态特点,适应一定的生长环境,并具有产漆量高、生漆品质好等特性。     

Swiss Mattenklee landraces,a distinct and diverse genetic resource of red clover (<Emphasis Type="Italic">Trifolium pratense</Emphasis> L.)

Genetic variability within and among 19 landraces and cultivars of red clover ( Trifolium pratense L.) was investigated by means of amplified fragment length polymorphism (AFLP) analysis in order to assess the potential value of Swiss Mattenklee landraces as genetic resources for plant breeding and the preservation of biodiversity. Populations were classified into three groups according to their origin and agronomic features: Mattenklee landraces (8), Mattenklee cultivars (8) and field clover cultivars (3). Analysis of molecular variance based on 276 polymorphic AFLP markers revealed 80% of total variability to be due to variability within populations while 12% were attributed to variability among groups. Stepwise discriminant analysis identified a subset of 126 AFLP markers which best separated individual plants into the three respective groups. Genetic distances between populations were considerably larger among groups than among populations within the same group, providing further evidence for the genetic distinction between Mattenklee landraces, Mattenklee cultivars and field clover cultivars. AFLP markers identified two landrace clusters, containing three and four populations respectively, which, together with one additional landrace, may sufficiently represent the genetic variability of all eight landraces investigated. The results of this study strongly suggest that Swiss Mattenklee landraces form a genetically distinct group of red clover. The data obtained provide criteria on how to efficiently manage, preserve and exploit Mattenklee germplasm.     

Amplified Fragment Length Polymorphism Fingerprinting of 16 Banana Cultivars (Musa cvs.)

Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits.     

Genetic relationships of Aglaonema species and cultivars inferred from AFLP markers

BACKGROUND AND AIMS: Aglaonema is an important ornamental foliage plant genus, but genetic relationships among its species and cultivars have not been reported. This study analysed genetic relatedness of 54 cultivars derived from nine species using amplified fragment length polymorphism (AFLP) markers. METHODS: Initially, 48 EcoRI + 2/MseI + 3 primer set combinations were screened, from which six primer sets that showed clear scoreable and highly polymorphic fragments were selected and used for AFLP reactions. AFLP fragments were scored and entered into a binary data matrix as discrete variables. Jaccard's coefficient of similarity was calculated for all pair-wise comparisons among the 54 cultivars, and a dendrogram was constructed by the unweighted pair-group method using the arithmetic average (UPGMA). KEY RESULTS: The number of AFLP fragments generated per primer set ranged from 59 to 112 with fragment sizes varying from 50 to 565 bp. A total of 449 AFLP fragments was detected, of which 314 were polymorphic (70 %). All cultivars were clearly differentiated by their AFLP fingerprints. The 54 cultivars were divided into seven clusters; cultivars within each cluster generally share similar morphological characteristics. Cluster I contains 35 cultivars, most of them are interspecific hybrids developed mainly from A. commutatum, A. crispum or A. nitidum. However, Jaccard's similarity coefficients among these hybrids are 0.84 or higher, suggesting that these popular hybrid cultivars are genetically much closer than previously thought. This genetic similarity may imply that A. nitidum and A. crispum are likely progenitors of A. commutatum. CONCLUSIONS: Results of this study demonstrate the efficiency and ease of using AFLP markers for investigating genetic relationships of ornamental foliage plants, a group usually propagated vegetatively. The AFLP markers developed will help future Aglaonema cultivar identification, germplasm conservation and new cultivar development.     

Comparative Assessment of SSR and AFLP Markers for Evaluation of Genetic Diversity and Conservation of Fig, <Emphasis Type="Italic">Ficus carica</Emphasis> L., Genetic Resources in Tunisia

This study characterises the genetic variability of fig, Ficus carica L., using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. It compares the efficiency and utility of the two techniques in detecting variation and establishing genetic relationships among Tunisian fig cultivars. Our results show that using both marker systems, the Tunisian fig germ plasm is characterised by having a large genetic diversity at the deoxyribonucleic acid level, as most of AFLP bands were detected and all SSR markers were polymorphic. In fact, 351 (342 polymorphic) and 57 (57 polymorphic) bands were detected using AFLP and SSR primers, respectively. SSR markers were the most polymorphic with an average polymorphic information content value of 0.94, while AFLP markers showed the highest effective multiplex ratio (56.9) and marker index (45.2). The effective marker index was recorded highest (4.19) for AFLP markers and lowest (0.70) for the SSR ones. Our results demonstrate that (1) independent as well as combined analyses of cluster analyses of SSR and AFLP fragments showed that cultivars are clustered independently from their geographical origin, horticultural classifications and tree sex; (2) the analysis of molecular variance allowed the partitioning of genetic variation within and among fig groups and showed greater variation within groups and (3) AFLP and SSR markers datasets showed positive correlation. This study suggests the SSR and AFLP markers are suitable for diversity analysis and cultivars fingerprinting. An understanding of the genetic diversity and population structure of F. carica in Tunisia can also provide insight into the conservation and management of this species.     

Characterization of two recessive genes controlling resistance to all races of bacterial spot in peppers

Bacterial spot, one of the most damaging diseases of pepper, is caused by Xanthomonas euvesicatoria. This pathogen has worldwide distribution and it is particularly devastating in tropical and sub-tropical regions where high temperatures and frequent precipitation provide ideal conditions for disease development. Three dominant resistance genes have been deployed singly and in combination in commercial cultivars, but have been rendered ineffectual by the high mutation rate or deletion of the corresponding cognate effector genes. These genes are missing in race P6, and their absence makes this race virulent on all commercial pepper cultivars. The breeding line ECW12346 is the only source of resistance to race P6 in Capsicum annuum, and displays a non-hypersensitive type of resistance. Characterization of this resistance has identified two recessive genes: bs5 and bs6. Individual analysis of these genes revealed that bs5 confers a greater level of resistance than bs6 at 25°C, but in combination they confer full resistance to P6 indicating at least additive gene action. Tests carried out at 30°C showed that both resistances are compromised to a significant extent, but in combination they provide almost full resistance to race P6 indicating a positive epistatic interaction at high temperatures. A scan of the pepper genome with restriction fragment length polymorphism and AFLP markers led to the identification of a set of AFLP markers for bs5. Allele-specific primers for a PCR-based bs5-marker have been developed to facilitate the genetic manipulation of this gene.     

Genetic variability of cultivated Chaenomeles speciosa (Sweet) Nakai based on AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis was performed to evaluate genetic relationships among 52 Chaenomeles speciosa accessions grown in China. A total of 208 polymorphic markers were generated from eight selective primer pair combinations. Genetic variations were remarkably observed in a wide range of dissimilarities, percent polymorphisms, and average polymorphism information. Genetic similarity values were calculated using Jaccard's coefficient between individuals of different flowering quince accessions; these values ranged from 0.296 to 0.931 with a mean of 0.597. Cluster analysis and principal coordinate analysis were then performed on the basis of AFLP profiles. In our generated dendrogram, accessions were clustered into seven major groups. Mantel's test was performed to determine cophenetic correlation (r = 0.9); this result indicated a very good fit of the dendrogram. We conducted analysis of molecular variance and found greater variations within cultivars than among cultivars. AFLP analysis results further revealed relevant information regarding the genetic background of flowering quince accessions essential for future breeding programs related to plant improvement.