AFLP Primer Pairs Research Articles

Molecular Marker Mapping of RSV4, a Gene Conferring Resistance to all Known Strains of Soybean Mosaic Virus

Soybean mosaic virus (SMV) is a prevalent viral pathogen of soybean [Glycine max (L.) Merr.] wherever it is grown. Several genes that confer resistance in soybean to soybean mosaic virus have been identified. One of these resistance loci, Rsv4, confers resistance to all the known strain groups of SMV. This study was conducted to determine the map position of the Rsv4 locus in the soybean genome. A population of 255 F2 individuals from the cross of the SMV resistant line LR2 (Rsv4) by the susceptible line Lee68 (rsv4) was evaluated in a mapping study. DNA from 12 to 15 F2 individuals being either homozygous resistant or susceptible were pooled to produce bulk resistant and bulk susceptible DNA samples. Parents and bulks were screened with 101 AFLP primer pairs and two linked polymorphisms were identified. A putative linked marker, amplified by the primers Mse‐AAA and Eco‐AAG, was converted to a restriction fragment length polymorphism (RFLP) clone and a single polymorphic band was mapped 4.8 cM away from Rsv4. The same band was mapped in a second population containing 400 mapped loci and it was determined that this marker was located on the soybean D1b linkage group. Subsequent mapping in the original Rsv4 mapping population showed that the Rsv4 locus is flanked by the microsatellite markers, Satt542 at 4.7 cM and Satt558 at 7.8 cM.

Breeding for ”low-gossypol seed and high-gossypol plants” in upland cotton. Analysis of tri-species hybrids and backcross progenies using AFLPs and mapped RFLPs

This work aims at breeding upland cotton [Gossypium hirsutum L., 2(AD)1 genome] with a reduced level of gossypol in the seeds for optimal food and feed uses, and a high gossypol level in the remaining organs for resistance to pests. Two tri-species Gossypium hybrids, (G. thurberi–G. sturtianum–G. hirsutum and G. hirsutum–G. raimondii–G. sturtianum) including G. sturtianum (2C1) as a donor, G. thurberi (2D1) and G. raimondii (2D5) as a bridge species, were created. Recurrent selection initiated with these tri-species hybrids produced backcross (BC) progenies expressing the ”low-gossypol seed and high-gossypol plant” trait at different levels. We used AFLP markers to assess the genetic similarity among the germplasm and RFLP probes to tag the introgression of specific chomosome segments from the parental species. Five pairs of AFLP primers generated 477 fragments, among which 417 (87.4%) were polymorphic. The genetic similarity between the upland cotton and the wild species ranged from 29.5 to 43.2%, while similarity reached 80% between upland cotton and BC3 plants. Introgression of species-specific AFLPs was evident from all the parental species and confirmed the hybrid origin of the analyzed progenies. Southern-blot analysis based on 49 RFLP probes allowed us to trace the introgression of parental DNA segments in the tri-species hybrids and in three generations of backcross. Introgression was evident from 11, 8 and 7 linkage groups of G. sturtianum, G. raimondii and G. thurberi respectively. The types of introgression revealed by RFLP probes are discussed, and breeding schemes to enhance recombination are proposed. The ability to trace DNA segments of known chromosomal locations from the donor G. sturtianum through segregating generations is a starting point to map the ”low-gossypol seed and high-gossypol plant” traits.

Diversity among Selected Vigna angularis (Azuki) Accessions on the Basis of RAPD and AFLP Markers

Genetic diversity within the azuki Vigna angularis (Willd.) Ohwi and Ohashi genepool has not been extensively examined with molecular markers. The objectives of this study were to: (i) use RAPD and AFLP analyses to assess the genetic similarity among selected azuki accessions from distinct production regions in Asia, and( ii) compare the utility of the two DNA marker systems for evaluating similarity. Results also will provide information for parental selection in a breeding program. Fifty‐seven RAPD and 214 AFLP bands were used to assess genetic diversity among 58 azukia acessions. Thirty percent of the RAPD and 18% of the AFLP amplification products were polymorphic among accessions. One AFLP primer pair detected a mean of 11.3 polymorphic bands per reaction versus a mean of 3.2 for each RAPD primer. On the basis of Jaccard's similarity coefficients (JSCs), higher level of molecular genetic variation was detected among accessions evaluated in this study than in other diversity assessments of azuki. Mean JSC based on AFLP data was significantly higher than that obtained from RAPD data (0.71 and 0.52, respectively), and results from phylogenetic analyses varied between DNA marker systems. A substantial difference in the number of polymorphic bands detected through AFLP versus RAPD analysis may have influenced these results since the ability to resolve genetic relationships among genotypes is related to the number of polymorphisms detected. Cluster analysis revealed groupings among accessions; however, no apparent correlations with putative geographic origin were detected. Results of this study suggest that the level of genetic diversity among selected azuki accessions should be sufficient for developing an intraspecific mapping population.

An Evaluation of the AFLP Fingerprinting Technique for the Analysis of Paternity in Natural Populations of Persoonia mollis (Proteaceae)

The accurate assignment of paternity in natural plant populations is required to address important issues in evolutionary biology, such as the factors that affect reproductive success. Newly developed molecular fingerprinting techniques offer the potential to address these aims. Here, we evaluate the utility of a new PCR-based multi-locus fingerprinting technique called Amplified Fragment Length Polymorphism (AFLP) for paternity studies in Persoonia mollis (Proteaceae). AFLPs were initially scored for five individuals from three taxonomic levels for 64 primer pairs: between species (P. mollis and P. levis), between subspecies (P. mollis subsp. nectens and subsp. livens), between individuals within a single population of P. mollis, as well as for a naturally pollinated seed from a single P. mollis subsp. nectens plant. Overall, 1164 fragments (24.6% of all fragments) were polymorphic between species, 743 (16.5%) between subspecies, 371 (8.6%) between individuals within a single population, and 265 (6.2%) between a plant and its seed. Within a single P. mollis population of 14 plants, 42 polymorphic fragments were scored from profiles generated by a single AFLP primer pair. The mean frequency of the recessive allele (q) over these 42 loci was 0.773. Based on these observations, it will be feasible to generate well over 100 polymorphic AFLP loci with as few as three AFLP primer pairs. This level of polymorphism is sufficient to assign paternity unambiguously to more than 99% of all seed in experiments involving small, known paternity pools. More generally, the AFLP procedure is well suited to molecular ecological studies, because it produces more polymorphism than allozymes or RAPDs but, unlike conventionally developed microsatellite loci, it requires no prior sequence knowledge and minimal development time.

Variation in Capsicum annuum revealed by RAPD and AFLP markers

Genetic relationships were examined among thirty-four pepper (Capsicum annuum) cultivars of different types. Two types of PCR-based markers were used, RAPD and AFLP, and their relative effectiveness was compared. A dendrogram based on RAPD markers separated the large-fruited sweet cultivars from the small-fruited pungent peppers, and the former group showed less divergence than the latter. The percentage of polymorphic markers was lower for AFLP than for RAPD markers (13 and 22% respectively). However, AFLP primers amplified on average six times more products than RAPD markers. The average numbers of polymorphic products per primer were 1.6 and 6.5 for RAPD and AFLP primers, respectively, i.e., AFLP primers were four times more efficient than RAPD primers in their ability to detect polymorphism in pepper. While four blocky type cultivars were indistinguishable by RAPD, two AFLP primer pairs were sufficient to distinguish the four cultivars from each other.

Rapid assessment of primer combinations and recovery of AFLP products using ethidium bromide staining

AFLP™ is a novel high-resolution fingerprinting method that can be used to delineate intraspecific relationships among a large variety of fungi and plants. We demonstrate that with the appropriate technical modifications, ethidium bromide staining and non-denaturing polyacryalmide minigels can be an inexpensive and time saving alternative for screening DNA samples for suitable AFLP primer pairs. Furthermore, the recovery of ethidium bromide stained polymorphic DNA fragments is not as tedious as the recovery of isotopic DNA fragments.

Introgression, tagging and expression of a leaf senescence gene in Festulolium

summaryA mutation of a gene (discovered in Festuca pratensis Huds. and designated sidy) confers indefinite greenness on senescing leaves. Via intergeneric hybrids with Lolium multiflorum L. and Lolium perenne L., the mutant gene (sidy) has been introgressed into a range of Lolium backgrounds. Using genomic in situ hybridization we have identified segments carrying sidy in recombinant chromosomes of Lolium–Festuca introgression lines. We also used L. perenne lines segregating 1:1 for the staygreen character to tag the gene with molecular markers. In two mapping populations a total of 84 genotypes were screened with isoenzymes, RAPD primers, RFLP probes and AFLP primer pairs. Over 180 polymorphic loci were identified, representing 10 linkage groups spanning 600 cM. Two AFLP markers are linked to sid at 4.6 and 14.9 cM, close enough to be usable for marker‐assisted selection. Introgression of sidy into Lolium temulentum L. resulted in the production of near‐isogenic inbreeding lines suitable for comparative studies of gene expression. Using a variation of the method of representational difference analysis a very small number of cDNAs have been identified as promising candidates for sid, or genes directly regulated by sid.