accurate identification becomes difficult. DNA fingerprinting techniques have been proven to be reliable for cultivar identification because the markers are not influenced by environmental factors, life stage, or type of plant tissue analyzed. The choice
Marie-José Côté and Lisa Leduc
L.S. Boiteux, M.E.N. Fonseca, and P.W. Simon
Seven plant genomic DNA purification protocols were evaluated for genetic fingerprinting analysis using six tissues obtained from inbred carrot (Daucus carota L.) lines. Evaluations included 1) DNA yield, 2) DNA purity, 3) DNA cleavage with HindIII, 4) DNA integrity, and 5) DNA suitability for amplification in a random amplified polymorphic DNA (RAPD) system. Significant differences were observed among tissues and purification methods for the total amount of DNA. An extraction method using CTAB buffer + organic solvents gave the best results in DNA yield, purity, and HindIII cleavage when compared with the other six nonorganic extraction methods. Of the tissues examined, flowers yielded the most DNA (average value = 115 ng of DNA/mg of fresh tissue); followed by seeds (54 ng·mg-1), fresh leaves (48 ng·mg-1), lyophilized leaves (40 ng·mg-1), calli (22 ng·mg-1), and tap roots (4 ng·mg-1). For most of the preparations, the DNA showed no traces of degradation. However, DNA preparations were not consistently accessible to HindIII cleavage in all tissue-extraction method combinations. Uncut DNA was observed chiefly in extractions from flowers and fresh leaves suggesting a tissue-specific adverse effect on restriction endonuclease activity. Differences in RAPD band (amplicon) intensity and number were observed across tissues and DNA extraction methods using identical PCR conditions for RAPD. Callus was the best type of tissue for RAPD-based fingerprinting yielding a consistently higher number of more intense amplicons when compared to the other tissues. In flowers and seeds, only DNA obtained with the CTAB extraction method could be amplified. Polymorphisms deviating from genetic expectations were mainly observed in root and fresh leaf DNA, indicating that some RAPD markers may not present satisfactory levels of reproducibility. Judicious and uniform selection of DNA purification method as well as tissue source for DNA extraction are, therefore, important considerations for reliable RAPD-based DNA fingerprinting analysis in carrot. In addition, our studies allowed the identification of a better combination of procedures for use in routine manipulations of carrot DNA such as RFLP-RAPD-based cultivar fingerprinting, molecular mapping, screening of transgenic plants, construction of genomic libraries, and gene cloning.
Mirko Siragusa, Fabio De Pasquale, Loredana Abbate, Letizia Martorana, and Nicasio Tusa
RAPD. The usefulness and reliability of these molecular markers has already been proven in several other lemon fingerprinting studies ( Capparelli et al., 2004 ; Fang and Roose, 1997 ). The data obtained have been used to find genetic relationships
Patrick J. Conner and Bruce W. Wood
Genetic variation among pecan [Carya illinoinensis (Wangenh.) C. Koch] cultivars was studied using randomly amplified polymorphic DNA (RAPD) markers. Using a combination of primers, a unique fingerprint is presented for each of the pecan genotypes studied. The genetic relatedness between 43 cultivars was estimated using 100 RAPD markers. Genetic distances, based on the similarity coefficient of Nei & Li, varied from 0.91 to 0.46, with an average value of 0.66 among all cultivars. The phenetic dendrogram developed from cluster analysis showed relatively weak grouping association. However, cultivars with known pedigrees usually grouped with at least one of the parents and genetic similarity estimates appear to agree with known genetic relationships.
Lisa J. Rowland, Smriti Mehra, Anik L. Dhanaraj, Elizabeth L. Ogden, Janet P. Slovin, and Mark K. Ehlenfeldt
Because randomly amplified polymorphic DNA (RAPD) is the only type of molecular marker that has been used extensively in blueberry (Vaccinium spp.) for mapping and DNA fingerprinting of cultivars, there is a need to develop a new, robust marker system. Expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants, were used to develop EST-PCR markers for blueberry. Thirty clones, picked at random from the cDNA library, were single-pass sequenced from the 5' and 3' ends. Thirty PCR primer pairs were designed from the ends of the best quality sequences that were generated and were tested in amplification reactions with genomic DNA from 19 blueberry genotypes, including two wild selections (the original parents of a mapping population), and 17 cultivars. Fifteen of the 30 primer pairs resulted in amplification of polymorphic fragments that were detectable directly after ethidium bromide staining of agarose gels. Several of the monomorphic amplification products were digested with the restriction enzyme AluI and approximately half resulted in polymorphic-sized fragments (cleaved amplified polymorphic sequences or CAPS markers). The polymorphic EST-PCR and CAPS markers developed in this study distinguished all the genotypes indicating that these markers should have general utility for DNA fingerprinting and examination of genetic relationships in blueberry. Similarity values were calculated based on the molecular marker data, and a dendrogram was constructed based on the similarity matrix. Coefficients of coancestry were calculated for each pair of genotypes from complete pedigree information. A fair correlation between similarity coefficients calculated from marker data and coefficients of coancestry was found.
Hong Xu, Diane J. Wilson, S. Arulsekar, and Alan T. Bakalinsky
Randomly amplified polymorphic DNA (RAPD) markers were generated for identifying grape (Vitis) rootstocks. Seventy-seven primers (10 bases long) were screened using CsCl-purified leaf DNA derived from several field samples of nine rootstocks sampled in successive years. Nine RAPD markers were detected from six primers and, in combination, distinguished all nine rootstocks tested. Because inconsistencies were encountered in performing the RAPD assay, sequence-specific primers were derived from cloned RAPD bands for use under more stringent amplification conditions. Southern hybridization analysis of the RAPD gels with cloned RAPD bands as probes revealed deficiencies of scoring RAPD bands based solely on ethidium bromide staining. In some cases, bands of the same size generated by the same primer in different rootstocks-normally scored as the same marker-failed to cross-hybridize, implying lack of homology between the bands. More commonly, bands scored as absent based on ethidium bromide staining were detected by hybridization. Six of the nine cloned RAPD bands were partially sequenced, and sequence-specific primer pairs were synthesized. Two primer pairs amplified a product the same size as the original RAPD band in all rootstocks, resulting in loss of polymorphism. Two other pairs of sequence-specific primers derived from the same marker failed to amplify the expected band consistently. Three of the most useful primer pairs amplified apparent length variants in some accessions and will have value as polymerase chain-reaction markers for fingerprinting.
Peter Boches, Lisa J. Rowland, Kim Hummer, and Nahla V. Bassil
Microsatellite markers for blueberry (Vaccinium L.) were created from a preexisting blueberry expressed sequence tag (EST) library of 1305 sequences and a microsatellite-enriched genomic library of 136 clones.
Microsatellite primers for 65 EST-containing simple sequence repeats (SSRs) and 29 genomic SSR were initially tested for amplification and polymorphism on agarose gels. Potential usefulness of these SSRs for estimating species relationships in the genus was assessed through cross-species transference of 45 SSR loci and cluster analysis using genetic distance values from five highly polymorphic EST-SSR loci. Cross-species amplification for 45 SSR loci ranged from 17% to 100%, and was 83% on average in nine sections. Cluster analysis of 59 Vaccinium species based on genetic distance measures obtained from 5 EST-SSR loci supported the concept of V. elliotii Chapm. as a genetically distinct diploid highbush species and indicated that V. ashei Reade is of hybrid origin. Twenty EST-SSR and 10 genomic microsatellite loci were used to determine genetic diversity in 72 tetraploid V. corymbosum L. accessions consisting mostly of common cultivars. Unique fingerprints were obtained for all accessions analyzed. Genetic relationships, based on microsatellites, corresponded well with known pedigree information. Most modern cultivars clustered closely together, but southern highbush and northern highbush cultivars were sufficiently differentiated to form distinct clusters. Future use of microsatellites in Vaccinium will help resolve species relationships in the genus, estimate genetic diversity in the National Clonal Germplasm Repository (NCGR) collection, and confirm the identity of clonal germplasm accessions.
Reynato P. Umali, Nanako Kameya, and Ikuo Nakamura
The banana (Musa sp., AAA) genome is continuously expanding due to the high frequency of somaclonal variation. Because of this increasing diversity, numerical and morphological methods of taxonomic and phylogenetic identification of banana cultivars became laborious, difficult, and often the subject of disagreements. The aim of this study, therefore, is to develop molecular tools for DNA fingerprinting that can discriminate Musa, AAA Cavendish subgroup cultivars. In this paper, we showed that the plastid-subtype identity (PS-ID) sequence of the noncoding region between rpl16 and rpl14 genes of plastid DNA was highly conserved except for single-base substitution and deletion. These differences separated the clones into three groups (G1, G2, and G3) and suggested that clones within groups are closely related maternally. Using arbitrary primer A13, we later identified negative RAPD markers A133.0 and A131.3 specifically for S4 (selection from Giant Cavendish subgroup, AAA) and S11 (`Morado' from `Red' and `Green Red' subgroup, AAA), respectively. Fragments corresponding to the missing bands were sequenced and used as templates to design new primers with overlapping sequences. Two of these primers, Ba3.0A and Ba1.3A, successfully generated positive markers consistently amplified as Ba3.0A0.8 and Ba1.3A0.6 for S4 and S11, respectively. It is proposed that the method just described can be a better alternative over screening more arbitrary primers in generating positive markers in cases when negative ones were already identified. Results of PS-ID subtype analysis likewise suggested potential use in identifying wild maternal progenitor in polyploid bananas.
Mohammad Sadat-Hosseini, Kourosh Vahdati, and Charles A. Leslie
growth regulators in the culture medium during this time. Table 2. Summary of ISSR primers for genetic analysis of plantlets derived from walnut somatic embryos. Fig. 3. ISSR fingerprint profile obtained with primer (AGTG)4 ( A ), (GA)8-TC ( B ), and CAC
Mario I. Buteler, Don R. LaBonte, and Robert L. Jarret
Microsatellites or simple sequence repeats (SSRs) were used to characterize 20 sweetpotato genotypes and to assign paternity for offspring from crosses among them. The PCR amplifications were performed with each of the sweetpotato genotypes and primers flanking a SSR loci previously characterized with the varieties Beauregard and Excel and 20 offspring from a cross among them. The PCR reaction products were separated in nondenaturing 12% acrylamide gels run at 25 V·cm–1 for 5 hours, and DNA fragments were visualized with silver staining. Gels were scanned on a flat bed scanner and analyzed using the Pro-RFLP software package. Three primer pairs were sufficient to produce an allelic profile capable of differentiating the 20 genotypes from each other. More than seven alleles/loci were found using each of the three primer pairs assayed. Occasionally primers produced allelic products clearly localized in two or three regions of the gel. These multiple loci segregated independently in a diploid fashion. This evidence suggests that there is not total homology among the three sweetpotato genomes.