The development and use of molecular markers for the detection and exploitation of DNA polymorphism is one of the most significant developments in the field of molecular genetics. The presence of various types of molecular markers, and differences in their principles, methodologies, and applications require careful consideration in choosing one or more of such methods. No molecular markers are available yet that fulfill all requirements needed by researchers. According to the kind of study to be undertaken, one can choose among the variety of molecular techniques, each of which combines at least some desirable properties. This article provides detail review for 11 different molecular marker methods: restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSRs), sequence characterized regions (SCARs), sequence tag sites (STSs), cleaved amplified polymorphic sequences (CAPS), microsatellites or simple sequence repeats (SSRs), expressed sequence tags (ESTs), single nucleotide polymorphisms (SNPs), and diversity arrays technology (DArT).

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An overview of molecular marker methods for plants

In less than a decade, the polymerase chain reaction (PCR) has evolved into the common denominator of molecular biology for nucleic acid research. The most important impact of PCR in plant molecular systematics has been rapid amplification of DNA regions, facilitating nucleotide sequencing (see Chapter 1). However, there are many other techniques aside from DNA sequencing in which PCR is appropriate and extremely useful. In this chapter we review these applications of PCR in plant systematics and highlight areas of research that have been underused in plant molecular systematics laboratories.

Contributions of PCR-Based Methods to Plant Systematics and Evolutionary Biology

Buffalograss, Buchloe dactyloides, is widely distributed throughout the Great Plains of North America, where it is an important species for rangeland forage and soil conservation. The species consists of two widespread polyploid races, with narrowly endemic diploid populations known from two regions: central Mexico and Gulf Coast Texas. We describe and compare the patterns of allozyme and RAPD variation in the two diploid races, using a set of 48 individuals from Texas and Mexico (four population samples of 12 individuals each). Twelve of 22 allozyme loci were polymorphic, exhibiting 35 alleles, while seven 10-mer RAPD primers revealed 98 polymorphic bands. Strong regional differences were detected in the extent of allozyme polymorphism: Mexican populations exhibited more internal gene diversity (He= 0.20, 0.19) than did the Texan populations (He= 0.08, 0.06), although the number of RAPD bands in Texas (n= 62) was only marginally smaller than in Mexico (n= 68). F-statistics for the allozyme data, averaged over loci, revealed strong regional differentiation (mean FRT=+ 0.30), as well as some differentiation among populations within regions (mean FPR=+ 0.09). In order to describe and compare the partitioning of genetic variation for multiple allozyme and RAPD loci, we performed an Analysis of Molecular Variance (AMOVA). AMOVA for both allozyme and RAPD data revealed similar qualitative patterns: large regional differences and smaller (but significant) population differences within regions. RAPDs revealed greater variation among regions (58.4% of total variance) than allozymes (45.2%), but less variation among individuals within populations (31.9% for RAPDs vs. 45.2% for allozymes); the proportion of genetic variance among populations within regions was similar (9.7% for RAPDs vs. 9.6% for allozymes). Despite this large-scale concordance of allozyme and RAPD variation patterns, multiple correlation Mantel techniques revealed that the correlations were low on an individual by individual basis. Our findings of strong regional differences among the diploid races will facilitate further study of polyploid evolution in buffalograss.

Evolutionary implications of allozyme and RAPD variation in diploid populations of dioecious buffalograss Buchloë dactyloides

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L sativa and 13 accessions of the wild species L serriola, L saligna, L virosa, L perennis, and L indica The same genotypes were analyzed as in a previous study that had utilized RFLP markers The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels

PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp

Seventeen olive (Olea europaea L.) cultivars, including oil and table olive cultivars originating from throughout the Mediterranean area, were screened using random amplified polymorphic DNA (RAPD) markers. The results indicate that a high degree of polymorphism is evident in the olive germplasm reexamined. Forty random decamer primers were screened; seventeen of these produced 47 reproducible amplification fragments useful as polymorphic markers. Each of the 17 cultivars can be discriminated with a few primers. Results were analyzed for similarity among the cultivars and a cluster analysis was performed. These analyses revealed two main groups: one comprising primarily small-fruited cultivars grown mainly for oil production, and the other characterized by having large fruit. There was no apparent clustering of olive cultivars according to their geographic origins. Olive (Olea europaea) is one of the most ancient cultivated fruit tree species in the Mediterranean basin. It is the only Mediterranean representative of the genus Olea, which includes 35-40 species distributed over tropical and southern Africa, south Asia, eastern Australia, New Caledonia, and New Zealand (Zohary and Hopf 1993). Traditionally, morphological and phonological traits are used to identify olive cultivars. More recently, workers have found isozymes to be useful for cultivar identification and determining patterns of relatedness among cultivars. Pontikis et al. (1980) identified 27 olive cultivars, mostly of Greek origin, using 16 enzyme systems in pollen. Trujillo et al. (1990) found that by using five pollen enzyme systems they could distinguish 134 of 155 cultivars. Ouazzani et al. (1993) distinguished 33 of 44 cultivars using 9 enzyme systems in leaf tissue. Although isozyme analysis has proved useful in olive, mainly due to the high level of isozyme polymorphism present in the species, direct analysis of DNA could increase considerably the number of markers produced. Furthermore, because isozymes are products of gene expression, differential expression by environment, tissue-specificit y, and other factors is common and may make interpretation of results difficult. Random amplified polymorphic DNA (RAPD) analysis, first described by Williams et al. (1990) and Welsh and McClelland (1990), has proven to be a useful tool for genetic typing and mapping. Bogani et al. (1994) described preliminary results of RAPD analyses of olive cultivars. They used 5 decamer nucleotide primers to generate RAPD polymorphisms among 11 olive cultivars, but found no consistent relationships. In this paper, we report the use of RAPD markers to distinguish among 17 olive cultivars, and we discuss the use of RAPDs to study relationships among cultivars of this species. Materials and Methods Plant materials. Seventeen Olea europaea cultivars were used in this study (Table 1). These were obtained from a collection mainor publication 26 Sept. 94. Accepted for publication 11 Jan 1995. We acknowledge K. Pinney for her capable technical assistance and S. for his advice on technique, J. Hormaza was supported by an INIA rom the Spanish Ministry of Agriculture, The cost of publishing this paper d in part by the payment of page charges. Under postal regulations, this fore must be hereby marked advertisement solely to indicate this fact. om Universita degli Studi di Catania, Istituto di Coltivazioni Arboree, Via 5, Catania Italy 95123. To whom reprint requests should be addressed. dress: Dept. Fruticultura, S.I.A.-D.G.A., Apartado 727, Campus de 0080 Zaragoza, Spain. tained in experimental orchards at the Univ. of California, Davis. DNA extraction and RAPD analysis. Young olive leaves were collected in Spring 1993 and stored at –70C before DNA extraction. Total DNA was extracted from leaf tissue following the CTAB method of Doyle and Doyle (1987) with minor modifications. Young leaves (5.0 g) were ground in liquid nitrogen and mixed with 20 ml of CTAB buffer (100 mM Tris-HCl pH 8, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 1% PVP, 0.2% β− mercaptoethanol, 0.1 % NaHSO4). Samples were incubated at 65C for 1 h, mixed with an equal volume of chloroform–isoamyl alcohol (24:1), and centrifuged at 2000× g for 15 min. The aqueous phase was recovered and mixed with two-thirds volume of isopropanol. The nucleic acid precipitate was recovered with a glass hook, washed with 10 ml of 10 mM ammonium acetate in 76% ethanol, dried overnight, and resuspended in 1 ml of modified TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA). Extracted DNA was diluted to 10 ng·μl and used for PCR amplification. Forty decamer oligonucleotides (Operon Technologies, Alameda, Calif.) were used for PCR amplifications following the procedure of Williams et al. (1990), with some modifications. Amplification reactions were carried out in 25-μl volumes containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.9 mM MgCl2, 0.00 1% gelatin, 100 μM each of dATP, dGTP, dCTP, and dTTP (Promega, Madison, Wis.), 0.4 μM primer, 0.75 units of AmpliTaq DNA polymerase (Perkin-Elmer-Cetus, Norwolk, Conn.) and 50 ng of genomic DNA. Each reaction mixture was overlaid with 25 μl of mineral oil to prevent evaporation. To destroy putative carryover products, reaction mixtures and mineral oil were placed on a UV (300 nm) transilluminator before addition of template DNA and DNA polymerase (Sarkar and Sommer, 1993). DNA amplification reactions were performed in a thermal cycler (PTC100; MJ Research, Watertown, Mass.) programmed for 1 cycle of 2 min at 94C followed by 40 cycles of 45 sec at 92C, 1 min at 36C, 2 min at 72C, for denaturing, annealing and primer extension, respectively. The last cycle was followed by incubation for 5 min at 72C; PCR products were stored at 4C before analysis. Amplification products were analyzed by gel electrophoresis in 2910 agarose (SeaKem; FMC, Rockland, Maine) in 1 x TBE buffer, stained with ethidium bromide, and photographed under UV light using Type 57 Polaroid film. Molecular sizes of amplification products were estimated using a 123 bp DNA ladder (Sigma Chemical Company, St. Louis). All reactions were repeated at least three times using different lots of Taq polymerase and only reproducible bands were used in further analyses. Each amplification fragment J. AMER. SOC. HORT. SCI. 120(3):538–542. 1995. Table 1. Olive cultivars included in this study and their country of origin.

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Random amplified polymorphic DNA analysis of olive (Olea europaea L.) cultivars

Twenty-one celery (Apium graveolens L. var. dulce) cultivars, one celeriac (var. rapaceum) and one annual smallage (var. secalinum) cultivar were screened for polymorphic RAPD (Random Amplified Polymorphic DNA) markers with 28 arbitrary 10-mer primers. Among a total of 309 bands observed, 29 (9.3%) were polymorphic in the 23 cultivars screened, but only 19 (6.1%) markers were polymorphic within the 21 type dulce cultivars. These markers were sufficient to distinguish each of the cultivars used. The average marker difference was 6.4 between two celery cultivars, 16.7 between celery and annual smallage, 14.7 between celery and celeriac, and 12.0 between annual smallage and celeriac. The celery cultivars surveyed were classified into three groups based on the marker differences. The relationship among the dulce-type cultivars concluded from this research is basically consistent with the known lineage of the cultivars and the previous study using stem protein and isozyme markers. RAPD technology provides a new alternative for cultivar identification and classification in celery.

X Yang

Papers

Identification and classification of celery cultivars with RAPD markers.