AFLP Analysis of Opium Poppy

There are several different DNA analysis procedures that have been used to identify and characterize plant Amplified restriction fragment length polymorphic (AFLP) DNA cultivars to determine genetic diversity. Each procedure analysis was performed on leaf samples of 40 accessions of opium poppy (Papaver somniferum L.) and two other control genera (Pahas it own requirements, sensitivity, and reliability. AFLP paver bracteatum Lindley and Papaver setigerum DC.) from a comis the one of the newer DNA analysis procedures, which mercial breeding collection held in Tasmania, Australia. A similarity combines assay flexibility with a high degree of sensitivdendrogram was produced on the basis of the analysis of all AFLP ity and reproducibility to yield significantly more inforbands that ranged between 66 and 367 base pairs (bp) seen on an mation about the plant genome under study than older autoradiogram from denaturing polyacrylamide sequencing electrotechniques (Lin et al., 1996). Studies of genetic diversity phoretic gels with three different primer pairs. It was necessary to based on AFLP DNA analysis have been applied to combine the analysis of all three primer pairs into a single dendrogram bacterial collections (Keim et al., 1997), fungal collecto ensure that replicate analyses of related populations were grouped tions (O’Neill et al., 1997), nematode populations (Semtogether. It was necessary to use more than one primer pair to resolve blat et al., 1998), and accessions from several plant genclearly the genetic relationship between the closely related plants described in this study; however, even a single primer pair could era including Brassica, potato (Solanum tuberosum L.), easily distinguish between populations of different poppy species. soybean [Glycine max (L.) Merr.], barley (Hordeum The application of AFLP DNA analysis for cultivar identification vulgare L.), wheat (Triticum aestivum L.), marijuana represents an efficient, reliable procedure for identifying opium poppy (Cannabis sativa L.), and lupines (Lupinus ssp.) (Brewer breeding lines in a definitive manner. This procedure required only et al., 1999; Brien et al., 1999; Griffiths et al., 1999; a small amount of leaf material for analysis and the degree of the Saunders et al., 1998; Travis et al., 1996). genetic relatedness was performed on autoradiographic banding patThe AFLP procedure is based on the digestion of terns with commercially available software. These data were used genomic DNA by endonuclease restriction enzymes folto generate a similarity dendrogram depicting the predicted genetic lowed by the ligation of adapters. The adapter sequence, relationship of the opium poppy cultivars. Opium poppy accessions restriction site, plus additional selected nucleotides are sharing common parental lines could be distinguished unambiguously from poppy accessions of more distant genetic background. The bandused as priming sites for PCR amplification. Increased ing patterns were reproducible, consistent within a genotype, and the selectivity to differentiate between closely related samDNA polymorphisms were frequent enough to be useful in characterples can be achieved by modifying the user selected nuizing genetic diversity in even closely related breeding lines. cleotides. To test the ability of AFLP markers to discriminate between the gene pools within P. somniferum and to test T plant species Papaver somniferum (poppy) is the potential application of genetic markers to identify grown commercially in several countries in secure poppy cultivars, DNA from 41 poppy accessions includenvironments to produce morphine for the world’s pharing two related species were subjected to AFLP analysis. maceutical industries. The Australian island state of MATERIALS AND METHODS Tasmania is a major supplier of morphine for these industries. Commercial poppy crops have been grown Plant Material in Tasmania since the 1970s with other major suppliers A selection of accessions of P. somniferum were chosen including India, Turkey and selected countries in Eufrom diverse poppy growing areas worldwide for this study rope. To remain competitive with other international (Table 1). In addition to samples of current and former compoppy industries, plant breeding programs in Tasmania mercial lines, representatives from land races and agro-botanihave steadily increased endogenous morphine concencal collections were also included. The hard seeded nondomestrations in poppy straw by conventional breeding methticated types from within the species P. somniferum were also ods. Although strides are being made in manipulating examined, both as an accession from a gene bank and as volunteer outcrosses found in commercial poppy fields. Accesalkaloid levels through traditional breeding programs, sions were chosen to represent the extensive genetic diversity the genetic origin and diversity of many of the accessions that can be found in P. somniferum. Individuals from each of in the breeding collection is not fully known. This study two related species, P. bracteatum and P. somniferum ssp. was initiated to evaluate the genetic diversity of the setigerum, were also included to compare and contrast with breeding populations currently in use in Tasmania to genetic variation within P. somniferum. The P. somniferum provide information on those lines with the greatest ssp. setigerum accession used in this study was found in a genetic heterogeneity. commercial poppy field in Tasmania, Australia. J.A. Saunders and M.J. Pedroni, Alternate Crops & Systems Lab, DNA Extraction USDA/ARS, Bldg. 50, Rm. 100, Beltsville, MD 20705; L.D.J. Penrose Fresh leaf tissue was taken from 60 individual poppy plants and A.J. Fist, Tasmanian Alkaloids Pty. Ltd., Box 130, Westbury, Tasmania 7303, Australia. Received 25 May 2000. *Corresponding shown in Table 1, and grown in the field or greenhouse under author ([email protected]). Abbreviations: AFLP, amplified restriction fragment length polymorphism; bp, base pair; PCR, polymerase chain reaction. Published in Crop Sci. 41:1596–1601 (2001).