Crop Science 43:1510-1515 (2003)
© 2003 Crop Science Society of America
PLANT GENETIC RESOURCES
RAPD Analysis of 54 North American Flax Cultivars
Yong-Bi Fu*,a,
Gordon G. Rowlandb,
Scott D. Duguidc and
Ken W. Richardsa
a Plant Gene Resources of Canada, Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, Canada S7N 0X2
b Crop Development Center, Univ. of Saskatchewan, 51 Campus Dr., Saskatoon, SK, Canada S7N 5A8
c Morden Research Station, Agriculture and Agri-Food Canada, Morden, MB, Canada R6M 1Y5
* Corresponding author (fuy{at}em.agr.ca)
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ABSTRACT
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Broadening the genetic base of linseed flax (Linum usitatissimum L.) cultivars to sustain improvement requires assessment of genetic diversity available in flax germplasm. The objective of this study was to analyze the genetic variation, genetic erosion, and genetic relationship of 54 North American flax cultivars by means of random amplified polymorphic DNA (RAPD) markers. The variations observed at the 84 polymorphic RAPD loci were relatively moderate with respect to primer, polymorphism, and cultivar. The proportions of fixed recessive RAPD loci for all the cultivars ranged from 36.9 to 59.2%, with an average of 45.3%. Genetic erosion in the century-long breeding programs was not statistically significant as revealed by the proportion of fixed recessive RAPD loci, but the trend appeared to be that 2.5% of variable RAPD loci were fixed over 100 yr. While some variable RAPD loci were fixed over different breeding periods, the genetic relatedness of the cultivars was reduced in the Canadian programs, but not in the U.S. programs. The genetic relationships of the cultivars inferred via RAPD similarity were largely consistent with known, but incomplete, pedigrees. Both Canadian and U.S. cultivars were intermixed in various groups without distinct separation and several genetically distinct cultivars (i.e., NDR 52, Vimy, Rocket, Norland, Dakota, and Marine) were identified.
Abbreviations: PGRC, Plant Gene Resources of Canada • NA, North American • AC, Agriculture Canada • CDC, Crop Development Center, University of Saskatchewan • RAPD, random amplified polymorphic DNA • PCA, principal component analysis • PCR, polymerase chain reaction • UBC, University of British Columbia
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INTRODUCTION
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PUBLIC LINSEED FLAX BREEDING in North America started in the early 1900s to meet the increased demand by the linseed oil industry (Dillman, 1953; Stoa, 1961; Lay and Hammond, 1984). The goal was to develop high yielding, wilt-resistant flax cultivars, as all known cultivars in North America were susceptible to the soil-borne fungus Fusarium oxysporum f. sp. lini (Bolley) Snyd. & Hans. The breeding efforts generated several well-adapted, wilt-resistant cultivars such as NDR 52, NDR 114, and Bison. By the late 1930s, flax rust casual organism [Melampsora lini (Ehrenb.) Desmaz.] became wide-spread and the breeding efforts were shifted to the development of cultivars with genetic resistance to both wilt and rust. While several wilt- and rust-resistant cultivars such as Royal and Renew were released, the breeding efforts continued into the 1980s as newer races of flax rust were found (Lay and Hammond, 1984; Kenaschuk and Rowland, 1995). Later released rust-resistant cultivars, such as Linott and Dufferin, have kept rust under control. In the last two decades, the breeding emphases have been directed more toward increasing the seed yield potential, decreasing days to maturity, and increasing seed oil concentration (Lay and Dybing, 1989; Kenaschuk and Rowland, 1995). These century-long breeding efforts have produced more than 70 registered flax cultivars and made significant impacts on North American (NA) linseed production.
Interestingly, however, no comprehensive studies have been made of genetic diversity on the flax germplasm released over the last century (Campbell et al., 1995; Fu et al., 2002a). It remains unknown how narrow the genetic base of NA flax breeding programs is and whether any significant genetic erosion has occurred during the breeding process. Also, the pedigree records are incomplete, particularly for those cultivars released before 1950, and no parentage analyses have been found (Lay and Dybing, 1989; Kenaschuk and Rowland, 1995). The genetic relatedness among some released cultivars remains unknown. The lack of genetic studies of flax germplasm using genetic markers has made it more difficult for plant breeders to broaden the genetic basis of their breeding materials for sustainable flax improvement (Carter, 1993; Rowland and Wilen, 1998).
Plant Gene Resources of Canada (PGRC; the Canadian national seed genebank) at Saskatoon maintains a flax collection consisting of 2813 active accessions of cultivated flax from 69 countries and 54 accessions of 26 wild species in the genus Linum (Diederichsen et al., 2000). To support the management and utilization of flax germplasm, the flax collection was characterized by means of RAPD technique (Williams et al., 1990) concurrently with its agrobotanic characterization during the summers of 1999 to 2001. The RAPD characterization generated 108 RAPD loci from 16 informative RAPD primers for each accession. These DNA fingerprints were analyzed to evaluate duplications in the germplasm collection, patterns of variation distribution, and effectiveness of sampling strategies for establishing flax core collections (Fu et al., unpublished). The objective of this study was to analyze the genetic variation, genetic erosion, and genetic relationship of 54 North American flax cultivars by means of RAPD markers.
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MATERIALS AND METHODS
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Selection of Flax Cultivars
Selection of 89 NA flax cultivars and breeding lines was first made from the PGRC flax database on the basis of passport and RAPD data. These accessions were then subjected to further selection based either on the year of their release (Kenaschuk and Comstock, 1974) or on unpublished pedigrees of Canadian flax varieties from E. Kenaschuk, G.G. Rowland, and S.D. Duguid, because some were breeding lines not released for commercial production. This selection generated 54 (28 Canadian and 26 U.S.) cultivars (Table 1) for this study. These cultivars were released from the major breeding efforts reflected in three breeding periods. They were Period 1 for the cultivars released before 1932, Period 2 from 1932 to 1980, and Period 3 after 1980 (Lay and Hammond, 1984; Lay and Dybing, 1989). These periods applied to both Canadian and U.S. breeding programs. It is worth noting that the selections in this study did not include Royal and Renew (dominant rust-resistant cultivars released in the 1940s), U.S. cultivars released after 1980, and several recently released Canadian cultivars (including solin cultivars), as they were not available in the PGRC flax collection. The records of release year for some cultivars such as Bison were found to be inconsistent. In this case, the earlier year was arbitrarily used.
RAPD Characterization
The RAPD characterization procedures used were the same as those previously applied for all the flax accessions held at PGRC and include seed planting in greenhouse, extraction of DNA from young leaves, PCR analysis of isolated DNAs using RAPD primers, and scoring of DNA fingerprints (Fu et al., 2002a). Following are brief descriptions of the major steps. Seeds of each accession listed in Table 1 were obtained from the PGRC flax collection and grown in the greenhouse at the Saskatoon Research Centre, Agriculture and Agri-Food Canada. Young leaves were collected from ten 3- to 4-wk-old seedlings, bulked for each accession, freeze-dried for 3 to 5 d, and stored at -80°C. DNA was extracted from 50 mg of dry tissue in a 2-mL Eppendorf tube by shaking with three glass beads, adding 1 mL of 95°C extraction buffer [0.1 M Tris-HCl (pH 8.0), 10 mM EDTA, 1 M KCl], and incubating at 80°C for 10 min with occasional agitation. The homogenate was centrifuged to remove cell debris. The supernatant was treated with 5 µL of 10 g L-1 RNase A and DNA was precipitated with isopropanol. The DNA was suspended in 50 µL of water and then quantified by fluorimetry with Hoechst 33258 stain (Sigma Chemical Co., St. Louis, Missouri, USA). Typical yields were 10 to 15 µg of DNA and all of the DNA samples were diluted to 2.5 ng µL-1.
Each PCR reaction contained 10 ng DNA as template, 4 U of Taq DNA polymerase (BRL, Mississauga, Ontario, Canada), 50 mM KCl, 2.5 mM MgCl2, 200 µM of each dNTP, and 0.2 µM decamer primer [University of British Columbia (UBC), Vancouver, British Columbia, Canada]. All PCR reactions were performed in the PTC-200 DNA Engine thermocycler (MJ Research, Watertown, MA, USA) with 1.5 min at 95°C, followed by 35 cycles of 20 s at 95°C, 1 min at 36°C (ramp 1°C/s), 1.5 min at 72°C, and a final 7 min cycle at 72°C. PCR products were separated in 2% (w/v) agarose gels in 1x TAE by electrophoresis at 100 V for 3 h. Gels were stained in ethidium bromide and photographed on a digital gel-documentation system (Stratagene, La Jolla, CA, USA).
RAPD analyses were performed with 16 informative UBC RAPD primers (UBC primers 73, 248, 290, 301, 336, 337, 365, 396, 403, 465, 548, 586, 601, 731, 775, and 790). These primers were selected from previous primer screenings and various tests of reproducibility as described in Fu et al. (2002a)(b). The analyses generated 84 polymorphic bands for all accessions. RAPD bands were scored independently by two individuals as present (1) or absent (0) for all accessions.
Data Analysis
In this analysis, each RAPD band was assumed to represent a unique genetic locus. The presence of a RAPD band was interpreted as either a heterozygote or dominant homozygote and the absence of a RAPD band as a recessive homozygote. As each accession was represented with a bulk sample of 10 individuals, the absence of a RAPD band at a locus would also mean a fixation of the recessive allele in the accession [with a frequency of the recessive allele greater than 0.9974 (0.951/2n, where n is the number of the individuals used); Fu, 2000]. Counting the number of fixed recessive RAPD loci for each accession generated the proportion of fixed recessive RAPD loci over the 84 polymorphic loci assessed. A higher proportion of fixed recessive loci obtained would mean lower genetic variation for the accession, when compared with another accession. Thus this measurement provides a simple means of comparing RAPD variations among various accessions, as no estimation of allele frequencies is required. The pattern and extent of RAPD variation were first examined with respect to primer, polymorphism, and cultivar. Then comparisons of RAPD variation were made among various groups of accessions representing three breeding periods and two countries. Later, a linear regression was made of the proportions of fixed recessive RAPD loci over the years of cultivar release to measure the change in genetic diversity in the flax cultivars released over the last 90 yr.
To understand the flax gene pool generated from the breeding programs over the different breeding periods, average RAPD similarities within a group of accessions representing three breeding periods and two countries were calculated from the 84 RAPD loci by the simple match formula (Apostol et al., 1993). These similarities measure the relative genetic relatedness among the accessions within a group. A higher similarity would mean more genetic relatedness among the accessions within the group.
Similarities among all pairs of the 54 accessions were also calculated by the simple match formula (Apostol et al., 1993). This similarity matrix was analyzed with NTSYS-PC 2.01 (Rohlf, 1997) and clustered with unweighted pair-group methods using arithmetic averages (UPGMA) algorithm to determine the genetic relationships among the 54 accessions. To assess the discrepancy between RAPD-based grouping and known pedigree, the RAPD similarity and coefficient of parentage were calculated for each pair of 23 Canadian cultivars with known pedigrees (i.e., those Canadian cultivars released in the second and third breeding periods) and their associations were assessed by a linear regression and plotting. The coefficient of parentage was calculated as described by Falconer (1988) with the following assumptions: (i) all ancestors without complete pedigree information are completely unrelated, (ii) all parents were completely inbred, and (iii) each parent of a biparental cross contributed equally to all progeny derived from the cross. To explore the grouping of these cultivars further, a principal component analysis (PCA) was conducted with SAS (SAS Institute, 1996) on the 54 cultivars using RAPD data as exploratory variables. Plots of the first three resulting principal components were made to assess the associations of various cultivars and identify genetically distinct cultivars.
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RESULTS AND DISCUSSION
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The selected 16 UBC primers generated a total of 84 polymorphic bands over the 54 flax cultivars with an average of five polymorphic bands per primer (Fig. 1A). The primer UBC 790 generated only two polymorphic bands and the primers UBC 73 and UBC 337 displayed nine polymorphic bands. These results agree with those reported previously from a separate, preliminary study (Fu et al., 2002a).
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Fig. 1. Extent of RAPD variations observed in 54 flax cultivars with respect to primer (A), polymorphism (B), and accession (C). A shows most RAPD primers generated 4 to 6 polymorphic bands. B displays most polymorphic RAPD bands were either frequently or infrequently detected in the cultivars. C indicates most cultivars had 43 to 51% of fixed recessive RAPD loci.
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The 84 polymorphic bands in the 54 cultivars appear to be either frequently or rarely present in the cultivars (Fig. 1B). There were 21 polymorphic bands (25%) present in most of the 54 accessions (with an occurrence frequency of 0.9 or greater) and 11 polymorphic bands present only in a few cultivars (with an occurrence frequency of 0.1 or lower). These patterns of RAPD variation were largely expected for the cultivars, as selection generally forces both dominant and recessive alleles toward fixation (Villand et al., 1998). There were two unique bands (UBC primers 336-250 and 465-770) observed in the cultivars Vimy and NDR 52, respectively. These unique bands could be used to distinguish these two cultivars from the other cultivars, if these loci can be proven to be homozygous by examining several plants from each of these cultivars.
The proportions of fixed recessive RAPD loci for all the cultivars ranged from 36.9 to 59.2% with an average of 45.3% (Fig. 1C). This average proportion of fixed loci was lower than those observed for all of the other cultivars in the PGRC collection (51.2% including fiber flax cultivars), higher than those for the flax landrace accessions assessed (42.7%), and comparable with those for the whole flax collection (45.8%; Fu et al., 2002a; Fu et al., unpublished). Thus, the overall RAPD variation present in these flax cultivars was relatively moderate.
Genetic Changes in Flax Breeding Programs
The regression of the proportions of fixed recessive RAPD loci over the registration years (Fig. 2) generated a linear regression coefficient of 0.025 (the proportion of loci per year), which was not statistically significant from zero (P > 0.26). A linear relationship was not obvious between the proportion of fixed recessive RAPD loci and the registration year of the NA flax cultivars released from 1908 to 1998 (Fig. 2). Linear regressions were also made separately on Canadian and U.S. cultivars. While the linear regression coefficient for the Canadian cultivars (0.072) was larger than that for the U.S. cultivars (0.023), none of them were statistically different from zero (P > 0.05).
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Fig. 2. Relationship between the proportion of fixed recessive RAPD loci and the registration year of the North American flax cultivars released from 1908 to 1998. The U.S. and Canadian cultivars were separately presented, but the presented regression line was made on all the cultivars.
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To allow a better understanding of the genetic changes in the breeding programs, average proportions of fixed recessive RAPD loci and average RAPD similarities were estimated for various groups of cultivars representing three breeding periods and two countries. While the obtained estimates (Table 2 and 3) did not differ significantly (P > 0.05) among various groups of cultivars, there were some obvious trends. Both Canadian and U.S. flax breeding programs fixed more variable RAPD loci from the 1930s to 1980, as revealed by an increased proportion of fixed recessive RAPD loci from 42.4 to 45.3% for the Canadian programs and from 43.6 to 46.8% for the U.S. programs (Table 2). This reflects the intensive selection for the development of rust-resistant cultivars during this period. Interestingly, however, such fixation was associated with a decrease in RAPD similarities (or genetic relatedness) of the cultivars released in the Canadian programs from 0.798 to 0.728 over the three breeding periods, compared with an increase from 0.747 to 0.769 in the U.S. programs (Table 3). This may reflect the fact that new germplasm, such as one parental plant of Vimy, was introduced to the Canadian breeding programs, rather than selections being made within the existing germplasm.
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Table 2. Average proportion (and standard deviation) of fixed recessive RAPD loci for flax cultivars of six groups representing three breeding periods of two countries (at diagonal) and average (and standard deviation) of the proportions of fixed recessive RAPD loci for cultivars of two corresponding groups (above diagonal).
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Table 3. Average RAPD similarity (and standard deviation) for flax cultivars of various groups representing three breeding periods of two countries.
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Genetic Relationships of Flax Cultivars
Similarities of all 54 cultivars reflected in the 84 RAPD loci were calculated and clustered into several groups (Fig. 3). First, there were no separate groupings for the Canadian and U.S. cultivars and they were largely intermixed in the various groups, confirming the exchange of germplasm within and between breeding programs of the two countries. Second, many of the groups obtained were consistent within known pedigrees. For example, ‘Somme’, ‘CDC Triffid’, and ‘AC Watson’ were derived from ‘NorLin’, thus grouped together. Similarly, the group of ‘Flanders’, ‘Redwood 65’, ‘Norstar’, ‘Redwood’, ‘Nored’, and Dufferin, were all related to Redwood; the group of ‘Ottawa 829C’, ‘Ottawa 770B’, and ‘Sheyenne’ were all related to Ottawa 770B; and the group of ‘AC Linora’, ‘NorMan’, and Linott were all related to Linott. Third, inconsistent groups also existed. For example, Bison, ‘Novelty’, and ‘B-5128’ were grouped together, unexpectedly deviating from the known pedigrees. ‘Andro’, ‘AC Carnduff’, and ‘AC Emerson’ were clustered as a group, which is difficult to explain from the known pedigrees. NDR 52, ‘CDC Bethune’, and Dakota appeared to be genetically distinct from the other cultivars. This was not expected for CDC Bethune since one of its parents is NorMan.
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Fig. 3. Genetic relationships of the 54 North American flax cultivars reflected in a dendrogram based on their RAPD similarities.
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These clustering results can be explained, at least in part, from the association found between RAPD similarity and parentage coefficient (Fig. 4). The RAPD similarities obtained were significantly correlated with the coefficients of parentage (P < 0.0007), implying that the RAPD-based groupings should be largely consistent with known pedigrees. However, the large prediction interval of the correlation at the 95% level (Fig. 4) indicates discrepancy could occur among RAPD-based clusters and known pedigrees. Such discrepancy also has been found in many related studies with RAPD and other molecular markers (Riedy et al., 1992; Messmer et al., 1993; Bernardo et al., 2000). Thus, the informativeness of the genetic relationships of cultivars inferred via molecular markers can be limited in some studies.
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Fig. 4. Association of RAPD similarity and coefficient of parentage for all the pairs of 23 Canadian flax cultivars with known pedigrees. The linear regression line and its prediction interval at the 95% level were shown in solid and dashed lines, respectively.
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Three principal components were obtained and accounted for 27.8% of the total RAPD variation (11.42, 8.51, and 7.87%, respectively). Although these components explained less than one third of the total variation, the results of the PCA were generally consistent with those obtained through the clustering analyses above. Plots of these components also revealed some genetically distinct cultivars. Three genetically distinct cultivars (Norland, AC Carnduff, and Vimy) were identified in the biplot of the first and second components (Fig. 5A) and another four (NDR 52, Rocket, Dakota, and Marine) in the biplot of the first and third component (Fig. 5B). Fig. 5B also displayed one distinct cluster consisting of CDC Triffid, Somme, NorLin, Andro, AC Watson, and AC Carnduff.
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Fig. 5. Associations of the 54 North American flax cultivars in the biplots of the first three principal components (PRIN1 versus PRIN2 in A and PRIN1 versus PRIN3 in B) that were estimated from the principal component analysis of their original RAPD data. Those labeled cultivars appear to be genetically distinct from the others.
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Implications for North American Flax Breeding
The RAPD analyses performed here did not include two dominant rust-resistant cultivars Royal and Renew, the U.S. cultivars released after 1980, and several recently released Canadian cultivars. Thus the results generated are not complete, nor comprehensive, for understanding the flax gene pool created by the NA breeding programs over the last century. However, the findings obtained are informative for the NA flax breeding programs in several aspects. First, the average proportion of fixed recessive RAPD loci observed for the NA flax cultivars was lower than those observed for the cultivars identified from the other countries (including fiber flax cultivars) and comparable with those assessed from the whole flax collection (Fu et al., unpublished). Thus the overall RAPD variation present in these cultivars was relatively moderate, although there are a few cultivars with relatively high proportions of fixed recessive RAPD loci (>54%) such as Dakota, Marine, and ‘McGregor’. Thus caution is needed in re-selection of these cultivars for special traits. Second, there was little or no evidence of genetic erosion in the century-long NA flax breeding programs. However, more fixed loci were observed in the Canadian, than U.S., programs, even with some introduction of new germplasm such as one parent of Vimy from Russia. Thus, directed effort to diversify the genetic base of breeding material and to seek a new source of favorable alleles would be still desirable. One possible source of useful alleles is from flax landraces, as they harbor more genetic variation than flax cultivars (Fu et al., 2002a). Third, the genetic relationships among the NA flax cultivars established with RAPD data not only show that the Canadian and U.S. cultivars were largely intermixed in various groups, but also revealed some genetically distinct cultivars. These findings should be useful to current and future breeding programs in selections of genetically distinct parents for germplasm development.
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ACKNOWLEDGMENTS
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We thank Mr. Gregory Peterson for his technical assistance on the RAPD characterization of the cultivars analyzed here and Dr. Bruce Coulman and Mr. Yasas Ferdinandez for their helpful comments on the manuscript.
Received for publication March 31, 2002.
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ABSTRACT
INTRODUCTION
