Introduction

Most plants are monoecious and in large part produce hermaphroditic flowers. These plants are expected to allocate resources to female and male function in such away that their fitness is maximized (Charnov 1982). Bothmale and female reproductive success contributes to total fitness. Another important feature of the breedingsystem of such plants is that they are often able to self-pollinate. It is known that for many species selfing produces offspring that have a lower fitness than out-crossed offspring (Charlesworth & Charlesworth 1987;Montalvo 1994).

Good estimates of female reproductive success inplants are relatively easily obtained by counting seed pro-duction. In contrast, selfing rates and male reproductivesuccess are often more difficult to determine.

We are interested in measuring the selfing rate in theself-compatible, insect-pollinated weed, Hound’s Tongue

Cynoglossum officinale, to determine its effect on seed quality and to establish levels of selfing and selectiveabortion (De Jong et al. 1990). Furthermore, we are inter-ested in developing a method to assign paternity tomeasure male reproductive success in relation to resourceallocation to male function.

Measurements of paternity and selfing are increasinglypossible by applying molecular biological techniques. Thebest estimates of paternity and selfing are obtained whenmany loci with multiple alleles are examined. A techniquethat optimizes the estimation of these measures is DNAfingerprinting (Jeffreys et al. 1985). In general other tech-niques, e.g. allozymes and RFLPs, provide far less information per assay then DNA fingerprinting. An addi-tional advantage of DNA fingerprinting (and RFLPs) isthat one membrane can be hybridized many times withdifferent probes (Epplen 1992).

At present, no molecular techniques are available tostudy C. officinale. Our aims were (i) to develop a DNAfinger-printing method for C. officinale, to screen for suitable probe/enzyme combinations, to develop a non-radioactive procedure for this examination, and (ii) toobtain measures of the selfing rate of C. officinale.

S H O R T C O M M U N I C A T I O N

Multilocus DNA fingerprints in the plant Cynoglossumofficinale L. and their use in the estimation of selfing

K . V R I E L I N G ,† ∗ P . S A U M I T O U - L A P R A D E , * E . M E E L I S , † J . T . E P P L E N ‡*Laboratoire de Génétique et Évolution des Populations Végétales, URA CNRS 1185, Université de Lille, Bâtiment SN2, F-59655Villeneuve d’Ascq Cedex, France, †Institute of Evolutionary and Ecological Sciences, Van der Klaauw Laboratory, PO Box 9516,2300 RA Leiden, the Netherlands, ‡Molecular Human Genetics, Ruhr-Universität, W-44780 Bochum, Germany

Summary

We have developed a nonradioactive oligonucleotide multilocus DNA fingerprintingmethod for Cynoglossum officinale. Of the 19 probes tested, six probes yielded bandingpatterns for all restriction enzymes used. All but one of the informative probes are repeatswith a four-base motif. Approximately 60% of the loci appeared to be polymorphic. Thesensitivity of the nonradioactive method was equal to that of the radioactive method. Inaddition, a new simple calculation method is presented to estimate selfing rates andapproximate 95% confidence limits from the DNA fingerprint profiles avoiding‘between-gel’ comparisons. The selfing rates differed significantly (as determined from95% confidence intervals) between naturally pollinated individuals of C. officinale with-in the experimental population. The estimates ranged from 0 to 70% selfing.

Keywords: multilocus DNA fingerprinting (GATA)4, synthetic oligonucleotide probes,Cynoglossum officinale, Hound’s-Tongue

Received 23 September 1996; revision accepted 7 January 1997

Molecular Ecology 1997, 6, 587–593

© 1997 Blackwell Science Ltd

Correspondence: Klaas Vrieling. Fax: + 31 71 5274900. E-mail:Vrieling@Rulsfb.LeidenUniv. ofnl

Material and Methods

Screening for useful probe/enzyme combinations by theradioactive approach

Leaf samples of 1 g fresh weight were taken from four toeight different genotypes of C. officinale and ground withliquid nitrogen. Extraction of the DNA followed a slightlymodified procedure described by Dellaporta et al. (1983).Addition of 2% PVP 40 (w/v) to the DNA-extractionbuffer was found to be optimal to complex the interferingphenolic compounds (Couch & Fritz 1990; Maliyakal1992). Approximately 4 µg of DNA was digested by one ofthe following restriction enzymes according to the manu-facturers’ instructions: MspI, HaeIII, RsaI, AvaI, EcoRI orBamHI (10 units/µg DNA). The digested DNA was pre-cipitated, dissolved in TE and electrophoresed on an 0.8%agarose gel for ≈ 20 h at 30 V. One gel contained EcoRI-,HaeIII-, RsaI-, MspI- and AvaI-digested DNA of at leastfour individuals, the other gel contained EcoRI-, HaeIII-and BamHI-digested DNA from eight individuals. Thetwo gels were dried and hybridized several times with dif-ferent synthetic radioactively labelled oligonucleotides(Table 1). A detailed description of the hybridization anddetection procedures has been given by Epplen (1992).

Non-radioactive DNA fingerprinting

After the screening of probes was completed, the mostinformative probe was chosen for testing a nonradioactive,chemolumninescent method. Approximately 4 µg of DNAof eight individuals was digested by EcoRI, HaeIII orBamHI (10 units/µg DNA). After electrophoresis on an0.8% agarose gel, the DNA was blotted on to a nylon membrane. The membrane was hybridized with a 5′digoxigenine-labelled (GATA)4 probe. Hybridization anddetection using chemoluminescence were carried out fol-lowing established methods (Epplen 1992).

Plant material

Rosette plants of C. officinale were collected in the autumnof 1993 from different populations in the dunes ofMeijendel near the Hague in the Netherlands. Eighteenplants were transferred to 10-L pots filled with dune sandand were placed outside in the experimental garden nearthe dunes. Plants were pollinated by the naturally occur-ring bumblebees. From all 18 parent plants some leaveswere collected and stored at – 80 °C prior to DNA isola-tion. All seeds were harvested and those from a selectionof plants were sown. After germination (100%), seedlingswere transferred to pots with a peatmould and kept in agreenhouse. About 1 g of fresh leaf tissue was harvestedfrom each offspring for DNA isolation. For all parents anda selected number of offspring families fingerprint profiles

were established from ≈ 4 µg of DNA digested with HaeIII(10 units/µg) and probed with nonradioactively labelled(GATA)4.

Analysis of the data

As comparisons between gels were not possible, digestedDNA of one family of offspring was always put on a geltogether with a sample of the mother to allow identifica-tion of nonmaternal bands (Fig. 1). In addition, all 18 parent plants were analysed on one gel for proper com-parison among the parents (Fig. 2).

An individual offspring was considered outcrossed if itshowed nonmaternal bands. It was assumed that non-maternal bands were not caused by mutations. If onlymaternal bands were present in the profile of an individ-ual offspring, it could not be immediately designated asselfed. Rather it could have been fertilized by a nonself

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 587–593

588 K . V R I E L I N G E T A L .

Fig. 1 DNA fingerprint patterns of a mother and 14 of her off-spring of Cynoglossum officinale. The DNA was digested withHaeIII and probed with a 5′ digoxigenine-labelled (GATA)4 probe.The parent plant is indicated with a P, outcrossed offsprings areindicated by an arrow.

gamete, which produces a banding pattern indiscerniblefrom that of a self-gamete. However, the probability that aparticular parent plant produces gametes which wouldyield patterns that are identical to those produced by themother’s gametes can be calculated. For this reason theparents were all run on one gel. The following method isanalogous to the methods developed by Shaw et al. (1981)and further elaborated by Cruzan et al. (1994). The band-ing patterns of all possible parent plants were comparedwith that of the mother plant, and for each plant the num-ber of nonmaternal bands was counted. Provided that thenonmaternal bands are not linked or heterozygous theprobability that a particular parent plant produces agamete indiscernible from a maternal gamete is 0.5n

(where n is the number of nonmaternal bands present inthe population). For each possible parent plant the proba-bility of producing gametes that were indistinguishablefrom a maternal gamete was calculated. Furthermore, ifwe assume that all plants produce an equal and largeamount of pollen (say m), then we can calculate the prob-ability that an offspring is selfed if it has a banding patternsimilar to that of a selfed offspring. The theoretical proba-bility of selfing (Os), given the fact that no nonmaternalbands are present in the offsprings profile is therefore:(number of self pollen grains)/(number of self pollengrains + number of pollen grains from all other parentsindiscernible from self pollen grains):

mOs =

m+(y1*m+y2*m+ . . . +yi*m)(1)

or

Os =1

(2)1+y1+y2+ . . . +yi

with the expectation of yi:

E(yi) = 0.5nji

for i parent plants, with the ith plant having nj non-maternal bands. The estimated fraction of selfing (Eps) isthen calculated as: observed fraction of offspring with nononmaternal bands*Os.

The 95% confidence limits were approximated by com-bining the variances of each possible father assuming abinomial distribution for the offspring indiscernible fromselfs. For father i with ji nonmaternal bands the expecta-tion of producing offspring with no nonmaternal bandsfor k offspring sampled from the mother is kpi with a vari-ance of kpi(1–pi) (with p = 0.5j).

These variances can be joined using the general form-ula for combining variances of independent variables:

var(Os)=var(yi)*(dOs)2+var(y2)*(dOs)2

+. . .+var(yi)*(dOs)2(3)

dy1 dy2 dyi

In our case Os is

Os(y1,y2, . . . , yi) =1

(4)(1+y1+y2+ . . . +yi)

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Probe MspI HaeIII RsaI AvaI EcoRI BamHI

(CA)8 – – – – –(GT)8 Smears but no bands visible(TC)8 10/5 4/0 –(GC)8 – – –(AGC)5 – – – – –(GTG)5 – – – – –(CAC)5 – – –(GAA)6 – – – – –(TCT)8

* – – – – –(TCT)6 – – –(GCC)5 – – –(TTTC)4 – – – – –(TTTC)5 – – – – –(TTTC)4 – – –(GGAT)4 8/0 16/10 8/4 8/3 16/5(GACA)4 7/3 13/9 9/7 9/5 14/8(GATA)4 12/5 27/20 20/14 12/8 23/15(CTAT)5CTA 16/11 11/8 10/1(CTAT)4CT 13/10 12/8 5/1GATAGACAGATA 7/2 27/18 12/10 12/6 19/15(TTAGGG)3 – – – – –(CT)4(CA)5 – – –

* A very weak signal is present perhaps due to wearing of the gel.

Table 1 Survey of simple repeatoligonucleotide/restrictionenzyme combinations used forgenerating multilocus fingerprintpatterns in Cynoglossum officinale.Two gels were hybridized severaltimes with different radioactivelylabelled probes. Gel I containedfour genotypes either digestedwith MspI, HaeIII, RsaI or EcoRIand three genotypes digestedwith AvaI. Gel II contained eightgenotypes either digested withHaeIII, EcoRI or BamHI. The firstnumber represents the totalnumber of readable bandpositions, the second number isthe number of band positionswhere variation betweenindividuals was observed. – , nodistinct bands, smear or veryvague pattern

with its first derivative beingdOs

=–1

(5)dyi (1+y1+y2+ . . . +yi)2

The variance of the fraction of yi is estimated as

var (xi) = pi(1–pi) (6)k

Substituting 5 and 6 in 3 yields

var(Os) =p1(1–p1) + . . . +

p1(1–p1) (7)k*(1+p1+p2+ . . . +pi)4 k*(1+p1+p2+ . . . +pi)4

which can be simplified to

var(Os) =(p1(1–p1) + p2(1–p2) + . . . + pi(1–pi)) (8)

k*(1 + p1 + p2 + . . . + pi)4

The variance of Eps is therefore calculated as: (observedfraction of offspring with no nonmaternal bands)2 * var(Os). The approximate 95% confidence limits wereobtained by multiplying the standard deviation of Eps by1.96.

Results

Screening of probes/enzymes

Of the 14 groups of radioactive probes used, five groupsgenerated informative patterns for all the restrictionenzymes used (Table 1). Distinguishing information was

© 1997 Blackwell Science Ltd, Molecular Ecology, 6, 587–593

590 K . V R I E L I N G E T A L .

Fig. 2 DNA fingerprint patterns of allparent plants of Cynoglossum officinale in theexperimental population. The DNA wasdigested with HaeIII and probed with a 5′digoxigenine-labelled (GATA)4 probe.

obtained with some ‘four-base motif’ probes associatedwith the HaeIII and EcoRI restriction enzymes (Table 1).Out of the 23 readable probe/enzyme combinations 21showed bands that were polymorphic. Overall 59% of thebands were polymorphic despite the small number ofplants used.

The commonly used probe (GATA)4 showed the high-est number of scorable bands and the highest number ofbands with variation between individuals (Fig. 3a). Twobase-pair and three base-pair motifs only yielded a read-able fingerprint pattern in one case. Generally, when aprobe gave a clear banding pattern with a particularrestriction enzyme, it was informative in combination withother restriction enzymes.

Nonradioactive visualization

The number of bands visible and readability increasedwhen a digoxigenine-labelled (GATA)4 probe was usedcompared to the radioactive probe (Fig. 3a,b). Intensebands appeared crisper and faint bands became more pro-nounced using chemoluminescence. Sensitivity of thechemoluminescent method appeared similar to that ofradioactive methods (Fig. 3a,b).

Percentage selfing

The selfing rate for individual plants in C. officinale variedfrom ≈ 0 to 0.70 (Table 2). The calculated 95% confidenceintervals indicate that selfing rates differed significantlybetween individuals (Table 2). The probability of selfing(Os), which was calculated from comparison of the profileof the mother with the profiles of all the possible parents,varied between 0.66 and 0.97 with an average of 0.85.

Discussion

For a many enzyme/probe combinations, informativebanding patterns were obtained. Furthermore, variation inbands between individuals was ubiquitous as ≈ 60% of thebands were polymorphic (Table 1). As plants were sam-pled from different populations within Meijendel,variation within natural populations is expected to besmaller than found in this experiment because the selfingrate is rather high and seed dispersal is limited (De Jong etal. 1990).

Using the nonradioactive method, thick bands becameless pronounced and faint bands became more pro-nounced as reported previously (Weising et al. 1991;Bierwerth et al. 1992). Non-radioactive patterns weretherefore easier to score. Of the two and three base repeatmotifs, only (TC)8 yielded a readable DNA fingerprintprofile. The results of this screening could be a roughguide for choosing the best candidates for screeningmicrosatellite markers in the C. officinale genome.

In general most bands are very close to each other, andhence between-gel comparison appeared to be impossiblefor large parts of the profiles. However, within-gel com-parison of profiles is possible (Figs 1 and 2). Therefore thismethod is not suitable for paternity analysis. Doubledigestion with restriction enzymes may offer better reso-lution and allow comparison between gels.

Because between-gel comparison is not possible, thecommonly used programs of Ritland (1990) for estimationof percentage selfing were not applicable. The method pre-sented here invokes a number of assumptions. First, it isassumed that all bands are unlinked. At present theknowledge is limited, but linkage between fingerprint lociin plants appears to be low (Dallas 1988; Weising et al.1995) depending on the number of chromosomes.

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Fig. 3 DNA fingerprint patterns of 10 genotypes ofCynoglossum officinale. The DNA was digested with HaeIII andprobed with (GATA)4. (A) Radioactive detection, (B) chemolumi-nescent detection. Note that different genotypes were used in Aand B.

Secondly, it is assumed that all loci are heterozygous.Estimates calculated for heterozygosity were higher than90% in humans (Jeffreys et al. 1985). As humans are oblig-ate outcrossers the level of heterozygosity for selfingplants might be substantially lower. In the worst case, if all(nonmaternal) bands are homozygous, all outcrossed andconsequently all selfed individuals are detected as such,provided that each genotype has a unique band or uniquecombination of bands.

In that case, because in the model we assume that allnonmaternal bands are heterozygous, a proportion of thesetruly selfed individuals are supposed to be undetectedoutcrosses and hence percentage selfing is under-estimated. In fact the error made in that case is 1–Os.Therefore, the error in the estimation of the selfing ratedue to the assumption of heterozygosity declines (i) withan increasing number of nonmaternal bands in the poten-tial fathers and (ii) with a decreasing number of potentialfathers. As many unique bands are present in our experi-mental C. officinale population (Fig. 2) and the populationis fairly small the average maximal error in the estimationof the selfing rate is 15% by assuming complete heterozy-gosity for nonmaternal bands if they are in fact allhomozygous.

The third assumption is that pollen production by allindividuals is equal. Pollen production per anther as wellas the total number of flowers produced can differbetween plants. However, no significant differences werefound in pollen production between genotypes of C. offici-nale (P. G. L. Klinkhamer & T. J. De Jong, personalcommunication).

Differences in number of flowers among the potentialfathers are expected to level out if the number of possible

fathers is large enough. The important factor is the num-ber of flowers (and therefore pollen produced) on themother plant in relation to the average number of flowersin the population. For plants with few flowers the Os willbe too high, and for large plants with many flowers theestimate will be too low. The number of flowers per plantvaries between 20 on small plants to 100 on large plants inthe field (Klinkhamer et al. 1989; Klinkhamer & De Jong1993). This fivefold difference would lead to an estimationof the probability of selfing that is 14% too low for smallplants and 6% too high for large plants with a calculatedOs of 0.9. If Os declines, the error increases rapidly. If Osis larger than 0.9, errors caused by the number of flowersare relatively small. The variance of Os is also influencedby differences in flower number. If the numbers of flowerson the parent plants are known, they can be used as correction factors for both Os and the variance of Os. Inaddition it is implicitly assumed that each pollen grain hasan equal probability of fertilizing an ovule or that a self-pollen grain has the same probability of reaching a floweras a pollen grain from the plant farthest away.Presumably, self-pollen has a much larger probability ofreaching a flower than outcross pollen in C. officinalebecause most pollinators visit multiple flowers on a plantin succession (Klinkhamer et al. 1989). Therefore, mostpollen transfer is within the plant and not between plants.Klinkhamer et al. (1989) found that on average seven flow-ers were visited in a row. If the carryover of pollen is low,it is expected that the amount of self pollen greatly exceedsthe amount of outcross pollen on stigmas. In general car-ryover is reported to be very low (Cresswell 1994;Cresswell et al. 1994). This would yield an underestimationof the selfing rate. The variance in this case will always bean overestimation.

Furthermore one has to take care that the Os calculatedfrom the parent gel takes into account only the region ofbands that is also readable in the progeny gel. Readabilityof the small fragments may differ especially between gels.

Despite the aforementioned assumptions, this methodoffers the advantage of estimating selfing without thenecessity of making comparisons between gels. The non-radioactive (GATA)4 probe combined with the restrictionenzyme HaeIII offers a valuable method of estimating self-ing rates in C. officinale because many highly variablebands are present in its pattern. The lowest number ofnonmaternal bands present for each possible pair of par-ents was four. That is a resolution which is not readilyobtained by allozymes or RFLPs. The major drawback ofDNA fingerprinting method is that locus heterozygositycannot readily be determined.

In this study the estimated selfing rates varied largelybetween genotypes and this variation is comparable withthat found in other insect pollinated species like Eichhorniapaniculata and Iris fulva and I. hexagona (Morgan & Barrett

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Table 2 Estimated percentage selfing (Eps) with 95% confi-dence limits of nine individual Cynoglossum officinale plants froman experimental garden population of 18 plants. Fnnm denotesthe fraction of offspring analysed that contained no nonmaternalbands. If a fingerprint profile of the particular offspring did notcontain nonmaternal bands then Os indicates the calculated prob-ability that such an offspring is selfed. See Material & Methods forfurther details. Different letters after the Eps column indicate sig-nificant differences based on the 95% confidence limits

OffspringMother analysed Fnnm Os Eps

2 14 0.714 0.974 0.696 ± 0.078 a10 22 0.682 0.926 0.631 ± 0.092 a11 34 0.471 0.870 0.409 ± 0.082 b13 12 0.417 0.933 0.389 ± 0.120 b17 12 0.500 0.713 0.356 ± 0.128 b9 24 0.333 0.859 0.286 ± 0.099 b

18 10 0.300 0.922 0.276 ± 0.137 b6 18 0.056 0.664 0.037 ± 0.093 c

12 21 0 0.750 0.000 ± 0.102 c

1990; Cruzan et al. 1994). An explanation for this large vari-ation in selfing rate might be due to differences in plantsize, because pollinator behaviour changes with the num-ber of flowers on a plant. The number of flowers visited insequence increases with plant size (Geber 1985;Klinkhamer et al. 1989; Harder & Barrett 1995) leading tothe expectation that selfing also increases with plant size(Klinkhamer et al. 1993; De Jong & Klinkhamer 1994;Harder & Barret 1995). The variation in size of the plantsin our experimental population might have been respon-sible for large differences in selfing rate between plants.

Acknowledgements

We thank three anonymous referees for constructive and detailedcomments on this manuscript. This research was supported bythe ‘Human capital and mobility fund’ of the EC to K. Vrieling.

References

Bierwerth S, Kahl G, Weigand F, Weising K (1992)Oligonucleotide fingerprinting of plant and. fungal genomes:A comparison of radioactive, colorigenic and chemilumines-cent detection methods. Electrophoresis, 13, 115–122.

Charlesworth D, Charlesworth B (1987) Inbreeding depressionand, its evolutionary, consequences. Annual Review of Ecologyand Systematics, 18, 237–268.

Charnov EL (1982) The Theory of Sex Allocation. PrincetonUniversity. Press, Princeton, NJ.

Couch JA, Fritz PJ (1990) Isolation of DNA from plants high inpolyphenolics. Plant Molecular Biology Reporter, 8, 8–12.

Cresswell JE (1994) A method for quantifying the gene flow thatresults from a single bumblebee visit using transgenic oilseedrape, Brassica napus L. cv. Westar. Transgenic Research, 3,134–137.

Cresswell JE, Bassom AP, Bell SA, Collins SJ, Kelly TB (1994)Predicted pollen dispersal by honey-bees and three species ofbumble-bees foraging on oil-seed rape: a comparison of threemodels. Functional Ecology, 9, 829–841.

Cruzan MB, Hamrick JL, Arnold ML, Bennett BD (1994) Matinsystem variation in hybridizing irises: effects of phenologyand floral densities on family outcrossing rates. Heredity, 72,95–105.

Dallas JF (1988) Detection of DNA ‘fingerprints’ of cultivated riceby, hybridization with a human minisatellite DNA probe.Proceedings of the National Academy of Sciences, 85, 6831–6835.

Dellaporta SL, Wood Jand, Hicks JB (1983) A plant DNAminipreperation: version II. Plant Molecular Biology Reporter, 1,19–21.

Epplen JT (1992) The methodology of multilocus DNA finger-printing using radioactive or nonradioactive oligonucleotideprobes specific for simple repeat motifs. In: Advances inElectrophoresis (eds Chrambach A, Dunn MJ, Radola BJ), Vol. 5,pp. 59–112. VCH, Weinheim.

Geber MA (1985) The relationship of plant size to self pollinationin Mertensia ciliata. Ecology, 66, 762–772.

Harder LD, Barrett SCH (1995) Mating costs of large floral dis-plays in hermaphroditic plants. Nature, 373, 512–515.

Jeffreys AJ, Wilson V, Thein SL (1985) Individual-specific ‘finger-prints’ of human DNA. Nature, 316, 76–79.

de Jong TJ, Klinkhamer PGL (1994) Plant size and. reproductivesuccess through female and male function. Journal of Ecology,82, 399–402.

de Jong TJ, Klinkhamer PGL, Boorman LA (1990) Cynoglossumofficinale L. Biological flora of the British Isles no. 170. Journal ofEcology, 78, 1123–1144.

Klinkhamer PGL, de Jong TJ, (1993) Attractiveness to pollinators:a plant’s dilemma. Oikos, 66, 180–184.

Klinkhamer PGL, de Jong TJ de Bruyn GJ, 1989) Plant size andpollinator visitation in Cynoglossum officinale. Oikos, 54,201–204.

Maliyakal EJ (1992) An efficient method for isolation of RNA andDNA from plants containing polyphenolics. Nucleic AcidsResearch, 20, 2381.

Montalvo A (1994) Inbreeding depression and maternal effects inAquilegia caerula, a partially selfing plant. Ecology, 75,2395–2409.

Morgan MT, Barrett SCH (1990) Outcrossing rates and correlatedmating within a population of Eichhornia paniculata(Pontederiaceae). Heredity, 64, 271–280.

Ritland K (1990) A series of FORTRAN computer programs forestimating plant mating system. Journal of Heredity, 81,235–237.

Shaw DV, Kahler AL, Allard RW (1981) A multilocus estimator ofmating system parameters in plant populations. Proceedings ofthe National Academy of Sciences USA, 78, 1298–1302.

Weising K, Beyermann B, Ramser J, Kahl G (1991) Plant DNAfingerprinting with radioactive and digoxigenated oligonu-cleotide probes complementary to simple repetitive DNAsequences. Electrophoresis, 12, 159–168.

Weising K, Nybom H, Wolff K, Meyer W (1995) DNAFingerprinting in Plants and Fungi. CRC Press, Boca Raton, AnnArbor, London, Tokyo.

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