Zea Mays L. GenomeThe Relationship Between Genetic and Physical Distances in the Cloned a1-sh2 Interval of the

L Civardi, Y Xia, KJ Edwards, PS Schnable, and BJ Nikolau

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Proc. Nati. Acad. Sci. USAVol. 91, pp. 8268-8272, August 1994Genetics

The relationship between genetic and physical distances in thecloned al-h2 interval of the Zea mays L. genome

(yeast artificial chromosomes/physical mapping/recombination rate/maize)

LAURA CIVARDI*, YuJI XIAt, KEITH J. EDWARDSt, PATRICK S. SCHNABLEt§, AND BASIL J. NIKOLAU*¶Departments of *Biochemistry and Biophysics, tZoology and Genetics, and §Agronomy, Iowa State University, Ames, IA 50011; and tZeneca Seeds, Jealott'sHill Research Station, Bracknell, Berkshire RG12 6EY, United Kingdom

Communicated by Arnel R. Hallauer, February 17, 1994 (received for review September 28, 1993)

ABSTRACT A 470-kb segment from the long arm ofchromosome 3 of Zea mays (inbred LH82), encompassing theal-sh2 interval, was cloned as a yeast artificial chromosome.Comparison of the sizes of the restriction fragments generatedfrom the cloned DNA fragment and from the DNA isolatedfrom the maize inbred line LH82 established the colinearity ofthe al-sh2 interval in these DNAs. By utilizing a chromosomefragmentation technique, a yeast artificial chromosome encom-passing the al-sh2 interval was separately fragmented at the a]and sh2 loci. Comparison of the sizes of these fragmentationproducts established the physical distance between the al andsh2 loci to be 140 kb. Furthermore, these fragmentationexperiments established the physical orientation of the a) andsh2 genes relative to the maize centromere. The molecularcloning of the contiguous region between the a) and sh2 locimade it possible to define the relationship between physical andgenetic distances over a relatively large segment of the maizegenome. In this interval, the relationship between physical andgenetic distances is 1560 kb/centimorgan, which compareswith 1460 kb/centimorgan for the entire maize genome, and217 kb/centimorgan for a 1-kb segment within the al locus.Therefore, these findings are consistent with the hypothesisthat genes per se are preferred sites for meiotic recombinationrather than the hypothesis that genes reside in large recombi-nationally active segments of the genome.

Meiotic recombination is an important force in the evolutionof eukaryotic organisms because of its role in generatinggenetic variability. In addition, meiotic recombination hasutility in basic research, where it is used to genetically mapgenomes, and in breeding, where the products of recombi-nation are used in the improvement of agriculturally impor-tant crops and livestock.The ability to accurately position a locus on a genetic map

may facilitate the molecular isolation of that locus basedsolely on its map position. "Map-based" cloning strategies,such as "chromosome walking," are currently being utilizedin a number of species, including the model plant Arabidopsisthaliana (1), which has a relatively small simple genome. Inaddition, chromosome walking has recently been demon-strated in a crop species with a large complex genome (2).Chromosome walking is facilitated, in part, by the identi-

fication of a molecular marker within a minimum physicaldistance from the gene of interest. Physical distances be-tween loci are usually estimated from their genetic distances.However, genetic and physical distances are not directlyproportional, because the rate of meiotic recombinationbetween loci (the measure of genetic distance) is dependentupon not only the physical distance between loci but also therate of recombination per unit physical length in the region ofthe chromosome being mapped, and this latter value varies

among subgenomic intervals (3-8). Hence, it is not possibleto accurately extrapolate the physical separation betweentwo loci based upon their relative positions on a genetic map,unless locally meaningful estimates of the ratio of genetic tophysical distances are available.

This manuscript describes the isolation of a 470-kb con-tiguous region of the long arm of chromosome 3 ofZea maysthat contains the two genetically and molecularly defined locia) and sh2. This achievement has enabled us to determine, inthe genome of a crop species, the relationship between thegenetic and physical distance over a molecularly clonedinterval larger than a single gene.

MATERIALS AND METHODSMaize Genetic Stocks. The two recessive alleles of al

utilized in this study, al-mum2 and al [here termed al::rdtfollowing the precedent set by Brown and Sundaresan (9)],have Mu) and rdt transposons inserted at nucleotides -97and 1084 of the al gene, respectively (10-12). These allelescondition a stable colorless phenotype in the absence of thetrans-activating regulatory transposons, MuDR and Dt,which were not present in the stocks used in this study.al-mum2 was obtained from pedigreed genetic stocks. The

sweet corn hybrid "Sweet Belle" (Asgrow Seed, Kalama-zoo, MI) is homozygous for the recessive reference allele sh2(data not shown), which arose in coupling with a Dt-responsive allele of al. There are two Dt-responsive allelesof the al locus, al-rdt and al-Cache. The pattern of muta-bility that resulted when Sweet Belle was crossed to a stockcarrying Dt and al-s (a stable allele that does not respond toDt) established that Sweet Belle is homozygous for the al-rdtallele (see figure 1 in ref. 9).Media. Yeasts were cultured in/on complete medium

[YPD: 2% (wt/vol) Bacto Peptone/1% yeast extract/2%(wt/vol) glucose] or selective media (AHC and SD) (13, 14).DNA Probes and Radioisotopic Labeling. DNA probes used

in this study were a 4.3-kb HindIII-EcoRI genomic fragmentcontaining the al gene (pALC2; ref. 15), the two EcoRIfragments from a 1.95-kb cDNA clone of the sh2 gene(pcSh2-la and pcSh2-lb; ref. 16). Probes for the left and rightarm of the vector pYAC4 were obtained as described (17).DNA was isolated from the B73 inbred line of maize using astandard isolation procedure (18). All DNAs were labeledwith [32P]dCTP by the random primer-extension method (19).

Screening of a Yeast Artificial Chromosome (YAC) Libraryof Maize. YAC clones containing al and sh2 sequences wereisolated from the maize YAC library constructed in thevector pYAC4 (20) at the Plant Biotechnology Section ofZeneca Seeds (21). The library was screened by a PCR-based

Abbreviations: YAC, yeast artificial chromosome; cM, centimor-gan(s); PFGE, pulsed-field gel electrophoresis.$To whom reprint requests should be addressed.

8268

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Proc. Natl. Acad. Sci. USA 91 (1994) 8269

approach (21) using primers obtained from the publishedsequences of al and sh2 (15, 16).

Isolation of gh Molecular Weight DNA and Southern BlotAnalysis. Maize high molecular weightDNA was isolated fromleaf protoplasts of the LH82 inbred line (Holden FoundationSeeds, Williamsburg, IA) as described (21). High molecularweight DNA was isolated from yeast strains harboring YACsas described (22). High molecular weight DNA, either prior toor after digestion with restriction endonucleases, was sepa-rated by pulsed-field gel electrophoresis (PFGE) with a Bio-Rad CHEF DRII apparatus, using the following conditions:1% agarose gel in 0.5x TBE (lx TBE = 89 mM Tris base/89mM boric acid/2 mM EDTA) at 200 V and 140C for 20-24 hrwith a switch time of 30 sec. DNA was transferred to a nylonmembrane (Magnagraph; Micron Separations, Westboro,MA) (23). Hybridization conditions were as described (24).

Construction of Fragmentation Plasmids for the al and sh2Loci. The plasmids used for chromosome fragmentation (25)are based on pBCL (26). The EcoRI site of pBCL wasdestroyed by EcoRI digestion, filled in by using the Klenowfragment of DNA polymerase I, and religated. The Alusequence was removed by replacing the Sca I-Sal I fragmentcontaining the Alu sequences and a portion of the ampicillin-resistance gene with the equivalent fragment from pBSK.The resulting plasmid was called pBLC-1.A fragmentation vector for the al locus was constructed by

cloning, in both orientations, the 1436-bp Sma I fragmentcontaining a portion of the a) gene into the Sma I site ofpBLC-1. The derived plasmids were called pA1(+) (with theinsert in a 5' -k 3' orientation relative to the telomere inpBLC-1) and pA1(-) (with the insert in a 3'-) 5' orientationrelative to the telomere in pBLC-1) (Fig. 1A). In a similarfashion, two fragmenting vectors were constructed for thesh2 gene using the 1000-bp EcoRI cDNA fragment frompcSh2-la. The derived plasmids were called pSh2(+) andpSh2(-) according to the definition for the al vectors (Fig.2A). Yeast transformation was carried out by a modifiedlithium acetate method (27).

RESULTSGenetic Characterization of the al-shz Interval of Chromo-

some 3. Intragenic recombination can be used to orient thedirection of transcription of genes relative to linked loci (3).Intragenic recombinants were recovered from the a] locusvia cross 1 by using a detasseled isolation plot procedure (28).Cross 1 was aJ-mum2 Sh2/al::rdt sh2 x al::rdt sh2/al::rdtsh2 (Sweet Belle).Most kernels from cross 1 were colorless (al-mum2/al::

rdt or al::rdt/al ::rdt). However, because all kernels from thiscross carry at least one dominant allele at all other locinecessary for anthocyanin biosynthesis (data not shown), rareintragenic recombination events between the two transposoninsertions at the a) locus that reconstitute a wild-type domi-nant allele (termedAl)') yield colored kernels (Al '/al::rdt). Ifa]is transcribed toward the Sh2 gene, intragenic recombinationwould have produced colored round kernels (Al 'Sh2/al::rdtsh2). In contrast, if a) is transcribed away from the sh2gene, the rare colored kernels would have been shrunken(Al'sh2/al::rdt sh2). Ten colored shrunken kernels wereobtained from a population of215,300 progeny ofcross 1. Testcrosses were used to confirm the genotypes of five of thesecolored shrunken kernels. The isolation of colored shrunkenkernels from cross 1 establishes that a) is transcribed awayfrom sh2 and toward the centromere of chromosome 3. Thisresult confirms that of Brown and Sundaresan (9). Based onthese results, the genetic distance between the transposoninsertions at a) is 0.0046 ± 0.0015 centimorgan (cM).The genetic distance between a) and sh2 was confirmed by

determining the number of genetic recombinants from cross

A pAl(-) e

SI Amp'/ori C

pAl (+)S .S

B

(kbi

485.0 -

388.0 -

291.0 -

l194.0 -

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1 2 3 4 5 6

_! f.1i Ml.i :4 . AfFt

LYS2 TEL

=o Si

c1 2 4 5 6

-* mm 4w - a-

FIG. 1. Fragmentation of YAC ASH-2 at al. (A) Schematic mapof fragmenting vectors for the a) locus. The vectors pAl(+) andpAl(-) were constructed. S, Sma I; Si, Sal I; TEL, telomere. (B)High molecular weight DNA was isolated from a yeast strainharboring YAC ASH-2 (lane 1) and from Lys+, Trp+, Ura- yeastcolonies selected from the transformation of the yeast strain harbor-ing YAC ASH-2 with linearized pAl(-) (lanes 2-6). DNA wasfractionated by PFGE and the gel was stained with ethidium bro-mide. (C) The gel shown inB was subjected to Southern blot analysisand probed with the al-specific sequence.

2. Genetic recombinants were isolated from cross 2 by virtueof their nonparental phenotypes (i.e., colorless round andcolored shrunken kernels). Cross 2 was Al Sh2/al: :rdt sh2 xal::rdt sh2/al::rdt sh2.One hundred thirty nine putative recombinants (30 colored

shrunken kernels and 109 colorless round kernels) wereisolated from a population of 67,000 progeny. The genotypesof =50%o of the kernels with nonparental phenotypes wereconfirmed by test crosses.Based on these crosses, 90o (9/10) and 30% (21/69) of the

colored shrunken and colorless round kernels, respectively,

A pSh2(-) -p5h2+Amp /or

pSh2(+IiFB

!kbk X2 3 4 5

* ..... :.:,f .....:..

485.0

388.0

291 0

194.097.0

c

IL.YS2 TEL

Dp 3 4 5 2 3 4 5

goso

FIG. 2. Fragmentation ofYAC ASH-2 at sh2. (A) Schematic mapof fragmenting vectors for the sh2 locus. The vectors pSh2(+) andpSh2(-) were constructed. E, EcoRI; SI, Sal I; TEL, telomere. (B)High molecular weight DNA was isolated from a yeast strainharboring YAC ASH-2 (lane 1) and from Lys+, Trp+, Ura- yeastcolonies selected from the transformation of the yeast strain harbor-ing YAC ASH-2 with linearized pSh2(-) (lanes 2-5). DNA wasfractionated by PFGE and the gel was stained with ethidium bro-mide. The gel shown inB was subjected to Southern blot analysis andprobed with a fragment specific for the 3' end ofthe sh2 gene (C) andwith a fragment specific for the 5' end of the sh2 gene (D).

Genetics: Civardi et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

were confirmed to have arisen via recombination or geneconversion events. These rates were used to calculate thenumber of true recombinants [(90% x 30) + (30%6 x 109) =60], and thus the genetic distance between a) and sh2 is 0.09± 0.01 cM (60/67,000), a value in close agreement withpublished values [e.g., E. H. Coe, Jr., personal communi-cation in (1983) Maize Newsletter 67, 133-166].

Identification and Characterization ofYAC Clones Contain-ing al and sh2. Four YAC clones were identified as contain-ing the al and/or sh2 sequences based upon their ability tobe amplified with a) - or sh2-derived PCR primers. Two oftheisolated clones (YACs 73i.E6 and 791.G7) hybridized to botha] and sh2 probes (data not shown), suggesting that these twoclones contain the entire genomic region between al and sh2.These two YACs will be referred to as YAC ASH-1 and YACASH-2, respectively. Analyses byPFGE showed that the twoYACs were approximately the same size (470 kb).Of the two remaining YAC clones, one of =70 kb (YAC

510.F2) hybridized only to the al-specific probe, and theother, of =250 kb (YAC 45.C5), hybridized only to thesh2-specific probe. They will be referred to as YAC Al andYAC SH2, respectively.

Restriction Analysis of YACs Containing al and sh2. Arestriction map (Fig. 3b) was constructed for YAC ASH-1using an indirect end-labeling method (19). In addition,restriction fragments released from YAC ASH-1 and YACASH-2 were detected by Southern blot analysis by using totalmaize DNA as probe. Due to the high content of repetitivesequences in the maize genome, all the restriction fragmentsgenerated from YAC ASH-1 and YAC ASH-2 were identi-fied. Comparisons of the sizes of the restriction fragmentsfrom YAC ASH-1 and YAC ASH-2 enabled us to constructa restriction map ofYAC ASH-2 (Fig. 3a) and to confirm therestriction map ofYAC ASH-1. These analyses revealed thatYAC ASH-1 and YAC ASH-2 were nearly identical, with theexception of a 20-kb deletion in YAC ASH-1 relative to YACASH-2. As shown in Fig. 4A, YAC ASH-1 and YAC ASH-2generate the identicalPme I fragments except the largestPmeI fragment contains a deletion of =20 kb in YAC ASH-1. This

Al

deletion is located near sh2, between the a) and sh2 genes(Fig. 3 a and b).

Restriction maps of YAC Al and YAC SH2 were alsoconstructed (Fig. 3 c and d). Comparison of all four restric-tion maps showed that YAC SH2 and YAC Al contain maizeDNA fragments internal to YAC ASH-1 and YAC ASH-2. Inaddition, the maize fragment cloned in YAC SH2 was colin-ear with the maize fragment cloned in YAC ASH-2, confirm-ing that 20 kb of maize DNA was deleted in YAC ASH-1.The integrity of the maize DNA cloned in YAC ASH-2 was

confirmed by identifying restriction fragments generated fromthe YAC and comparing their sizes to the homologous frag-ments in the maize inbred line LH82, which is the source oftheDNA cloned in this YAC (21). Southern blot analysis with theSh2-la probe identified the identicalPme I and Sfi I fragmentsin the maize and YAC DNA (Fig. 4B). In addition, restrictionfragments hybridizing to a) are identical in YAC ASH-2 andthe maize genomic DNA (data not shown).The data reported here demonstrate the fidelity of the

maizeDNA fragment cloned inYAC ASH-2, particularly, theregion between the al and sh2 sequences.

Molecular Positioning of a) and sh2 on YAC ASH-2. Highmolecular weight DNA isolated from a yeast strain harboringYAC ASH-2 was digested to completion with a variety ofrestriction enzymes, and the products were fractionated byPFGE, transferred to nylon membrane, and sequentiallyhybridized with a)- and sh2-specific probes. These analysesunambiguously placed the a) and sh2 genes between restric-tion sites identified in Fig. 3a, thus placing these genesbetween 110 and 190 kb from each other.A more accurate determination of the positions and orien-

tations of the a) and sh2 genes on YAC ASH-2, and thus onthe maize genome, was obtained by chromosome fragmen-tation (25). Fragmentation was carried out with the vectorsthat contain a telomeric sequence and the yeast L YS2 gene asa selectable marker.

Fragmentation ofYAC ASH-2 at the a) locus was done bytransforming with the vectors pAl(+) and pAl(-) that hadbeen linearized by digestion with Sal I to expose the a) andtelomeric sequences. Yeast cells containing the fragmenta-

Sh2

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N A A P P NA

-1 III I I I

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FIG. 3. Restriction maps of YACs containing a) and/or sh2 sequences. Restriction maps were constructed (see Results) for YAC ASH-2(a), YAC ASH-1 (b), YAC Al (c), and YAC SH2 (d) by using the rare-cutting enzymes Asc I (A), Not I (N), Pac I (P), Pme I (Pm), and SfiI (S). The dotted lines connecting YAC ASH-1 and YAC ASH-2 delineate the location ofthe 20-kb deletion identified in the maize DNA fragmentcloned in YAC ASH-1. The horizontal lines above YAC ASH-2 define the 60-kb Sfi I-Pme I and the 20-kb Sfi I-Pac I fragments that containthe a) and the sh2 genes, respectively.

d cI

5O kbII

8270 Genetics: Civardi et al.

Proc. Natl. Acad. Sci. USA 91 (1994) 8271

A

Pme I Sfi I

r- CM - Ckb in (I en in

<

B

Pme I Sfi I

1) C Aw c(N NCU l UC/)E E <

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Pm SIa

sh2

p P NA P

TI I 1 'I I I

194.0 -

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49.0-99^

FIG. 4. Fidelity of the al-sh2 interval cloned in YAC ASH-2. (A)

High molecular weight DNA isolated from yeast strains harboringYAC ASH-i and YAC ASH-2 was digested with Pine I or Sfi The

resulting restriction fragments were fractionated by PFGE, subjectedto Southern blot analysis, and probed with total maize DNA. (B)

High molecular weight DNA isolated from the maize inbred line

LH82 and from a yeast strain harboring YAC ASH-2 was digestedwith Pine I and Sfi The resulting restriction fragments were

separated by PFGE, subjected to Southern blot analysis, and probedwith a sh-specific probe.

tion product were initially selected on minimal medium

lacking lysine and tryptophan.

With vector pAl(-) (Fig. 1A) --500 transformants per pg

of transforming DNA were obtained. One hundred colonies

with the Lys+, Trp+ phenotype were further screened on

selective medium lacking lysine, tryptophan, and uracil to

determine their phenotype. Approximately 35% of the Lys+,Trp+ colonies required uracil to grow, thus, indicating the

loss of the noncentromeric end of YAC ASH-2 that carries

the URA3 gene.

High molecular weight DNA was isolated from some of the

Lysui, Trpit,Urao transformants and separated by PFGE(Fig. 1B). This DNA was subsequently transferred to a nylon

membrane and probed with the al-specific probe (Fig. 1C).This analysis revealed that all the selected yeast strainscontained a fragmented YAC ofb70 kb. As expected, theseYACs contained the a) gene but had lost the sh2 gene (data

not shown). Therefore, the a] gene is positioned 70 kb from

the centromeric end of YAC ASH-2 and is oriented with the

3' end closest to the centromere of the YAC.

The identical procedure was utilized to position the sh2

gene on YAC ASH-2. Yeast cells containing YAC ASH-2

were transformed with the linearized vectors pSh2(-) or

pSh2(+). The vector pSh2(-) (Fig. 2A) gave rise to Lys+,Trp+ colonies at a frequency of ----200 transformants per pg of

transforming DNA. About 46% of these colonies were found

to be also Ura, demonstrating that they had lost the non-centromeric end of YAC ASH-2. PFGE analysis of highmolecular weight DNA isolated from some of these Lys+,Trp+, Ura colonies showed the presence of a common new

fragmented YAC of 7210kb (Fig. 2B). The YACs produced

by fragmentation at the sh2 gene hybridized to thesh2-gaprobe (Fig. 2C), which corresponds to the 3' end of the sh2

cDNA clone, but did not hybridize to the sh2-1b probe (Fig.

FIG. 5. Physical distance between a) and sh2. The molecularanalysis ofYAC ASH-2 and intragenic recombination analysis at a)revealed that the a) and sh2 genes are transcribed toward thecentromere of chromosome 3 of maize and are located 140 kb fromeach other.

2D), which corresponds to the 5' end ofthe Sh2 cDNA clone.A DNA fingerprint analysis ofthe fragmented YAC (data notshown) demonstrated that no internal deletion had occurredin the YACs during the fragmentation procedure and that thefragmented product indeed contained the entire region be-tween the al and sh2 genes as in the original YAC ASH-2.Therefore, the sh2 gene is in the same orientation as the a)gene (i.e., the 3' end of sh2 is closest to the centromeric endof the YAC) and is located 210 kb from the centromeric endof YAC ASH-2.

Therefore, because the a) and sh2 genes are 70 kb and 210kb from the centromeric end of YAC ASH-2, respectively,the physical distance between these two genes is 140 kb.Furthermore, based on the relative orientation of the genes,as determined both by fragmentation of YAC ASH-2 and byintragenic recombination at a), and because a] is proximal tosh2, the orientation and physical arrangement of these twogenes on chromosome 3 is as shown in Fig. 5.

DISCUSSIONThe Rate of Meiotic Recombination Is Variable Across a

Genome. For brevity in the following discussion, we denotethe ratio of the genetic and physical distance for a givengenomic interval as p. Genetically, the entire genome ofmaize contains at least 2061 cM [E. H. Coe, Jr., personalcommunication in (1983) Maize Newsletter 67, 133-166] (butmay be as large as 3000 cM), which encompasses 3 x 106 kbofDNA (29). Therefore, over the entire genome, the value ofp is 1460 kb/cM. However, a number of studies have sug-gested that the value of p is not uniform throughout the entiregenome (refs. 3-5 and 30-32, and J. L. Kermicle cited in ref.33). These studies have established that the value of p withindefined loci is one to two orders ofmagnitude smaller than theaverage value of p over the entire genome. Plant genes,therefore, serve as recombination "hot spots." The exis-tence of hot spots implies that the genome also containsrecombination "cold spots." In fact, data exist to supportthis view; in a number of organisms p is very high in thevicinity of centromeres (6-8, 34). Hence, it is unclear fromthese studies whether genes are hot spots for recombinationper se or whether genes occur in areas of the genome that arethemselves hot spots for recombination.

Determination of the value of p in specific chromosomalsegments has several practical implications. For example, thevalue of p greatly affects the feasibility of chromosomewalking. In addition, it affects the amount oflinkedDNA thatis inadvertently transferred during classical backcrossingprocedures designed to move desirable alleles (such as dis-ease and insect resistance) from unadapted to elite germplasm (i.e., linkage drag).Attempts to estimate the relationship between genetic and

physical distances over subgenomic intervals have beenhampered because until recently it has not been possible toaccurately determine physical distances larger than severaltens of kilobases. Recently, it has become possible to clone

TEL

/I -

Genetics: Civardi et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

large contiguous segments (contigs) of plant genomes byusing YACs. Analyses of such contigs can provide accuratedeterminations of the physical distances between molecularmarkers.

Determination of p Across the al-shz Interval. The studydescribed herein has determined the value of p over arelatively large physical distance in the maize genome. FourYAC clones that contain the al and/or sh2 genes wereisolated from a maize genomic YAC library; one of theseYACs (ASH-2) contained both genes and was shown toencompass the entire interval between al and sh2.By independently fragmenting YAC ASH-2 at the al and

sh2 loci and determining the sizes of the resulting recombina-tion products, we ascertained that the al and sh2 loci areseparated by 140 kb ofDNA. By comparison, we establishedthat the a) and sh2 loci are separated by 0.09 ± 0.01 cM.Therefore, the value ofpfor the al-sh2 interval is 1560 kb/cM.

In addition, we measured the value of p for a 1-kb intervaldefined by two transposon insertions within the a] gene.Genetically, this interval spans 0.0046 ± 0.0015 cM; there-fore, the value of p is 217 kb/cM. Although this value of p issomewhat less than previously reported (9), it is significantlydifferent from the value of p for the al-sh2 interval. Thus thea] locus appears to be more recombinationally active thanthe genome as a whole, and more significantly, it is morerecombinationally active than the neighboring 140-kb intervalbetween the a] and sh2 loci. These findings are consistentwith the hypothesis that recombination occurs at higher ratesin genes per se rather than because genes are most likely tobe found in large recombinationally active chromosomalsegments.The al-shz Interval Is a Model for the Fine Structure

Analysis of Meiotic Recombination in the Maize Genome. Themolecular isolation of the contiguous segment of the chro-mosome encompassing the a) and sh2 loci has long-termimplications for elucidating the interrelationships among re-combination, genome organization, and genome function.These relationships have profound evolutionary implica-tions. For example, intragenic recombination results in thecreation of chimeric alleles. Thus, the evolutionary advan-tages of creating additional genetic diversity in a populationmay have fostered mechanisms that preferentially selectgenes as sites for recombination.

In addition, the physical mapping of the interspersion ofgenes, repetitive, middle-repetitive, and low-repetitive se-quences, in the al-sh2 interval can be correlated with meioticrecombination breakpoints. This type of high-resolutionphysical mapping of recombination breakpoints may uncoverthe molecular features associated with meiotic recombinationhot and cold spots. However, recombination events can onlybe scored when they occur in a marked heterozygote. Be-cause a chromosomal segment undergoes different rates ofrecombination depending upon the particular heterozygotecombination used, absolute values of p for a given chromo-somal segment cannot be determined. For example, p istypically lower when estimated using transposon-insertionmutant alleles of a given locus than when estimated usingalleles carrying point mutations (5, 30, 32, 35). Hence, thevalue of p for the al-sh2 interval is dependent' upon knowncis-acting factors and as yet unidentified cis- and trans-actingfactors specific to the particular heterozygous combinationused in this study.

We thank the following individuals: Dr. Curt Hannah and Dr. AlfonsGierl for the gift of the sh2 and a) clones, respectively; Shane Heinenand John VanDiepen for technical assistance; Lei Zhang for assistingwith the confirmation of meiotic recombinants; and Homer Caton of

AgriPro Seeds for providing access to maize ear drying facilities. Thisresearch was supported by the Midwest Plant Biotechnology Consor-tium, Pioneer Hi-Bred International, Cargill Hybrid Seeds, and theBiotechnology Office of Iowa State University. Y.X. is a graduatestudent at the Iowa State University Interdepartmental GeneticsProgram. This is Journal Paper J-15564 of the Iowa Agriculture andHome Economics Experiment Station, Ames, Iowa and Project 3125.

1. Gibson, S. I. & Somerville, C. (1992) in Methods in Arabidopsis Re-search, eds. Koncz, C., Chua, N. & Schell, J. (World Scientific, RiverEdge, NJ), pp. 119-143.

2. Martin, G. B., Brommonschenkel, S. H., Chunwongse, J., Frary, A.,Ganal, M. W., Spivey, R., Wu, T., Earle, E. D. & Tanksley, S. D. (1993)Science 262, 1432-1436.

3. Dooner, H. K., Weck, E., Adams, S., Ralston, E., Favreau, M. &English, J. (1985) Mol. Gen. Genet. 2W0, 240-246.

4. Nelson, 0. E. (1968) Genetics 6C, 507-524.5. Freeling, M. (1977) Genetics 89, 211-224.6. Tanksley, S. D., Ganal, M. W., Prince, J. P., deVicente, M. C., Boni-

erbale, M. W., Broun, P., Fulton, T. M., Giovannoni, J. J., Grandillo,S., Martin, G. B., Messeguer, R., Miller, J. C., Miller, L., Paterson,A. H., Pineda, O., R6der, M. S., Wing, R. A., Wu, W. & Young, N. D.(1992) Genetics 132, 1141-1160.

7. Roberts, P. A. (1965) Nature (London) 205, 725-726.8. Lambie, F. J. & Roeder, G. S. (1986) Genetics 114, 769-789.9. Brown, J. & Sundaresan, V. (1991) Theor. Appl. Genet. 81, 185-188.

10. O'Reilly, C., Shepherd, N. C., Pereira, A., Schwarz-Sommer, Z., Ber-tam, I., Robertson, D. S., Peterson, P. A. & Saedler, H. (1985) EMBOJ. 4, 877-882.

11. Shepherd, N. S., Sheridan, W. F., Mattes, M. G. & Deno, G. (1988) inPlant Transposable Elements, ed. Nelson, 0. (Plenum, New York), pp.137-148.

12. Brown, J. J., Mattes, M. G., O'Reilly, C. & Shepherd, N. S. (1989) Mol.Gen. Genet. 215, 239-244.

13. Brownstein, B. H., Silverman, G. A., Little, R. D., Burke, D. T., Kors-meyer, S. J., Schlessinger, D. & Olson, M. V. (1989) Science 244,1348-1351.

14. Sherman, F. (1991) in Guide to Yeast Genetics and Molecular Biology,eds. Guthrie, C. & Fink, G. R. (Academic, San Diego), pp. 3-21.

15. Schwarz-Sommer, Z., Shepherd, N., Tacke, E., Gierl, A., Rohde, W.,Leclercq, L., Mattes, M., Berndtgen, R., Peterson, P. A. & Saedler, H.(1987) EMBO J. 6, 287-294.

16. Bhave, M. R., Lawrence, S., Barton, C. & Hannah, L. C. (1990) PlantCell 2, 581-588.

17. Ausubel, F. M., Brent, R., Kingston, R. E. & Moore, D. D., eds. (1988)Current Protocols in Molecular Biology (Greene & Wiley-Interscience,New York).

18. Dellaporta, S. J., Wood, J. & Hicks, J. B. (1983) Plant Mol. Biol. Rep.1, 19-21.

19. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13.20. Burke, D. T., Carle, G. F. & Olson, M. V. (1987) Science 236, 806-812.21. Edwards, K. J., Thompson, H., Edwards, D., deSaizieu, A., Sparks, C.,

Thompson, S. A., Greenland, A. J., Eyers, M. & Schuch, W. (1992)Plant Mol. Biol. 19, 299-308.

22. Albertsen, H. M., Abderrahim, H., Cann, H. M., Dausset, J., LePaslier,D. & Cohen, D. (1990) Proc. Natl. Acad. Sci. USA 87, 4256-4260.

23. Sambrook, J., Fritsch, E. F. & Maniatis, T., eds. (1989) MolecularCloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plain-view, NY), 2nd Ed.

24. Helentjaris, T., Weber, D. & Wright, S. (1985) Plant Mol. Biol. 5,109-118.

25. Vollrath, D., Davis, R. W., Connelly, C. & Hieter, P. (1988) Proc. Natl.Acad. Sci. USA 85, 6027-6031.

26. Lewis, B. C., Shah, N. P., Braun, B. S. & Denny, C. T. (1992) Genet.Anal. Technol. Appl. 9, 86-90.

27. Gietz, D., St. Jean, A., Woods, R. A. & Schiestl, R. H. (1992) NucleicAcids Res. 20, 1425.

28. Peterson, P. A. (1978) in Maize Breeding and Genetics, ed. Walden,D. B. (Wiley, New York), pp. 601-631.

29. Galbraith, D. W., Harkins, K. R., Maddox, J. M., Ayres, N. M.,Sharma, D. P. & Firoozabady, E. (1983) Science 220, 1049-1051.

30. Dooner, H. K. (1986) Genetics 113, 1021-1036.31. Wessler, S. R. & Varagona, R. (1985) Proc. Natl. Acad. Sci. USA 82,

4177-4181.32. Sachs, M., Dennis, E., Gerlach, W. & Peacock, W. J. (1986) Genetics

113, 449-467.33. Dooner, H. K., Keller, J., Harper, E. & Ralston, E. (1991) Plant Cell 3,

473-482.34. Moore, G., Gale, M. D., Kurata, N. & Flavell, R. B. (1993) Biol

Technology 11, 584-589.35. Dooner, H. K. & Kermicle, J. L. (1986) Genetics 113, 135-143.

8272 Genetics: Civardi et al.