Proc. Nati. Acad. Sci. USA Vol. 91, pp. 8268-8272, August 1994 Genetics The relationship between genetic and physical distances in the cloned 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's Hill Research Station, Bracknell, Berkshire RG12 6EY, United Kingdom Communicated by Arnel R. Hallauer, February 17, 1994 (receivedfor review September 28, 1993)

ABSTRACT A 470-kb segment from the long arm of among subgenomic intervals (3-8). Hence, it is not possible chromosome 3 of Zea mays (inbred LH82), encompassing the to accurately extrapolate the physical separation between al-sh2 interval, was cloned as a yeast artificial chromosome. two loci based upon their relative positions on a genetic map, Comparison of the sizes of the restriction fragments generated unless locally meaningful estimates of the ratio of genetic to from the cloned DNA fragment and from the DNA isolated physical distances are available. from the maize inbred line LH82 established the colinearity of This manuscript describes the isolation of a 470-kb con- the al-sh2 interval in these DNAs. By utilizing a chromosome tiguous region of the long arm of chromosome 3 ofZea mays fragmentation technique, a yeast artificial chromosome encom- that contains the two genetically and molecularly defined loci passing the al-sh2 interval was separately fragmented at the a] a) and sh2. This achievement has enabled us to determine, in and sh2 loci. Comparison of the sizes of these fragmentation the genome of a crop species, the relationship between the products established the physical distance between the al and genetic and physical distance over a molecularly cloned sh2 loci to be 140 kb. Furthermore, these fragmentation interval larger than a single gene. experiments established the physical orientation of the a) and sh2 genes relative to the maize centromere. The molecular cloning of the contiguous region between the a) and sh2 loci MATERIALS AND METHODS made it possible to define the relationship between physical and Maize Genetic Stocks. The two recessive alleles of al genetic distances over a relatively large segment of the maize utilized in this study, al-mum2 and al [here termed al::rdt genome. In this interval, the relationship between physical and following the precedent set by Brown and Sundaresan (9)], genetic distances is 1560 kb/centimorgan, which compares Mu) with 1460 kb/centimorgan for the entire maize genome, and have and rdt transposons inserted at nucleotides -97 217 kb/centimorgan for a 1-kb segment within the al locus. and 1084 of the al gene, respectively (10-12). These alleles Therefore, these findings are consistent with the hypothesis condition a stable colorless phenotype in the absence of the that genes per se are preferred sites for meiotic recombination trans-activating regulatory transposons, MuDR and Dt, rather than the hypothesis that genes reside in large recombi- which were not present in the stocks used in this study. nationally active segments of the genome. al-mum2 was obtained from pedigreed genetic stocks. The sweet corn hybrid "Sweet Belle" (Asgrow Seed, Kalama- is for the recessive allele sh2 Meiotic recombination is an important force in the evolution zoo, MI) homozygous reference of eukaryotic organisms because of its role in generating (data not shown), which arose in coupling with a Dt- genetic variability. In addition, meiotic recombination has responsive allele of al. There are two Dt-responsive alleles utility in basic research, where it is used to genetically map of the al locus, al-rdt and al-Cache. The pattern of muta- genomes, and in breeding, where the products of recombi- bility that resulted when Sweet Belle was crossed to a stock nation are used in the improvement of agriculturally impor- carrying Dt and al-s (a stable allele that does not respond to tant crops and livestock. Dt) established that Sweet Belle is homozygous for the al-rdt The ability to accurately position a locus on a genetic map allele (see figure 1 in ref. 9). may facilitate the molecular isolation of that locus based Media. Yeasts were cultured in/on complete medium solely on its map position. "Map-based" cloning strategies, [YPD: 2% (wt/vol) Bacto Peptone/1% yeast extract/2% such as "chromosome walking," are currently being utilized (wt/vol) glucose] or selective media (AHC and SD) (13, 14). in a number ofspecies, including the model plantArabidopsis DNA Probes and Radioisotopic Labeling. DNA probes used thaliana (1), which has a relatively small simple genome. In in this study were a 4.3-kb HindIII-EcoRI genomic fragment addition, chromosome walking has recently been demon- containing the al gene (pALC2; ref. 15), the two EcoRI strated in a crop species with a large complex genome (2). fragments from a 1.95-kb cDNA clone of the sh2 gene Chromosome walking is facilitated, in part, by the identi- (pcSh2-la and pcSh2-lb; ref. 16). Probes for the left and right fication of a molecular marker within a minimum physical arm of the vector pYAC4 were obtained as described (17). distance from the gene of interest. Physical distances be- DNA was isolated from the B73 inbred line of maize using a tween loci are usually estimated from their genetic distances. standard isolation procedure (18). All DNAs were labeled However, genetic and physical distances are not directly with [32P]dCTP by the random primer-extension method (19). proportional, because the rate of meiotic recombination Screening of a Yeast Artificial Chromosome (YAC) Library between loci (the measure of genetic distance) is dependent of Maize. YAC clones containing al and sh2 sequences were upon not only the physical distance between loci but also the isolated from the maize YAC library constructed in the rate ofrecombination per unit physical length in the region of vector pYAC4 (20) at the Plant Biotechnology Section of the chromosome being mapped, and this latter value varies Zeneca Seeds (21). The library was screened by a PCR-based

The publication costs of this article were defrayed in part by page charge Abbreviations: YAC, yeast artificial chromosome; cM, centimor- payment. This article must therefore be hereby marked "advertisement" gan(s); PFGE, pulsed-field gel electrophoresis. in accordance with 18 U.S.C. §1734 solely to indicate this fact. $To whom reprint requests should be addressed.

8268 Downloaded by guest on September 23, 2021 Genetics: Civardi et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8269

approach (21) using primers obtained from the published A pAl(-) e LYS2 TEL sequences of al and sh2 (15, 16). SI Amp'/ori Si Isolation of gh Molecular Weight DNA and Southern Blot C =o Analysis. Maize high molecular weight DNA was isolated from pAl (+) leaf protoplasts of the LH82 inbred line (Holden Foundation S .S Seeds, Williamsburg, IA) as described (21). High molecular weight DNA was isolated from yeast strains harboring YACs B c as described (22). High molecular weight DNA, either prior to 1 2 4 5 6 or after digestion with restriction endonucleases, was sepa- (kbi 1 2 3 4 5 6 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/89 485.0 - _! f.1i Ml :4 mM boric acid/2 mM EDTA) at 200 V and 140C for 20-24 hr 388.0 - .i . AfFt with a switch time of 30 sec. DNA was transferred to a nylon 291.0 - membrane (Magnagraph; Micron Separations, Westboro, l194.0 - MA) (23). Hybridization conditions were as described (24). 97.0 - -* mm 4w - a- Construction of Fragmentation Plasmids for the al and sh2 48.5 - Loci. The plasmids used for chromosome fragmentation (25) are based on pBCL (26). The EcoRI site of pBCL was destroyed by EcoRI digestion, filled in by using the Klenow fragment of DNA polymerase I, and religated. The Alu FIG. 1. Fragmentation of YAC ASH-2 at al. (A) Schematic map was the Sca I-Sal of fragmenting vectors for the a) locus. The vectors pAl(+) and sequence removed by replacing I fragment pAl(-) were constructed. S, Sma I; Si, Sal I; TEL, telomere. (B) containing the Alu sequences and a portion of the ampicillin- High molecular weight DNA was isolated from a yeast strain resistance gene with the equivalent fragment from pBSK. harboring YAC ASH-2 (lane 1) and from Lys+, Trp+, Ura- yeast The resulting plasmid was called pBLC-1. colonies selected from the transformation ofthe yeast strain harbor- A fragmentation vector for the al locus was constructed by ing YAC ASH-2 with linearized pAl(-) (lanes 2-6). DNA was cloning, in both orientations, the 1436-bp Sma I fragment fractionated by PFGE and the gel was stained with ethidium bro- containing a portion of the a) gene into the Sma I site of mide. (C) The gel shown in B was subjected to Southern blot analysis pBLC-1. The derived plasmids were called pA1(+) (with the and probed with the al-specific sequence. insert in a 5' -k 3' orientation relative to the telomere in pBLC-1) and pA1(-) (with the insert in a 3'-) 5' orientation 2. Genetic recombinants were isolated from cross 2 by virtue relative to the telomere in pBLC-1) (Fig. 1A). In a similar of their nonparental phenotypes (i.e., colorless round and fashion, two fragmenting vectors were constructed for the colored shrunken kernels). Cross 2 was Al Sh2/al: :rdt sh2 x sh2 gene using the 1000-bp EcoRI cDNA fragment from al::rdt sh2/al::rdt sh2. pcSh2-la. The derived plasmids were called pSh2(+) and One hundred thirty nine putative recombinants (30 colored pSh2(-) according to the definition for the al vectors (Fig. shrunken kernels and 109 colorless round kernels) were 2A). Yeast transformation was carried out by a modified isolated from a population of67,000 progeny. The genotypes lithium acetate method (27). of =50%o of the kernels with nonparental phenotypes were confirmed by test crosses. RESULTS Based on these crosses, 90o (9/10) and 30% (21/69) ofthe colored shrunken and colorless round kernels, respectively, Genetic Characterization of the al-shz Interval of Chromo- some 3. Intragenic recombination can be used to orient the direction of transcription of genes relative to linked loci (3). A pSh2(-) - IL.YS2 TEL Intragenic recombinants were recovered from the a] locus p5h2+Amp /or via cross 1 by using a detasseled isolation plot procedure (28). Cross 1 was aJ-mum2 Sh2/al::rdt sh2 x al::rdt sh2/al::rdt sh2 (Sweet Belle). pSh2(+IiF Most kernels from cross 1 were colorless (al-mum2/al:: B c D rdt or al::rdt/al ::rdt). However, because all kernels from this 2 3 4 5 p 3 4 5 2 3 4 5 cross carry at least one dominant allele at all other loci !kbk X rare necessary for anthocyanin biosynthesis (data not shown), * ..... :.:,f.....:.. intragenic recombination events between the two transposon insertions at the a) locus that reconstitute a wild-type domi- 485.0 goso nant allele (termedAl)') yield colored kernels (Al '/al::rdt). Ifa] 388.0 is transcribed toward the Sh2 gene, intragenic recombination 291 0 would have produced colored round kernels (Al 'Sh2/al::rdt 194.0 sh2). In contrast, if a) is transcribed away from the sh2 97.0 gene, the rare colored kernels would have been shrunken (Al'sh2/al::rdt sh2). Ten colored shrunken kernels were obtained from a population of215,300 progeny ofcross 1. Test FIG. 2. Fragmentation ofYAC ASH-2 at sh2. (A) Schematic map crosses were used to confirm the genotypes of five of these of fragmenting vectors for the sh2 locus. The vectors pSh2(+) and colored shrunken kernels. The isolation of colored shrunken pSh2(-) were constructed. E, EcoRI; SI, Sal I; TEL, telomere. (B) kernels from cross 1 establishes that a) is transcribed away High molecular weight DNA was isolated from a yeast strain from sh2 and toward the centromere of chromosome 3. This harboring YAC ASH-2 (lane 1) and from Lys+, Trp+, Ura- yeast colonies selected from the transformation ofthe yeast strain harbor- result confirms that of Brown and Sundaresan (9). Based on ing YAC ASH-2 with linearized pSh2(-) (lanes 2-5). DNA was these results, the genetic distance between the transposon fractionated by PFGE and the gel was stained with ethidium bro- insertions at a) is 0.0046 ± 0.0015 centimorgan (cM). mide. The gel shown inB was subjected to Southern blot analysis and The genetic distance between a) and sh2 was confirmed by probed with a fragment specific for the 3' end ofthe sh2 gene (C) and determining the number of genetic recombinants from cross with a fragment specific for the 5' end of the sh2 gene (D). Downloaded by guest on September 23, 2021 8270 Genetics: Civardi et al. Proc. Natl. Acad. Sci. USA 91 (1994) were confirmed to have arisen via recombination or gene deletion is located near sh2, between the a) and sh2 genes conversion events. These rates were used to calculate the (Fig. 3 a and b). number of true recombinants [(90% x 30) + (30%6 x 109) = Restriction maps of YAC Al and YAC SH2 were also 60], and thus the genetic distance between a) and sh2 is 0.09 constructed (Fig. 3 c and d). Comparison of all four restric- ± 0.01 cM (60/67,000), a value in close agreement with tion maps showed that YAC SH2 and YAC Al contain maize published values [e.g., E. H. Coe, Jr., personal communi- DNA fragments internal to YAC ASH-1 and YAC ASH-2. In cation in (1983) Maize Newsletter 67, 133-166]. addition, the maize fragment cloned in YAC SH2 was colin- Identification and Characterization of YAC Clones Contain- ear with the maize fragment cloned in YAC ASH-2, confirm- ing al and sh2. Four YAC clones were identified as contain- ing that 20 kb of maize DNA was deleted in YAC ASH-1. ing the al and/or sh2 sequences based upon their ability to The integrity ofthe maize DNA cloned in YAC ASH-2 was be amplified with a)- or sh2-derived PCR primers. Two ofthe confirmed by identifying restriction fragments generated from isolated clones (YACs 73i.E6 and 791.G7) hybridized to both the YAC and comparing their sizes to the homologous frag- a] and sh2 probes (data not shown), suggesting that these two ments in the maize inbred line LH82, which is the source ofthe clones contain the entire genomic region between al and sh2. DNA cloned in this YAC (21). Southern blot analysis with the These two YACs will be referred to as YAC ASH-1 and YAC Sh2-la probe identified the identical Pme I and Sfi I fragments ASH-2, respectively. Analyses by PFGE showed that the two in the maize and YAC DNA (Fig. 4B). In addition, restriction YACs were approximately the same size (470 kb). fragments hybridizing to a) are identical in YAC ASH-2 and Of the two remaining YAC clones, one of =70 kb (YAC the maize genomic DNA (data not shown). 510.F2) hybridized only to the al-specific probe, and the The data reported here demonstrate the fidelity of the other, of =250 kb (YAC 45.C5), hybridized only to the maize DNA fragment cloned in YAC ASH-2, particularly, the will region between the al and sh2 sequences. sh2-specific probe. They be referred to as YAC Al and Molecular Positioning of a) and sh2 on YAC ASH-2. High YAC SH2, respectively. molecular weight DNA isolated from a yeast strain harboring Restriction Analysis of YACs Containing al and sh2. A YAC ASH-2 was digested to completion with a variety of restriction map (Fig. 3b) was constructed for YAC ASH-1 restriction enzymes, and the products were fractionated by using an indirect end-labeling method (19). In addition, PFGE, transferred to nylon membrane, and sequentially restriction fragments released from YAC ASH-1 and YAC hybridized with a)- and sh2-specific probes. These analyses ASH-2 were detected by Southern blot analysis by using total unambiguously placed the a) and sh2 genes between restric- maize DNA as probe. Due to the high content of repetitive tion sites identified in Fig. 3a, thus placing these genes sequences in the maize genome, all the restriction fragments between 110 and 190 kb from each other. generated from YAC ASH-1 and YAC ASH-2 were identi- A more accurate determination of the positions and orien- fied. Comparisons of the sizes of the restriction fragments tations of the a) and sh2 genes on YAC ASH-2, and thus on from YAC ASH-1 and YAC ASH-2 enabled us to construct the maize genome, was obtained by chromosome fragmen- a restriction map of YAC ASH-2 (Fig. 3a) and to confirm the tation (25). Fragmentation was carried out with the vectors restriction map ofYAC ASH-1. These analyses revealed that that contain a telomeric sequence and the yeast L YS2 gene as YAC ASH-1 and YAC ASH-2 were nearly identical, with the a selectable marker. exception of a 20-kb deletion in YAC ASH-1 relative to YAC Fragmentation of YAC ASH-2 at the a) locus was done by ASH-2. As shown in Fig. 4A, YAC ASH-1 and YAC ASH-2 transforming with the vectors pAl(+) and pAl(-) that had generate the identical Pme I fragments except the largestPme been linearized by digestion with Sal I to expose the a) and I fragment contains a deletion of =20 kb in YAC ASH-1. This telomeric sequences. Yeast cells containing the fragmenta- Al Sh2 SfA1| > Pmd Sf1 20I1 Pod L1- A

PP PN A N A A P P N A P P PN A A P NA N PP A P A NPP PP I I I lI I I I I I lI I III II II11 I I I I I I I II II I II l l S S S S S Pm S Pm Pm Pm S S SS wz If Pms 1 I : A 01 pH AN A A P PlInI0 PP PH A A P NA N PP A P A NPP PP I , JI I I V IIi ba; ILL I I I I I I I I I I I s TI S 8 S S S Pm S Pm Pm Pm S S S S C I4 A ., elpH it1 - S AS

cIN A A P P NA P P PA APNA N P P A I I I _ d I I I II I I II -1 II I I I I I I I I 5O kb S S S S S s S Pm S Pm le II 6Mu

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 Sfi I (S). The dotted lines connecting YAC ASH-1 and YAC ASH-2 delineate the location ofthe 20-kb deletion identified in the maize DNA fragment cloned 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 contain the a) and the sh2 genes, respectively. Downloaded by guest on September 23, 2021 Genetics: Civardi et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8271

A B 140 kb

Pme I Sfi I Pme I Sfi I al sh2

r- 1) C wA c( CM - C N N CEN AN A A Fp P NA P TEL CU l kb in (I en in UC/) - E E < /I < I I I1 I' Pm S TI I a

FIG. 5. Physical distance between a) and sh2. The molecular 194.0 - analysis of YAC ASH-2 and intragenic recombination analysis at a) 4',, revealed that the a) and sh2 genes are transcribed toward the centromere of chromosome 3 of maize and are located 140 kb from 146.0- each other. 2D), which corresponds to the 5' end ofthe Sh2 cDNA clone. 97.0 - A DNA fingerprint analysis ofthe fragmented YAC (data not shown) demonstrated that no internal deletion had occurred 49.0-99^ in the YACs during the fragmentation procedure and that the fragmented 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 end of the YAC) and is located 210 kb from the centromeric end of YAC ASH-2. Therefore, because the a) and sh2 genes are 70 kb and 210 FIG. 4. Fidelity of the al-sh2 interval cloned in YAC ASH-2. (A) kb from the centromeric end of YAC ASH-2, respectively, High molecular weight DNA isolated from yeast strains harboring the physical distance between these two genes is 140 kb. YAC ASH-i and YAC ASH-2 was digested with Pine I or Sfi The Furthermore, based on the relative orientation of the genes, resulting restriction fragments were fractionated by PFGE, subjected as determined both by fragmentation of YAC ASH-2 and by to Southern blot analysis, and probed with total maize DNA. (B) intragenic recombination at a), and because a] is proximal to High molecular weight DNA isolated from the maize inbred line sh2, the orientation and physical arrangement of these two LH82 and from a yeast strain harboring YAC ASH-2 was digested genes on chromosome 3 is as shown in Fig. 5. with Pine I and Sfi The resulting restriction fragments were separated by PFGE, subjected to Southern blot analysis, and probed with a sh-specific probe. DISCUSSION

tion product were initially selected on minimal medium The Rate of Meiotic Recombination Is Variable Across a lacking lysine and tryptophan. Genome. For brevity in the following discussion, we denote With vector pAl(-) (Fig. 1A) --500 transformants per pg the ratio of the genetic and physical distance for a given interval as p. of transforming DNA were obtained. One hundred colonies genomic Genetically, the entire genome of maize contains at least 2061 cM H. Coe, Jr., personal with the Lys+, Trp+ phenotype were further screened on [E. communication in selective medium lacking lysine, tryptophan, and uracil to (1983) Maize Newsletter 67, 133-166] (but may be as as 3000 encompasses x kb determine their phenotype. Approximately 35% of the Lys+, large cM), which 3 106 ofDNA (29). Therefore, over the entire genome, the value of Trp+ colonies required uracil to grow, thus, indicating the p is 1460 a sug- loss of the noncentromeric end of YAC ASH-2 that carries kb/cM. However, number of studies have gested that the value of p is not uniform throughout the entire the URA3 gene. genome (refs. 3-5 and 30-32, and J. L. Kermicle cited in ref. High molecular weight DNA was isolated from some of the 33). These studies have established that the value of p within Lysui, Trpit,Urao transformants and separated by PFGE defined loci is one to two orders ofmagnitude smaller than the (Fig. 1B). This DNA was subsequently transferred to a nylon average value of p over the entire genome. Plant genes, membrane and probed with the al-specific probe (Fig. 1C). therefore, serve as recombination "hot spots." The exis- This analysis revealed that all the selected yeast strains tence of hot spots implies that the genome also contains containedb70a YAC of kb. As fragmented expected, these recombination "cold spots." In fact, data exist to support YACs contained the a) gene but had lost the sh2 gene (data this view; in a number of organisms p is very high in the not shown). Therefore, the a] gene is positioned 70 kb from vicinity of centromeres (6-8, 34). Hence, it is unclear from the centromeric end of YAC ASH-2 and is oriented with the these studies whether genes are hot spots for recombination 3' end closest to the centromere of the YAC. per se or whether genes occur in areas ofthe genome that are The identical procedure was utilized to position the sh2 themselves hot spots for recombination. gene on YAC ASH-2. Yeast cells containing YAC ASH-2 Determination of the value of p in specific chromosomal were transformed with the linearized vectors pSh2(-) or segments has several practical implications. For example, the pSh2(+). The vector pSh2(-) (Fig. 2A) gave rise to Lys+, value of p greatly affects the feasibility of chromosome Trp+ colonies at a frequency of ----200 transformants per pg of walking. In addition, it affects the amount oflinked DNA that transforming DNA. About 46% of these colonies were found is inadvertently transferred during classical backcrossing to be also Ura, demonstrating that they had lost the non- procedures designed to move desirable alleles (such as dis- centromeric end of YAC ASH-2. PFGE analysis of high ease and insect resistance) from unadapted to elite germ molecular weight DNA isolated from some of these Lys+, plasm (i.e., linkage drag). Trp+, Ura colonies showed the presence of a common new Attempts to estimate the relationship between genetic and fragmented YAC of 7210kb (Fig. 2B). The YACs produced physical distances over subgenomic intervals have been by fragmentation at the sh2 gene hybridized to thesh2-ga hampered because until recently it has not been possible to probe (Fig. 2C), which corresponds to the 3' end of the sh2 accurately determine physical distances larger than several cDNA clone, but did not hybridize to the sh2-1b probe (Fig. tens of kilobases. Recently, it has become possible to clone Downloaded by guest on September 23, 2021 8272 Genetics: Civardi et al. Proc. Natl. Acad. Sci. USA 91 (1994) large contiguous segments (contigs) of plant genomes by AgriPro Seeds for providing access to maize ear drying facilities. This using YACs. Analyses of such contigs can provide accurate research was supported by the Midwest Plant Biotechnology Consor- determinations of the physical distances between molecular tium, Pioneer Hi-Bred International, Cargill Hybrid Seeds, and the markers. Biotechnology Office of Iowa State University. Y.X. is a graduate Determination of p Across the al-shz Interval. The study student at the Iowa State University Interdepartmental Genetics described herein has determined the value of p over a Program. This is Journal Paper J-15564 of the Iowa Agriculture and relatively large physical distance in the maize genome. Four Home Economics Experiment Station, Ames, Iowa and Project 3125. YAC clones that contain the al and/or sh2 genes were 1. Gibson, S. I. & Somerville, C. (1992) in Methods in Arabidopsis Re- isolated from a maize genomic YAC library; one of these search, eds. Koncz, C., Chua, N. & Schell, J. (World Scientific, River YACs (ASH-2) contained both genes and was shown to Edge, NJ), pp. 119-143. encompass the entire interval between al and sh2. 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) By independently fragmenting YAC ASH-2 at the al and Science 262, 1432-1436. sh2 loci and determining the sizes of the resulting recombina- 3. Dooner, H. K., Weck, E., Adams, S., Ralston, E., Favreau, M. & tion products, we ascertained that the al and sh2 loci are English, J. (1985) Mol. Gen. Genet. 2W0, 240-246. separated by 140 kb of DNA. By comparison, we established 4. Nelson, 0. E. (1968) Genetics 6C, 507-524. that the a) and sh2 loci are separated by 0.09 ± 0.01 cM. 5. Freeling, M. (1977) Genetics 89, 211-224. 6. Tanksley, S. D., Ganal, M. W., Prince, J. P., deVicente, M. C., Boni- Therefore, the value ofpforthe al-sh2 interval is 1560 kb/cM. erbale, M. W., Broun, P., Fulton, T. M., Giovannoni, J. J., Grandillo, In addition, we measured the value of p for a 1-kb interval S., Martin, G. B., Messeguer, R., Miller, J. C., Miller, L., Paterson, defined by two transposon insertions within the a] gene. A. H., Pineda, O., R6der, M. S., Wing, R. A., Wu, W. & Young, N. D. Genetically, this interval spans 0.0046 ± 0.0015 cM; there- (1992) Genetics 132, 1141-1160. the value of is 217 this value of is 7. Roberts, P. A. (1965) Nature (London) 205, 725-726. fore, p kb/cM. Although p 8. Lambie, F. J. & Roeder, G. S. (1986) Genetics 114, 769-789. somewhat less than previously reported (9), it is significantly 9. Brown, J. & Sundaresan, V. (1991) Theor. Appl. Genet. 81, 185-188. different from the value of p for the al-sh2 interval. Thus the 10. O'Reilly, C., Shepherd, N. C., Pereira, A., Schwarz-Sommer, Z., Ber- a] locus appears to be more recombinationally active than tam, I., Robertson, D. S., Peterson, P. A. & Saedler, H. (1985) EMBO the genome as a whole, and more significantly, it is more J. 4, 877-882. interval 11. Shepherd, N. S., Sheridan, W. F., Mattes, M. G. & Deno, G. (1988) in recombinationally active than the neighboring 140-kb Plant Transposable Elements, ed. Nelson, 0. (Plenum, New York), pp. between the a] and sh2 loci. These findings are consistent 137-148. with the hypothesis that recombination occurs at higher rates 12. Brown, J. J., Mattes, M. G., O'Reilly, C. & Shepherd, N. S. (1989) Mol. in genes per se rather than because genes are most likely to Gen. Genet. 215, 239-244. be found in large recombinationally active chromosomal 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, segments. 1348-1351. The al-shz Interval Is a Model for the Fine Structure 14. Sherman, F. (1991) in Guide to Yeast Genetics and Molecular Biology, Analysis of Meiotic Recombination in the Maize Genome. The eds. Guthrie, C. & Fink, G. R. (Academic, San Diego), pp. 3-21. molecular isolation of the contiguous segment of the chro- 15. Schwarz-Sommer, Z., Shepherd, N., Tacke, E., Gierl, A., Rohde, W., Leclercq, L., Mattes, M., Berndtgen, R., Peterson, P. A. & Saedler, H. mosome encompassing the a) and sh2 loci has long-term (1987) EMBO J. 6, 287-294. implications for elucidating the interrelationships among re- 16. Bhave, M. R., Lawrence, S., Barton, C. & Hannah, L. C. (1990) Plant combination, genome organization, and genome function. Cell 2, 581-588. These relationships have profound evolutionary implica- 17. Ausubel, F. M., Brent, R., Kingston, R. E. & Moore, D. D., eds. (1988) Current Protocols in Molecular Biology (Greene & Wiley-Interscience, tions. For example, intragenic recombination results in the New York). creation of chimeric alleles. Thus, the evolutionary advan- 18. Dellaporta, S. J., Wood, J. & Hicks, J. B. (1983) Plant Mol. Biol. Rep. tages of creating additional genetic diversity in a population 1, 19-21. may have fostered mechanisms that preferentially select 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. genes as sites for recombination. 21. Edwards, K. J., Thompson, H., Edwards, D., deSaizieu, A., Sparks, C., In addition, the physical mapping of the interspersion of Thompson, S. A., Greenland, A. J., Eyers, M. & Schuch, W. (1992) genes, repetitive, middle-repetitive, and low-repetitive se- Plant Mol. Biol. 19, 299-308. quences, in the al-sh2 interval can be correlated with meiotic 22. Albertsen, H. M., Abderrahim, H., Cann, H. M., Dausset, J., LePaslier, D. & Cohen, D. (1990) Proc. Natl. Acad. Sci. USA 87, 4256-4260. recombination breakpoints. This type of high-resolution 23. Sambrook, J., Fritsch, E. F. & Maniatis, T., eds. (1989) Molecular physical mapping ofrecombination breakpoints may uncover Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plain- the molecular features associated with meiotic recombination view, NY), 2nd Ed. hot and cold spots. However, recombination events can only 24. Helentjaris, T., Weber, D. & Wright, S. (1985) Plant Mol. Biol. 5, a 109-118. be scored when they occur in marked heterozygote. Be- 25. Vollrath, D., Davis, R. W., Connelly, C. & Hieter, P. (1988) Proc. Natl. cause a chromosomal segment undergoes different rates of Acad. Sci. USA 85, 6027-6031. recombination depending upon the particular heterozygote 26. Lewis, B. C., Shah, N. P., Braun, B. S. & Denny, C. T. (1992) Genet. combination used, absolute values of p for a given chromo- Anal. Technol. Appl. 9, 86-90. 27. Gietz, D., St. Jean, A., Woods, R. A. & Schiestl, R. H. (1992) Nucleic somal segment cannot be determined. For example, p is Acids Res. 20, 1425. typically lower when estimated using transposon-insertion 28. Peterson, P. A. (1978) in Maize Breeding and Genetics, ed. Walden, mutant alleles of a given locus than when estimated using D. B. (Wiley, New York), pp. 601-631. alleles carrying point mutations (5, 30, 32, 35). Hence, the 29. Galbraith, D. W., Harkins, K. R., Maddox, J. M., Ayres, N. M., Sharma, D. P. & Firoozabady, E. (1983) Science 220, 1049-1051. value of p for the al-sh2 interval is dependent' upon known 30. Dooner, H. K. (1986) Genetics 113, 1021-1036. cis-acting factors and as yet unidentified cis- and trans-acting 31. Wessler, S. R. & Varagona, R. (1985) Proc. Natl. Acad. Sci. USA 82, factors specific to the particular heterozygous combination 4177-4181. used in this study. 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, We thank the following individuals: Dr. Curt Hannah and Dr. Alfons 473-482. Gierl for the gift ofthe sh2 and a) clones, respectively; Shane Heinen 34. Moore, G., Gale, M. D., Kurata, N. & Flavell, R. B. (1993) Biol and John VanDiepen for technical assistance; Lei Zhang for assisting Technology 11, 584-589. with the confirmation of meiotic recombinants; and Homer Caton of 35. Dooner, H. K. & Kermicle, J. L. (1986) Genetics 113, 135-143. Downloaded by guest on September 23, 2021