Analysis and Mapping of Gene Families Encoding ß-1,3-Glucanases of Soybean

Corresponding author: Harry T. Horner, Department of Botany, Iowa State University, Ames, IA 50011-1020., hth{at}iastate.edu (E-mail)

Communicating editor: V. SUNDARESAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Oligonucleotide primers designed for conserved sequences from coding regions of ß-1,3-glucanase genes from different species were used to amplify related sequences from soybean [Glycine max (L.) Merr.]. Sequencing and cross-hybridization of amplification products indicated that at least 12 classes of ß-1,3-glucanase genes exist in the soybean. Members of classes mapped to 34 loci on five different linkage groups using an F₂ population of 56 individuals. ß-1,3-Glucanase genes are clustered onto regions of five linkage groups. Data suggest that more closely related genes are clustered together on one linkage group or on duplicated regions of linkage groups. Northern blot analyses performed on total RNA from root, stem, leaf, pod, flower bud, and hypocotyl using DNA probes for the different classes of ß-1,3-glucanase genes revealed that the mRNA levels of all classes were low in young leaves. SGlu2, SGlu4, SGlu7, and SGlu12 mRNA were highly accumulated in young roots and hypocotyls. SGlu7 mRNA also accumulated in pods and flower buds.

THE ß-1,3-glucanases (EC 3.2.1.39) are hormonally and developmentally regulated plant hydrolytic enzymes found during anther and coleoptile development, pollen tube growth, in endosperm cells, and in the end walls of sieve elements. They also are induced upon pathogen infection or by environmental stresses (ABELES et al. 1970 ; BOLLER 1987 ; MAUCH et al. 1988 ; CASTRESANA et al. 1990 ; TAKEUCHI et al. 1990 ; BREDERODE et al. 1991 ; DONG et al. 1991 ; LEAH et al. 1991 ; WARD et al. 1991 ; SCHRODER et al. 1992 ; BEERHUES and KOMBRINK 1994 ; BEFFA et al. 1996 ). Five distinct classes and a total of 12 ß-1,3-glucanase genes have been identified in tobacco (FELIX and MEINS 1986 ; VAN DEN BULCKE et al. 1989 ; LINTHORST et al. 1990 ; ORI et al. 1990 ; PAYNE et al. 1990 ; BUCCIAGLIA and SMITH 1994 ). The best characterized are the isoforms that are induced by either pathogen infection or ethylene treatment. They exhibit developmental regulation (FELIX and MEINS 1986 ) and appear to be localized primarily in vacuoles (VAN DEN BULCKE et al. 1989 ). The second class, which includes the pathogenesis-related proteins PR-2, PR-N, PR-O, and PR-35 (KAUFFMANN et al. 1987 ; VAN DEN BULCKE et al. 1989 ), also are induced by pathogens and appear to be localized in the extracellular spaces. The third class, which includes the pathogenesis-related protein PR-Q' (PAYNE et al. 1990 ), also is induced by pathogens and seems to be localized in extracellular spaces. The members of the fourth class, including sp41a and sp41b, accumulate to high levels in the transmitting tract of the style and are not pathogen inducible (ORI et al. 1990 ). The fifth class is a secreted ß-1,3-glucanase that is expressed in the anther tapetum (BUCCIAGLIA and SMITH 1994 ) and is involved in tetrad callose wall dissolution. Related monocot ß-1,3;1,4-glucanases encode proteins similar to ß-1,3-glucanases. The function of these latter proteins is to catalyze the hydrolysis of ß-1,3;1,4-glucans, which are major components of endosperm cell walls (FINCHER et al. 1986 ).

Although many physiological studies have been conducted, little work has been done on the genetic analysis of ß-1,3-glucanase genes. Seven ß-1,3-glucanase genes were located on the long arm of chromosome 3 in barley (LI et al. 1996 ). Six were clustered in a region <20 cM in length. The mode of regulation of these genes is unknown. Genes on the same chromosome seemed to be closely related to one another but substantially different from those on other chromosomes (MUTHUKRISHNAN et al. 1984 ).

Characterization of the ß-1,3-glucanase genes of soybean will provide information about these gene families and their evolution in a diploidized polyploid. We report the cloning of ß-1,3-glucanase genes, the characterization of mRNA accumulation patterns of these genes, and their chromosomal localization in the genome. The linkage relationships of ß-1,3-glucanase genes accounting for mRNAs accumulating in different organs, sequence relationships among the genes found at various loci, and the genomic organization of these loci suggest mechanisms underlying ß-1,3-glucanase gene regulation, evolution, and possible functions of multiple gene loci.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material and nucleic acid manipulations:
Plant tissues for nucleic acid isolation were collected from the soybean cultivar Minsoy (PI 27890) grown in the greenhouse or growth chamber. Material for RNA extraction was immediately frozen in liquid nitrogen and stored at -80° until used. Soybean genomic DNA was extracted from young leaves (KEIM et al. 1988 ). Total DNA (10 µg) was digested and subjected to Southern blotting (SAMBROOK et al. 1989 ). Total RNA was extracted using guanidinium isothiocyanate extraction (CHOMCZYNSKI and SACCHI 1987 ), electrophoresed on formaldehyde-agarose gels, and blotted onto Zeta Probe nylon membranes (Bio-Rad, Richmond, CA; SAMBROOK et al. 1989 ). Equal amount of RNA loading was verified by probing blots with 18S rRNA (data not shown). Blots were prehybridized for 3 hr at 65° in 5x SSC, 2% SDS, 5x Denhardt's solution, and 0.1 mg/ml herring sperm DNA. Hybridization was carried out overnight at 65° in the same solution. Blots were washed at 60° with 2x SSC + 0.4% SDS and 1x SSC + 0.4% SDS at 60° before autoradiography exposure.

Genetic mapping of ß-1,3-glucanase genes:
Mapping was conducted using a Glycine max (L.) Merr. x G. soja (Zieb. & Zucc.) population containing 56 individuals (KEIM et al. 1990 ). Parental DNA was digested with 14 restriction endonucleases (AccI, AluI, BclI, BamHI, EcoRI, EcoRV, DraI, HaeIII, HhaI, HindIII, HinfI, RsaI, SspI, and TaqI). Each polymorphism was mapped using MapMaker (LANDER et al. 1987 ). The Kosambi (KOSAMBI 1944 ) mapping function was selected, and a minimum LOD score of 3 was required for a two-point linkage.

PCR amplification, cloning, and sequence analysis:
Sequences encoding ß-1,3-glucanase were amplified from soybean genomic DNA and cDNA (prepared from flower bud RNA) using primers for conserved sequences of the ß-1,3-glucanases of other species (SIMMONS 1994 ). To obtain cDNA, 0.1 µg mRNA was prepared from flower buds. The mRNA was incubated with 5 µM random hexamers; 1 mM each of dATP, dCTP, dGTP, and dTTP; and 2 units/µl Rnase inhibitor (Promega, Madison, WI) in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 2.5 mM MgCl₂ for 30 min at 37°, and then for an additional 45 min at 37° after the addition of 20 units/µl superscript reverse transcriptase (BRL Life Technology, Rockville, MD). After heating at 65° for 3 min, cDNA was used in the PCR experiments. Each 25-µl reaction contained 60 ng of soybean genomic DNA or 20 ng of flower cDNA; 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 3 mM MgCl₂; 200 µM (each) dATP, dCTP, dGTP, and dTTP; 2.5 units of Taq polymerase (BRL Life Technology); and 2 µM (each) PCR primer. PCR amplifications were performed under the following conditions: 96° for 1 min, 44° for 1 min, and 72° for 1 min for 4 cycles, followed by 94° for 1 min, 55° for 1 min, and 72° for 1 min for 30 cycles. The 5' primer was 5' CGCGGNGTNTGYTAYGG 3'; the 3' primer was 5' CGCGGCCANCCNSWYTC 3' (where N is A, C, G, T; Y is C, T; S is C, G; and W is A, T). The regions used correspond to amino acids 37–41 and 276–282 of the mature protein (SIMMONS 1994 ). An aliquot of each PCR product was analyzed by agarose gel electrophoresis. A 700-bp fragment was cut from the gel and subjected to another round of PCR using identical conditions. Aliquots of these PCR products were digested with restriction enzymes recognizing 4-bp sites, and the digestion products were compared with undigested DNA on agarose gels. The PCR products were cloned into the pGEM-T vector (Promega), and ~280 clones were tested by dot blot analysis (SAMBROOK et al. 1989 ) to determine if they cross-hybridized.

Clones were sequenced on both strands using an automated sequencer. Two to five sequences were obtained for each class. DNA sequence analysis was carried out with the DNAsis (Hitachi, San Francisco, CA) and GCG (Genetics Computer Group, Madison, WI) sequence analysis packages. Alignment of sequences was done using CLUSTAL (THOMPSON et al. 1994 ). Phylogenetic analysis of amino acid sequences was performed using PAUP version 3.0 (SWOFFORD 1991 ).

BAC library screening:
The soybean bacterial artificial chromosomal (BAC) library (MAREK and SHOEMAKER 1996 ) was replicated onto nylon membranes (Zeta Probe GT, Bio-Rad). The membranes were screened using two ß-1,3-glucanase gene probes, SGlu2 and SGlu5.

The sequence data presented in this article have been submitted to the GenBank Data Libraries under the accession nos. AF034106, AF034107, AF034108, AF034109, AF034110, AF034111, AF034112, AF034113, AF0341114, AF034115, AF034116, and AF034117.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of soybean ß-1,3-glucanase genes and sequence analysis:
PCR amplification of soybean genomic DNA using degenerate primers designed from conserved regions of other ß-1,3-glucanases resulted in the production of an ~700-bp band. The band was excised from the gel and subjected to a further round of PCR. We then asked whether the PCR product consisted of a mixture of DNA sequences, which would be consistent with the amplification of multigene families. The PCR product was digested with HaeIII, AluI, RsaI, MseI, and Sau3AI. Each restriction digestion yielded a series of fragments whose molecular weights summed to a value greater than that of the original PCR product (data not shown). The presence of a heterogeneous PCR product suggested the involvement of multigene families.

The PCR product was cloned, and ~280 clones were analyzed. The clones were grouped into 12 classes on the basis of cross-hybridization results under stringent hybridization conditions (0.1x SSC/0.1x SDS/60° wash).

PCR experiments using cDNA as a template also generated an ~700-bp band, indicating that the targeted genomic coding regions of the members of the multigene families may not be interrupted by introns.

BAC library screening identified 15 BACs representing Sglu5 and 5 BACs representing Sglu2. The copy number of ß-1,3-glucanase gene sequences within each BAC was estimated by digesting the BACs with restriction enzymes not having recognition sites within the ß-1,3-glucanase probe sequences and by hybridizing with Sglu2 and Sglu5 probes (results not shown). Sglu2 BACs each contained two copies of the sequence. This result agreed with the prediction of two to four copies based on genomic Southern hybridization patterns (results not shown). The class 5 BACs each appeared to have one to two copies of the class 5 ß-1,3-glucanase sequence. This result agreed with the prediction of two copies based on genomic Southern hybridization.

Three to five clones from each class were sequenced on both strands, and the deduced amino acid sequences of the representative clones from each class are shown (Figure 1). Among the sequenced clones, classes 2 and 7 showed heterogeneity (two subclasses). The deduced protein contains 30 amino acids that are identical among all 12 soybean classes and classes I, II, III, IV, and V of tobacco ß-1,3-glucanases (Figure 1). These include conserved acidic amino acids, glutamic acid residues 96 and 245, and tryptophan residues 74 and 248. Glutamic acid 245 and tryptophan 248 are surrounded by highly conserved amino acids that are similarly conserved in plant and yeast glucanases (MACGREGOR and BALLANCE 1991 ; VARGHESE et al. 1994 ).

View larger version (151K): In this window In a new window Download PPT slide   Figure 1. Alignment of deduced amino acid sequences of ß-1,3-glucanases from soybean. Arrows indicate locations of PCR primers used to amplify ß-1,3-glucanase sequences. Dashed regions indicate gaps in sequences introduced to maximize alignment. Asterisks represent conserved residues. Conserved tryptophans are marked by triangles. Identical or functionally conserved amino acids surrounding acidic residues are marked with hyphens on the top. Periods and colons represent different degrees of conservative substitution. Tobacco classes I, II, III, IV, and V amino acid sequences were included for comparison. GenBank database accession numbers for these genes are as follows: class I, M20619; class II, M60462; class III, X54456; class IV, X54430; and class V, Z28697.

Sglu9 is probably a pseudogene because it contains multiple stop codons; however, it did show strong similarity with Sglu8. Sglu8 and Sglu9 clones differ at only 6 positions in 700 bases of overlapping sequence. Pairwise comparisons between different classes revealed that amino acid identities ranged from 11.1 to 81.8%; similarities ranged from 35.2 to 99.9%. Subclasses 7a and 7b differed at only six positions at the DNA sequence level and showed 98.3% amino acid identity.

Phylogenetic analysis of ß-1,3-glucanase-coding regions:
To determine the relationships of the 12 classes of soybean ß-1,3-glucanases to the five classes of ß-1,3-glucanase genes from tobacco, a parsimony analysis was conducted. Amino acid sequences were aligned using CLUSTAL (THOMPSON et al. 1994 ) and then analyzed using PAUP version 3.0 (SWOFFORD 1991 ). Figure 2 shows the unrooted consensus tree (from 100 bootstrap replicates) that groups four soybean classes (SGlu1, SGlu8, SGlu4, and SGlu12) as a branch with tobacco class I; five classes (SGlu3, SGlu5, SGlu6, SGlu10, and SGlu11) are grouped closely to acidic ß-1,3-glucanases (class II) and stylar acidic glucanases (class IV). The structurally similar, pathogen-induced acidic glucanases (class II) and stylar acidic glucanases (class IV) are grouped into one branch. This result agrees with a previous report (BUCCIAGLIA and SMITH 1994 ). SGlu2 is grouped closely to class III. SGlu7a and 7b and Tag1 (class V) are grouped together, suggesting that SGlu7 could be an anther-specific ß-1,3-glucanase.

View larger version (16K): In this window In a new window Download PPT slide   Figure 2. Phylogenetic analysis of selected plant ß-1,3-glucanases. Deduced amino acid sequences were aligned, and an unrooted tree was established using parsimony analysis. Tobacco classes I, II, III, IV, and V are included for comparison.

Mapping of 12 classes of ß-1,3-glucanase genes:
An F₂ population derived from an interspecific cross was used to determine the genomic location of 12 soybean classes of ß-1,3-glucanase genes. Clones representing separate classes were hybridized to Southern blots of soybean genomic DNA digested with 14 restriction enzymes to identify polymorphisms and to estimate gene family copy number. Coding regions of genes hybridized to two to seven fragments, indicating that most ß-1,3-glucanase class probes are members of a supergene family with classes that range in size from two members (SGlu7) to as many as seven members (SGlu6). The average number of fragments detected suggests the presence of >40 genes.

The segregation analysis of 56 F₂ individuals of the F₂ population placed the 11 classes into 5 of 26 linkage groups, B1, J, K, L, and N1 (Figure 3). All these linkage groups contained more than two loci of ß-1,3-glucanases. Two classes, SGlu4 and SGlu12, cosegregated with the pea ß-1,3-glucanase gene involved with fungal resistance (CHANG et al. 1992 ).

View larger version (23K): In this window In a new window Download PPT slide   Figure 3. Distribution of ß-1,3-glucanases on soybean genetic map. SGlu loci indicated by family designators (1–12) are shown on right of each linkage group. B162 and PEG488 are previously mapped RFLP markers. B1, J, K, L, and N1 are the names of the linkage groups (SHOEMAKER et al. 1996B ).

Some class-specific probes (SGlu2, SGlu3, SGlu5, and SGlu9) detected multiple polymorphic fragments. These mapped to different loci, indicating that some gene families are dispersed. SGlu7a and b were not mapped because no polymorphisms were detected between parental DNA digested with any of the 14 restriction enzymes.

In the soybean, large domains of different linkage groups seem to have been derived from the same ancestral linkage group through duplication (SHOEMAKER et al. 1996A , SHOEMAKER et al. 1996B ). The different domains contain homologous members of the same gene families. Some of the ß-1,3-glucanase gene loci (e.g., SGlu2) lie within these duplicated regions (Figure 3). For example, two markers (pB162 and PEG488) detected loci on linkage groups L and N1. On both linkage groups, these markers also are linked to multiple SGlu2 loci (Figure 3). This result suggests that these multiple ß-1,3-glucanase gene loci have arisen during evolution via duplications of large chromosomal regions in which linkage relationships between ß-1,3-glucanase genes and the other genes have been maintained.

Nucleotide sequences, phylogenetic relationships, and chromosomal locations:
Twelve ß-1,3-glucanase genes used in the mapping project also were sequenced and analyzed, and the extent to which ß-1,3-glucanase genes mapped to individual loci is correlated to their sequence relationships (Figure 3). The sequenced region covers ~60% of the coding region, including the proposed catalytic sites. The 12 ß-1,3-glucanase sequences show an average nucleotide sequence identity of 64.8% (range 21.4–95%), an average amino acid sequence identity of 36.2% (range 11.1–81.8%), and an average amino acid sequence similarity of 70.2% (range 35.2–99.9%). Those ß-1,3-glucanases that mapped together always showed >88% nucleotide sequence identity. For example, Sglu5 and Sglu11 both mapped to a locus on linkage group B. As Figure 2 and Figure 3 show, sequence-similar genes are clustered on one linkage group or on duplicated regions of linkage groups; e.g., Sglu1, 4, 8, and 12 group into tobacco class I and are all mapped on linkage group K.

mRNA accumulation patterns of different classes:
To study mRNA accumulation patterns of 12 classes, the presence of mRNAs corresponding to each class was analyzed by RNA blot analyses using class-specific probes under stringent conditions (Figure 4). The mRNA levels of all classes were quite low in young leaves. However, SGlu2, SGlu4, SGlu7, and SGlu12 mRNA were highly accumulated in young roots and hypocotyls. SGlu7 mRNA also was accumulated in the pod and flower bud. We were unable to detect mRNA from SGlu1, SGlu3, SGlu8, SGlu9 (pseudogenes), or Sglu11 genes on RNA gel blots. The lack of detectable mRNA corresponding to these four classes suggests that these genes were not expressed, expressed at low levels, or expressed at a developmental stage or condition not tested in this study. In addition, it appears that SGlu5, SGlu6, and Sglu10 probes hybridize to mRNA of different sizes. This may indicate multiple starts or stops of transcription, differential processing of transcripts, or transcripts from multiple genes.

View larger version (60K): In this window In a new window Download PPT slide   Figure 4. mRNA accumulation patterns from 12 classes of ß-1,3-glucanase genes. RNA gel blot hybridization analysis illustrates the pattern of ß-1,3-glucanase mRNA accumulation in young roots, stems, leaves, pods, flower buds, and hypocotyls. mRNA transcripts are ~1.2 kb.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The goal of this study was an analysis of ß-1,3-glucanase gene families in the soybean. Through exhaustive cross-hybridizations and sequence analyses, we identified and classified ß-1,3-glucanase gene families of moderate size into 12 classes. We then analyzed sequence relationships among these genes and determined their genomic locations.

Using an F₂ population, 45 restriction fragment length polymorphisms were mapped with 14 restriction enzymes and 12 ß-1,3-glucanase probes. We resolved 34 distinct ß-1,3-glucanase loci on five different linkage groups. Some loci contained clusters of ß-1,3-glucanase genes. It is likely that at least some genes will be tandemly arranged in a cluster, as observed with other multigene families (YAMAGUCHI-SHINOZAKI et al. 1989 ; SUTLIFF et al. 1991 ; KANAZIN et al. 1996 ; SULLIVAN et al. 1996 ). Tandem arrays provide a template for recombination events, including unequal crossing over and gene conversion, which can lead to expansion and further diversification of the sort that is apparent among the classes we have cloned (for review see MAEDA and SMITHIES 1986 ; CLEGG et al. 1997 ).

The distribution of ß-1,3-glucanase genes may protect against catastrophic losses of ß-1,3-glucanase genes by unequal crossing over or gene slippage that might occur if ß-1,3-glucanase genes were clustered at a single locus (DOVER 1993 ). The existence of multiple ß-1,3-glucanase genes might also promote the development and maintenance of structural and functional diversity in the ß-1,3-glucanase gene families.

Our data show that some ß-1,3-glucanase genes are organized into duplicated regions in the genome (Figure 3). These gene loci are probably related evolutionarily and probably have arisen during evolution via duplications of large chromosomal regions. However, retrotransposition and duplication of individual genes also might have occurred during the evolution of ß-1,3-glucanase gene families since SGlu9 mapped as a single locus on linkage groups B1 and J.

In higher plants, ß-1,3-glucanases are encoded by gene families of considerable complexity (LINTHORST et al. 1990 ; XU et al. 1992 ). The significance of the gene multiplicity is unclear. Such multiplicity could provide more flexibility for spatial and temporal regulation of diverse functions of ß-1,3-glucanase: microsporogenesis, pollen tube growth, senescence, and disease resistance described previously, or "pathogenesis-related" ß-1,3-glucanases might be expressed either constitutively or inducibly, and there may be requirements for different responses in different tissues. It may also reflect functional differences between closely related proteins. Multiplicity of ß-1,3-glucanase functions might confer advantages to plants by providing several lines of defense against invading microorganisms. Also, the diversity of ß-1,3-glucanases, as well as their organ specificity and developmental and differential expression patterns, may indicate that this enzyme has additional, as yet unidentified, biological functions in plant growth and development.

In an effort to group 12 classes of soybean ß-1,3-glucanases into previously described glucanase classes (SHINSHI et al. 1988 ; LINTHORST et al. 1990 ; ORI et al. 1990 ; PAYNE et al. 1990 ; BUCCIAGLIA and SMITH 1994 ), we used parsimony analysis to determine their relationship (Figure 2). Our results are consistent with classifications reported previously (classes I–V; Figure 2) on the basis of sequence similarity. Whether this classification accurately reflects functionally distinct classes of ß-glucanases in soybean remains to be demonstrated.

Sequence and phylogenetic analyses of the ß-1,3-glucanase genes showed that members of the same ß-1,3-glucanase class, as well as closely related classes, often mapped to the same linkage group or to duplicated regions of linkage groups. All class I ß-1,3-glucanases mapped on linkage group K, all class III ß-1,3-glucanases mapped to paralogous regions on linkage groups L and N1, and all class II and IV ß-1,3-glucanases mapped on linkage groups B1 and J. Other examples of genes with similar functions and expression patterns that reside on the same linkage group can be found among gene families involved in the regulation of floral identity and cell differentiation (PICKETT and MEEKS-WAGNER 1995 ) and disease-resistance genes (KANAZIN et al. 1996 ).

The multigene families encoding the ß-1,3-glucanases are large; all the member genes clearly have a common ancestral origin but have undergone considerable divergence such that individual genes encoding proteins share 11.1–81.5% amino acid identity. Three classes of tobacco ß-1,3-glucanase cDNA clones differ at only 18 positions in 1055 bases of overlapping sequence (SHINSHI et al. 1988 ). The high homology level of the tobacco ß-1,3-glucanases is maintained by intergenomic DNA exchange between genes (SPERISEN et al. 1991 ). In the soybean, 2 classes of ß-1,3-glucanase genes (SGlu8 and SGlu9) differ at only 6 positions in 700 bases. How this high level of homology is maintained is unknown.

Our results indicate that in the soybean, pathogen invasion is not a prerequisite for expression of some of the ß-1,3-glucanase genes. SGlu2, SGlu4, SGlu7, and SGlu12 mRNA were accumulated in young roots and hypocotyl. These genes could represent a form of defense against possible threats from rhizosphere microbes (MEMELINK et al. 1990 ). These results do not preclude a role for these ß-1,3-glucanases in defense against microorganism infection. Preemptive or proactive expression of the enzymes (independent of microbial attack) could provide some measure of insurance against infection during the crucial phases of the life cycle in which plant tissues are susceptible to pathogen attack (KNOGGE et al. 1987 ).

The 12 classes of ß-1,3-glucanase genes are distributed in clusters, and two of them (Sglu 4 and Sglu 12; Figure 3) mapped at the same location as a known pathogen-induced gene (the ß-1,3-glucanase gene expressed in pea pods challenged with pathogen Fusarium solani f. sp. mapped to this location; CHANG et al. 1992 ). Our findings demonstrate that ß-1,3-glucanase genes with similar sequences are clustered. Duplications of large chromosomal domains followed by extensive gene duplication and divergence and duplication of individual genes are involved in the evolution of the ß-1,3-glucanase gene families.

	FOOTNOTES

¹ Present address: Developmental Cell and Molecular Biology Group, Duke University, Durham, NC 27708.

	ACKNOWLEDGMENTS

We thank Kim Lewers for help with mapping and C. Baldwin for help with screening the BACs library; D. Grant and Laura Marek for helpful discussions during the course of this work; L. Hadwiger for pea ß-1,3-glucanase cDNA clone; Y. Takeuchi for PEG488 cDNA clone from soybean; and J. T. Colbert, A. G. Smith, and M. P. Timko for critically reviewing the manuscript. This is Journal Paper J-17722 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa (Projects 3236 and 3352), and from the U.S. Department of Agriculture (USDA), Agricultural Research Service, Corn Insect and Crop Genetics Research Unit. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by Iowa State University or the USDA and does not imply its approval to the exclusion of other products that may also be suitable.

Manuscript received October 8, 1998; Accepted for publication June 2, 1999.

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