The Journal of Heredity 75:365-370. 1984.

Chromosomal locations of twelveisozyme loci in Pisum sativum

ABSTRACT: Approximate chromosomal locations of 12 loci specifying electrophoretlc en-zyme variants are described in the garden pea (Pisum sativum L.). The enzyme loci aredistributed on five of the seven chromosomes. The position of the loci on chromosomes2 and 3 are such that most of the known markers on these chromosomes will exhibit link-age with at least one of the isozyme loci. Several of the loci studied code for enzymes thathave isozymic counterparts in other compartments of the cell. In order to distinguishamong the genes coding these isozymes we have added a suffix to the locus designationcorresponding to the Intracellular location of its product.

N. F. Weeden

G. A. Marx

The authors are affiliated with the Department ofHorticultural Sciences, New York Agricultural Ex-periment Station, Cornell University, Geneva, NY14456. Invaluable technical assistance was provided inthese studies by A. C. Emmo. The authors also thankD. W. Barton and R. W. Robinson for their helpfulcomments and reviews.© 1984, American Genetic Association.

ISOZYMES are distinguishable forms of anenzyme often found within the same cell. Inmany cases the variability can be attributedto different alleles at a single locus, identifyingthe variants as a specific subclass of isozymescalled allozymes. The simple genetic basis ofallozymic forms permits their use as geneticmarkers for a number of applications24.Mapping of these "isozyme loci" in maize9,tomatoes18, barley6, wheat and relatedspecies13'14 has resulted in the formation ofpartial linkage maps, extremely useful toolsfor the breeder and plant geneticist. Conspi-cuously missing from the list of plants inves-tigated are representatives from the Legumi-nosae, a major family of flowering plants bothin terms of number of taxa and importance toagriculture. Initial genetic studies of the iso-zymes in soybeans10", alfalfa17, and beans3-26

have been published, but the loci have not beenmapped.

For several reasons the garden pea is highlysuited for the development of an enzymelinkage map. It already possesses many chro-mosomal markers that facilitate mapping ofisozyme loci; it is a convenient plant for geneticstudies (short generation time, naturallyself-pollinated and easily cross pollinated, di-verse germplasm collections available); and itmay possibly serve as a model system for othercommercially important legumes. At present,only one isozyme locus has been mapped onthe pea genome, Lap-1, which codes for themore anodal leucine aminopeptidase isozymeobserved after starch gel electrophoresis of raw

extracts2. However, a large number of poly-morphic isozyme loci have been identified27,potentially enough to form an enzyme linkagemap covering the entire genome should the locibe distributed randomly. We have thereforeinitiated a program to systematically map allknown polymorphic enzyme loci in the pea.We report here approximate map positions of12 isozyme loci located on five differentchromosomes.

Materials and Methods

Inbred lines of Pisum satioum with knownmorphological markers were selected from thepea collection available at the New York StateAgricultural Experiment Station, Geneva,NY. Lines were screened for isozyme pheno-type, and appropriate crosses were made withsuitable marker lines. Three crosses were se-lected for further screening of F2 populations,the three families being designated A, E, andG. The A family consisted of seed from six Fiplants produced in a cross between linesB77-254 and A78-237. Three of the F, indi-viduals were generated using B77-254 as thefemale parent; the other three were from areciprocal cross. The F2 population, E, wasderived from the cross A1078-234 X B77-257while G was derived from the cross B78-288X A1078-236. The relevant morphologicalmarkers possessed by each line are given inTable I.

Linkage between loci was calculated usingthe tables published by Allard1 and computer

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programs "F2" and "Progeny" developed atthe New York State Agricultural ExperimentStation.

Samples were prepared for electrophoresisusing two extraction buffers as describedpreviously30. Samples extracted in the tris-HC1 buffer were placed on a pH 8.1 tris-ci-trate/lithium borate system21. The phosphateextraction buffer was used for samplessubjected to electrophoresis on either a pH 6.5histidine7 or a pH 6.1 citrate/N-(3-amino-propyl)-morpholine8 gel system.

The assays for NADP-specific isocitratedehydrogenase (IDH), acid phosphatase(AcP), 6-phosphogluconate dehydrogenase(6PGD), and leucine aminopeptidase (LAP)were identical to or slight modifications ofthose described by Shaw and Prasad22. Theassays for aspartate aminotransferase (AAT),shikimate dehydrogenase (SKDH), aldolase(Aldo), and phosphoglucomutase (PGM)have been described previously31. Methyl-umbelliferyl esterase (Est) was assayed asdescribed by Bender et al.4. The enzymesAAT, PGM, LAP, and SKDH were assayedon anodal slices cut from gels run using thetris-borate buffer system. The histidine buffersystem was used for 6PGD, and the citrate/N-(3-aminopropyl)-morpholine buffer systemwas used to resolve the IDH, AcP, and Estisozymes.

Reagents were obtained from SigmaChemical Company, St. Louis, Missouri, ex-cept for the N-(3-aminopropyl) morpholine,which was purchased from Aldrich ChemicalCompany, Milwaukee, Wisconsin.

Results

All plants remained healthy throughout theexperiment and seed set was excellent forfamilies A and E. Some sterility was encoun-tered among plants in the G population. If thissterility was caused by differences in parentalkaryotype such differences did not appear tointerfere with the mapping of isozyme loci, forthe chromosomal markers and isozyme phe-

Table I. Relevant morphological marker genesin parental lines

Line

A1078-234

A1O78-236A78-237B77-254B77-257B78-288

Marker genes*

/', s, wb, k, st, b, n,fa.le, cp, gp, wlo, tl, r

k, M, st, b, le, wlos, wb, k, st, b, tl, rPur, oh, Pu, gpNp,Btd, ar, U, PI

FIGURE 1 Mitochondrial aspartate amino-transferase (AAT-3) phenotypes after electro-phoresis of leaf extracts. The fast allozyme (F) hasmobility approximately intermediate between theplastid isozyme (AAT-2) and the cytosolic form(AAT-4). The slow alloxyme (S) is slower than butpartially overlaps AAT-4. The heterozygous pat-tern is designated H. Arrow indicates direction ofmigration.

notypes gave normal segregation ratios, andunexpected linkages between morphologicalmarkers were not observed. Segregation ratiosfor the reciprocal crosses in family A gavesimilar ratios (data not shown); therefore, thetwo data sets were combined for linkageanalysis.

Phenotypes observed after electrophoresis

Isocitrate dehydrogenase: A single band ofIDH activity was observed in extracts frominbred lines. Crosses made between lines ex-hibiting forms with different electrophoretic

©

ORIGIN-

o

J EST-1

EST-2

^ } EST-3

' ^ && •«&• I EST-41 2 3 *

FIGURE 2 Pea leaf esterase xymograms asvisualized using the florogenic substrate methyl-umbelliferyl acetate. Column 1 represents an ex-tract containing the slow alloxyme of each esterase.Column 2 depicts the pattern observed in a plantheterozygous at Est-1, Est-3, and Est-4 (Est-2 ishomoxygous for the slow allele). The third trackshows the fast allozyme for all esterases except forEst-2, which is slow.

mobilities gave progeny with a three-bandedphenotype, reflecting the dimeric structure ofthis enzyme. In the crosses reported here themobilities of the two homodimeric forms didnot differ enough to permit the resolution ofthe intermediate heterodimer.Thus, heterozy-gous plants displayed a single, wide band afterelectrophoresis.

Aspartate aminotransferase: Cell frac-tionation studies of the AAT isozymes in thepea indicated that the third most anodal set ofallozymes, AAT-3 (Figure 1), representedmitochondrial forms. In a previous paper31 themitochondrial specific forms were labeled'AAT-2' because the most anodal set of AATisozymes (AAT-1 in Figure 1) could not beseen. The fast variant possessed a mobility

• For description of characters see Blixt5

FIGURE 3 Variation observed in both isozymes of 6PGD. The phenotypes (6PGD-1/6PGD-2) cor-responding to the labeled tracks are: (a) F/H, (b) H/H, (c) S/S, (d) S/H, and (e) H/F, where F •= fast al-lozyme, S •» slow allozyme, H = heterozygous phenotype. Doubly heterozygous individuals expresssix bands: the four homodimeric forms and two intragenic heterodimers.

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relative to the borate front (R/) of 0.32.Theslow form, partially obscured by cytosolicAAT isozyme, had an Rj of 0.25. Heterozy-gous individuals exhibited a wide blur withfainter staining of the homodimeric forms.

Leucine aminopeptidase: Several investi-gators16'20 have described polymorphism attwo leucine aminopeptidase loci, Lap-1 andLap-2, the loci being numbered in order ofdecreasing migration rate of the respectiveisozymes. Scandalios and Espiritu20 observedthat the two aminopeptidascs they isolatedfrom pea extracts could be distinguished onthe basis of substrate specificity. Althoughboth isozymes could cleave either leucyl oralanyl a-naphthylamide, one was more activewith the former substrate while the other ex-hibited the opposite preference. When we as-sayed duplicate slices from a pH 8.1 gel forleucine aminopeptidase and alanyl amino-peptidase activity we found that both LAP-1and LAP-2 isozymes were more active withthe leucyl a-naphthylamide substrate. How-ever, a third set of bands, partially overlappingthe LAP-2 allozymes, were visualized whenalanyl a-naphthylamide was used as the sub-strate. These alanyl aminopeptidase bands areproducts of a locus distinct from Lap-1 andLap-2 although the chromosomal location ofthis locus has not been determined (Weeden,unpub.). All three aminopeptidases can beclearly separated using the histidine gel sys-tem; however, the resolution of the LAP-1allozymes is poorer on this gel.

Esterases: We found that the esterases inyoung leaf tissue were best observed using theflorogenic substrate 4-methylumbelliferylacetate. The citrate/3 amino-morpholinesystem resolved four areas of esterasc activity,one cathodal and three anodal (Figure 2). Thecathodal esterase, Est-4, gave relatively blurrybands, and the heterozygous pattern couldonly be interpreted as a broad smear.

6-Phosphogluconate dehydrogenase: Twoloci are responsible for the 6PGD activityobservable in the pea leaf extracts28. The moreanodal isozyme (6PGD-1) is localized in theplastid compartment while 6PGD-2 is cyto-solic. Plants heterozygous at cither locus ex-hibit intragenic heterodimers (Figure 3)concordant with the dimeric structure of thisenzyme23. Hybrid enzymes containing one6PGD-1 subunit and one 6PGD-2 subunitwere not observed.

Phenotypes for shikimate dehydrogenase,phosphoglucomutase, aldolase, and acidphosphatase were described previously31-32.

Linkages observed

Tables II and III present the segregationdata for individual loci and joint segregation

Table LL Segregation of allelei at loci involved in mapping experiment*

Family

A

E

G

Locus

PurOhSWbK

StBGpRTl

IdhSkdhAldo-pAat-mPgm-p

Lap-1Acp-1Acp-2Acp-36pgd-p

WbKStBGp

RTlBtPgm-pLap-1

Acp-1Acp-26pgd-cEst-4

DArKhtSt

BIdhAat-mSkdhPgm-p

Lap-1Lap-2Acp-1Acp-2Acp-3Est-4

No.

recessiveor slow

1923152225

2221201924

2023222823

2421211723

3133363132

2628372532

27292928

11896

12

139

1088

988876

observed with designated phenotype

heterozygous

37

4542354045

3737333139

55

5256

58495752

20232017

182125132020

dominantor fast

4461696359

6263646524

1919131716

2326211923

8582818584

8434773226

31293128

2926303229

26118

1216

13107

109

13

X2

0.890.252.280.041.02

0.060.0

0.060.251.42

0.450.382.313.141.60

1.211.781.080.490.29

0.180.842.080.180.40

0.110.773.381.110.67

0.280.760.150.22

0.130.040.081.800.40

1.440.200.800.804.32

1.200.442.551.060.672.54

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data between pairs of loci that gave significantdeviations from random assortment. Themorphological characters used for mappingthe isozyme loci have well-characterizedphenotypes and are specified by single loci(Table I) that have been located on the chro-mosomal map of the pea5. The enzyme locimentioned in the tables can be placed in fivelinkage groups, corresponding to regions ofchromosomes 1, 2, 3, 5, and 7 (Figure 4).

from Wb. Linkage was not observed betweenPgm-p and Skdh in family A or betweenPgm-p and Ar in family G.

Chromosome 3

Leucine aminopeptidase: Both isozymes ofLAP are specified by genes located on chro-mosome 3. The tight linkage between Lap-1and B was demonstrated previously2. Our data

confirm this observation and indicate thatLap-1 is on the centromere side of B. The genecoding the LAP-2 isozyme, designated Lap-2,appears to be located near M at the oppositeend of the chromosome from B. Relativelyclose linkage (9 percent recombination) wasobserved between M and Lap-2 in family G,and significant deviations from random as-sortment also were seen between Lap-2 and st(families E and G) and Acp-3 (family G). The

Chromosome 1

Isocitrate dehydrogenase: The locus codingsubunits of this enzyme, Idh, showed closelinkage with two marker loci on chromosome1: Pur and D. Each marker locus exhibitedabout 5 percent recombination with Idh. An8 percent recombination frequency has beenreported between Pur and D, suggesting thatIdh may be located between these two; how-ever, a three-point test involving the loci wasnot performed in our experiments.

Chromosome 2

Shikimate dehydrogenase: In the A popu-lation (84 individuals) no recombinants werefound between Skdh and Oh, the locus con-trolling a reddish-brown pigment in the testa.The positioning of Skdh on chromosome 2 wasfurther substantiated in population G indi-cating a linkage between this locus and Ar,also located on that chromosome.

Aspartate aminotransferase: The locusspecifying mitochondrial AAT, Aat-m, alsowas assigned to chromosome 2 by virtue of itslinkage with Skdh in both populations A andG, with Oh in A and with Ar in G. The twoindependent determinations of linkage be-tween Skdh and Aat-m gave recombinationfrequencies of 24 percent and 18 percent(Table III). In G a three-point test suggestedthat the sequence Aat-m—Skdh—Ar is thecorrect order of the genes, with Aat-m posi-tioned toward the end of the chromosome.

Aldolase: Variation in the plastid specificaldolase was produced by alleles at locusAldo-p, near the end of the known linkagemap. In population A, Aldo-p showed linkagewith both Skdh and Aat-m, with the relativerecombination frequencies indicating a geneorder of Skdh—Aat-m—Aldo-p. These re-sults confirm an earlier report of possiblelinkage between Skdh and Aldo-p31.

Phosphoglucomutase: The gene coding theplastid specific form of phosphoglucomutase,Pgm-p, exhibited linkage with S, K, and Wbloci mapping near the middle of chromosome2. The relative recombination frequencies inthe possible three-point tests suggested thatPgm-p was located on the opposite side of K

Table HI. Joint segregation data for pairs of loci exhibiting significant deviationsfrom random assortment

Loci

Chromosome 1Pur.IdhD.Idh

Chromosome 2Oh:Aat-mOh.SkdhSkdh:Aat-mAat-nvAldo-pSkdh:Aldo-p

Ar:Aat-mAr.SkdhSkdh:Aat-mS:Pgm-pWb:Pgm-pK: Pgm-p

S:WbS:KWb:KWb:Pgm-pK:Pgm-pWb:K

Chromosome 3St:Acp-3St.BSt: Lap-1Acp-3: Lap-1B:Acp-3B: Lap-1

St:Lap-2St.BM:Lap-2M:Acp-3Lap-2:Acp-3

St:Lap-2St:Acp-3St:Lap-lSt:BAcp-3: Lap-1B:Acp-3B:Lap-1

Family

AG

AAAAA

GGGAAA

AAAEEE

AAAAAA

EEGGG

GGGGGGG

N

6340

8483847675

343440838484

848484

109108115

668383416757

86116373434

39364039363539

No.

-/-

159

152315187

150

131722

151418162223

121321

110

620

002

3717130

observed with designated- / H

21

60959

332254

139

5

5440

12

025

633

261

- / +

00

20413

406000

014107

19

1561

12

916641

108441

12

H / -

61015

3

6

3

3

H/H

292420

15

7

11

11

phenotypes*H/+

433

2

2

4

6

+/-

11

130206

636

1060

711793

10

57

2356

19

2011861

5086539

+/H

3219

3341

444

16136

434042

3942

25

318

2725

26

20183

151715

31417

+/+

1310

151911118

4100

151616

625855313275

1854

72

181

1369

344

995

22180

Recomb.fracL

55

27

241742

30191816134

71413231115

19212036192

43259

2035

379

24273034

2

SE

2.83.5

5.5—3.93.45.6

9.17.44.84.33.92.1

3.14.14.04.53.23.7

5.35.14.87.05.21.5

6.54.84.97.67.6

9.25.07.68.66.89.41.8 -

• Designations: recessive phenotype or homozygous slow = —; heterozygous - H; dominant phenotype orhomozygous fast =» +

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d . .Pur- • '**h

2 3 4-rAldo-p -j-Lap-2

- -Aat-m

oh- Skdhar- •

wb-

k- •

Pust

: • Acp-3

- -Pgrn-p

fa- •

Np-

le-

5 6

CP+ wlo--

g p - •

• Acp-1

pi - -

•+• 6 pgd-c

u - .

B t - -

Acp-2

Ett-4

FIGURE 4 Relative map positions on the seven chromosomes of Pisum sativum of the marker lociand the 12 isozyme loci examined in this study. Marker loci are shown to the left of the chromosomewhile the uoxyme loci are labeled in bold print to the right of the chromosome.

fewer number of recombinants observed be-tween M and Acp-3 than between Lap-2 andAcp-3 (family G, Table III) suggests thatLap-2 is situated distal to M on this arm ofchromosome 3.

Acid phosphatase-3: This isozyme also iscoded by a locus on chromosome 3. The locusis close to St, near the middle of the knownlinkage map for this chromosome. The dataplace Acp-3 on the M side of St. Both Acp-3

Table m . Continued

Loci

Chromosome 5Gp: Acp-1Gp:6pgd-c6pgd-c:Acp-lGp: Acp-1Gp:6pgd-c6pgd-c:Acp-l6pgd-c:Acp-l

Chromosome 7R:T1R.Acp-2Tl:Acp-2R:TlR:Acp-2R:Est-4

Tl:Acp-2Tl:Est-4Bt:Acp-2Bt:Est-4Acp2:Est-4

Family

AAAEEEG

AAAEEE

EEEEG

N

838484

11511611640

837375

110105101

10710810510527

No.

-/-

161217211721

7

1900

261

16

2175

160

observed with designated- / H

4859

1384

045069

71016140

- / +

0012200

013170

190

190

1247

H / -

4

61

9

38

0

H/H

31

4119

23

3932

9

phenotypes*H/+

4

92

0

1

810

1

+ / -

41006

1200

321122

2812

243

24125

+/H

3331

1484392

3729

5524138

3103136

3

+/+

262321292922

5

2463

301026

2181723

2

Recomb.fract.

10231017261512

415192

1822

1523403216

SE

3.45.12.43.84.62.63.9

2.24.53.61.14.24.6

2.73.45.85.35.5

and St exhibit linkage with Lap-1 and Lap-2,permitting most of the known linkage map tobe spanned by the three isozyme loci.

Chromosome 5

Acid phosphatase-1: The gene coding themost anodal of the acid phosphatases exhibitednonrandom assortment with Gp on chromo-some 5 in both populations A and E. The re-combination frequencies observed betweenthese two loci in both crosses were very similar(Table III).

6-Phosphogluconate dehydrogenase: Thesubunits of the cytosolic isozyme of 6PGD-2also are specified by a gene linked with Gp.Both three-point crosses involving the loci Gp,Acp-1, and 6pgd-c indicate a gene order ofGp—Acp-1—6pgd-c. Again, recombinationfrequencies between 6pgd-c and the other twoloci were quite repeatable. An absence oflinkage between 6pgd-c and Cp in populationE suggests that both 6pgd-c and Acp-1 lie onthe opposite side of Gp from Cp. Preliminarydata indicating linkage between 6pgd-c andFs32 are consistent with such a position for6pgd-c.

Chromosome 7

Acid phosphatase-2: In leaf tissue this locusproduces an isozyme of relatively weak ac-tivity. The pattern of segregation at Acp-2closely followed segregation seen at the Tl andR loci on chromosome 7.

Esterase-4: This locus also exhibited linkagewith Tl and R. In a four-point cross with Tl,R, and Acp-2, the Est-4 locus appeared to befarther from the Tl—R region than Acp-2(recombination frequencies: Est-4, Tl = 23percent; Acp-2, 77 = 15 percent). The se-quence of Tl and R relative to Acp-2 couldnot be reliably determined from the data dueto the tight linkage between the former twoloci; however, Est-4 also showed linkage withBt located near the lower end of the chromo-somal linkage map, indicating that Acp-2 andEst-4 are located between Tl and Bt (Figure4).

Discussion

Our results provide approximate map lo-cations of 11 previously unmapped isozymeloci in Pisum and confirm the map position ofLap-1, the only previously mapped isozymelocus. The consistency of the results in threeindependent crosses indicated that the devia-tions from random assortment were not causedby irregularities at meiosis or pseudolinkagedue to heterogeneity of chromosome structurebetween parental lines. Similar to results in

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maize9, the isozyme loci in the pea do not ap-pear to cluster within the genome. Not onlyare the 12 loci distributed over five chromo-somes but the map distances between loci onthe same choromosome appear to be relativelylarge. The most closely linked enzyme loci,Acp-1 and 6pgd-c, showed a recombinationfrequency of 10 percent while the three loci onchromosome 3 (Lap-1, Acp-3, and Lap-2)

span nearly the entire linkage group. In con-trast, Tanksley and Rick reported that nearly30 percent of the isozyme loci in the tomatowere associated into tight clusters25.

The arrangement of the isozyme loci onchromosomes 2 and 3 are especially fortunate,for nearly every gene on these chromosomeswill exhibit linkage with an isozyme marker.This distribution should prove extremelyuseful for mapping and for marking charactersthat are difficult or inconvenient to score di-rectly.

Although the four loci coding mitochondrialor plastic specific enzymes or isozymes are alllocated on chromosome 2, the possibility thatsuch a grouping reflects a complex of such lociis incompatible with other results. The sig-nificant distances between the loci precludesthe possibility that these genes may be underthe control of a single cis-acting regulator. Inaddition, two other loci coding plastid specificproteins, Aat-p and 6pgd-p, do not exhibitlinkage with markers on chromosome 2 or witheach other (unpub. data). Loci coding mito-chondrial specific enzymes in maize also ap-pear not to be linked15.

The designations used for loci specifyingmitochondrial and plastid specific isozymesrepresents a deviation from the terminologyused in maize and the tomato9-18. Such achange was initiated because recent studieshave demonstrated that the subcellular com-partmentation of isozymes in many systems(e.g., phosphoglucomutase, aspartate ami-notransferase, malate dehydrogenase, andglucose phosphate isomerase) is an importantand predictable characteristic of these systems(see reviews by Gottlieb12, Newton15, andWeeden29). Since comparison of linkagegroups can only be accomplished when ho-mologous loci are being considered it is im-perative to determine the subcellular local-

ization of isozymes, especially those known tohave organelle specificities, and to conve-niently mark their localization. The designa-tion of loci coding plastid specific isozymeswith a suffix "p", cytosolic with a "c" andmitochondrial with an "m" would serve thispurpose. Standard numbering protocol couldbe used to distinguish multiple isozymes in thesame subcellular compartment.

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11. and . Electrophoretic measurementof genetic variation within the named soybeangermplasm. Genetics 100^27.1982.

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