association of isozymes with a reciprocal ... staining methods used were those of brewer and sing...

Copyright 0 1984 by the Genetics Society of America

ASSOCIATION OF ISOZYMES WITH A RECIPROCAL TRANSLOCATION IN CULTIVATED RYE

(SECALE CEREALE L.) ANA M. FICUEIRAS, MARlA T. GONZALEZ-JAEN, JULIO SALINAS’ AND

CESAR BENITO

Departamento de Genitica, Facultad de Biologk, Universidad Complutense, Madrid-3, Spain

Manuscript received July 7, 1984 Revised copy accepted August 17, 1984

ABSTRACT

In rye (Secale cereale L. cv. “Ailis”) the progeny of a cross between a structural heterozygote for a reciprocal translocation (involving the I R chromosome) and a homozygote for the standard chromosome arrangement were analyzed for the electrophoretic patterns of eight different leaf isozymes and also for their meiotic configuration at metaphase 1.--The Got-3 and Mdh- 26 loci are linked to each other and also to the reciprocal translocation. The Mdh-26 locus is located in the interstitial segment of the 3Rq chromosome arm, with an estimated distance of 8 cM to the breakpoint. Therefore, the reciprocal translocation involves the I R and 3R chromosomes.--Also, the Mdh-I and 6-Pgd-2 loci are linked (16 zk 3 cM) and have been located on the 2Rq arm. Finally, the Per-3 and P e r 4 loci are located on the 2Rp chromosome arm at an estimated distance of 26 zk 4 cM.

OLYMORPHJSM for reciprocal translocations has been reported in several P plant species (BURNHAM 1956). One of the most obvious consequences of a reciprocal translocation is the suppression or reduction of crossing over in the structural heterozygote at the interstitial zone; thus, the allelic combinations located in the recombination-sheltered zones of the reciprocal translocations would remain unchanged (GRANT 1956). It is likely that particular gene combinations would prove favorable, and in this case they would remain un- altered in the populations (DOBZHANSKY 1970). Certain alleles could become associated with the rearranged chromosomal segments (HEDRICK, JAIN and HOLDEN 1978; ELLSTRAND and LEVIN 1980). Consequently, the association of alleles with reciprocal translocations is expected to be nonrandom (HEDRICK, JAIN and HOLDEN 1978).

In the case of rye, a number of cultivars showing polymorphism for reciprocal translocations have been observed; in these cultivars the structural heterozygote frequencies occur between 1.4 and 4.7% (FIGUEIRAS, CANDELA and LACADENA 1983). In the case of “Ail&” cultivar this frequency is surprisingly high (1 5-20s) and shows many different reciprocal translocations (CANDELA, FIGUEIRAS and LACADENA 1979).

PEREZ DE LA VEGA and ALLARD (1984) and VAQUERO, VENCES and PEREZ ’ Present address: Laboratoire de GGnGtique Mol6culaire. lnstitut Jacques Monod, Universite de Paris VI1

Tour 43, 2 Place Jussieu, 75005 Paris, France.

Genetics 109 177-193 January, 1985

178 A. M. FIGUEIRAS ET AL.

DE LA VEGA (1982) described the monogenic control and the dimeric behavior of the GOT-3, PGI-1 and MDH-1 isozymes. They also reported that the isozymes PGM-1, MDH-2, ALK-3, PER-2, PER-3 and PER-4 were monomers under monogenic control in rye.

The chromosomal location of the structural genes for these isozymes have been determined by different authors in a number of rye cultivators (“Impe- rial,” “King 11” and “Dakold”) by TANG and HART (1975), HART (1979), RAO and RAO (1980), CHOJECKI and GALE (1982), BARBER et al. (1968), BERGMAN and MAAN (1973) and SALINAS and BENITO (1983, 1984a,b,c) (Table 1).

In rye, SALINAS and BENITO (1984~) suggested the occurrence of a duplication for the Mdh-2 locus, on the basis that the different alleles for this system can be located in both the IR and the 3R chromosomes (Table 1).

In the present work we have tried to relate particular structural translocations with specific isozymes in order to elucidate the possible relevance of such associations in the maintenance of chromosomal polymorphism for reciprocal translocations in natural plant populations.

MATERIALS AND METHODS

Different cultivars of rye Secale cereale L. were used. In the case of the cv. Ail& (polymorphic for reciprocal translocations) a heterozygous plant for a reciprocal translocation (HT, I I V + 5” at metaphase 1 (MI) was crossed with another plant of the same cultivar, homozygous for the standard chromosome arrangement (HM, 7” at MI). Both the chromosome constitution and the isozyme patterns were analyzed in the progeny of this cross.

In addition, plants from the cultivars Rafia (RA) and Riodeva (RI) (the latter was an inbred line with 20 generations of selfing), both having the standard chromosome constitution, were crossed. The offsprings of two crosses, (RA X RI)-I and (RA X RI)-2, were analyzed for linkage relationships among isozyme loci.

The cytological study was made on the pollen mother cells (PMCs) at MI following the aceto- orcein stain procedure (SHARMA and SHARMA 1965). Those plants showing l” + 5” at MI were classified as structural heterozygotes for the reciprocal translocation, and the plants showing 7” were classified as structural homozygotes.

To analyze whether the nucleolar organizer chromosome ( I R ) was involved in the interchange of the structural heterozygote, PMCs at diplotene-diakinesis were stained with ammonium-ferric- sulfate and hematoxylin in order to make the nucleolus visible and ascertain whether or not it was associated with the quadrivalent.

As an approach to the recognition of the remaining chromosome complement, the C-banding technique was used with PMCs at MI (GIRALDEZ and ORELLANA 1979) following the chromosome classification proposed by ORELLANA and GIRALDEZ ( 1 98 1) and the chromosome nomenclature of SYBENGA (1983).

The seeds of the three different progenies were allowed to germinate, and the biochemical analyses were carried out with the 12-day-old leaves. The following isozymic systems were studied using 12% starch gels: glutamate oxaloacetate transaminase (GOT), phosphoglucomutase (PGM), phosphoglucoisomerase (PGI), esterase (EST), using Tris-citric acid (0.015 M, pH 7.75) as the gel buffer and NaOH-boric acid (0.3 M, pH 8.6) as the electrode buffer; malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6-PGD), alkaline phosphatases (ALK) and cathodal peroxidases (PER) using Tris-citric acid (0.043 M, pH 7.0) as the electrode buffer and histidine (0.006 M, pH 7.0) as the gel buffer. Each crude extract was analyzed simultaneously by all of these systems using I-cm thick gels, sliced into 2-mm slabs before staining.

The staining methods used were those of BREWER and SING (1970) for PGM, PGI, MDH and 6-PGD; SHAW and KOEN (1968) for EST, ALK and PER and the method of SELANDER et al. (1971) for GOT. In each case minor modifications were introduced.

ISOZYMES AND TRANSLOCATIONS IN RYE

TABLE 1

Chromosomal location of isozyme structural genes in Secale cereale L. cu. Imperial, cu. King I1 and cu. Dakold

179

Enzymes

Chromosome or chromo-

some arm of Genes genome R References

Glucosephosphate isomerase- 1"

Malate dehydrogenase-2 Glutamate dehydrogenase-1 Leaf peroxidase 6-Phosphogluconate dehydro-

Esterase- 1 a

genase-2"

Glutamate oxaloacetate trans-

Malate dehydrogenase-2 Alcohol dehydrogenase-1"

Phosphoglucomutase-I

aminase-3"

Endosperm peroxidase- 1 6-Phosphogluconate dehydro-

Glucose-6-phosphate dehydro-

Aminopeptidase- 1

6-Phosphogluconate dehydro-

Embryo plus scutellum peroxi-

Glutamate oxaloacetate trans-

Esterase-2 Endosperm peroxidase-2,3,4 Acid phosphatase

genase- 1 a

genase

genase- 1

dase

aminase-2"

Alkaline phosphatase Endopeptidase- 1 Glutamate oxaloacetate trans-

aminase- 1 '

Gpi-I

Mdh-2a

LPer- 1,2,3,4 Gdh-1

6-Pgd-2

Est-I

Got-3

Mdh-26 Adh-1

Pgm-1

EPer-1 6-Pgd - 1 a

G-6-Pd-4

Amp-I

6-Pgd-lb

SPer-1,2

Got-2

Est-2 EPer-2,3,4 Acph

Alk-3 EP- I Got-I

HART (1 979), CHOJECKI and

SALINAS and BENITO (1 984c) SALINAS and BENITO (1 983) SALINAS and BENITO (1 984b) SALINAS and BENITO (1 983)

BARBER et al. (1 968), BERGMAN

HART (1 975), TANG and HART

SALINAS and BENITO (1984~) TANG and HART (1 975), HART

J. SALINAS and C. BENITO, un-

SALINAS and BENITO (1 98413) RAO and RAO (1 980), SALINAS

SALINAS and BENITO (1 983)

GALE (1982)

and MAAN (1973)

(1975)

(1 979)

published results

and BENITO (1 983)

TANG and HART (1 975), HART

RAO and RAO (1 980). SALINAS

SALINAS and BENITO (1 984b)

HART (1 975), TANG and HART

BERGMAN and MAAN (1 973) SALINAS and BENITO (1984b) HART (1979), SALINAS and BEN-

ITO (1 984a) SALINAS and BENITO (1 984a) HART (1 979) TANG and HART (1 975), HART

(1 979)

and BENITO (1983)

(1 975)

(1 979)

p, Short arm; q, long arm; p? or q?, chromosome arm assigned by homoeology with hexaploid

a Dimeric enzymes. wheat.

The crude extracts were always absorbed in paper wicks (Whatman N3) and inserted in hori- zontal gels. The gels for GOT, PGM, PGI and EST were electrophoresed at a constant voltage of 280 V for 3 hr; the gels for MDH, 6-PGD, ALK and PER at a constant voltage of 150 V for 5 hr.


a b e ' ,

?R @ & 4R ,

4 1 -.

2 R 2R 4 R

6

5R 6 R ? R 3 2 1 3 3 3 4 4 4 3 3 3 FIGURE 1 .-a, PMC at diplotene-diakinesis with hematoxylin of the structural heterozygote

(HT) used in the cross H T X HM. Arrow indicates the nucleolus associated to the quadrivalent. b, MI cell with C-banding of the structural heterozygote (HT) used in the cross HT X HM. c, The five different bivalents of the H T plant. Arrows indicate the bivalent 2R is heterozygote for a telomeric C-band. d, 6-Phosphogluconate dehydrogenase (6-PGD) zymograms shown by (1) structural heterozygote (HT), parental genotype (12); (2) structural homozygote (HM), parental genotype ( 1 1); (3) the progeny of the cross H T X HM; (4) homozygous plants for the slow allele (2) of 6-PGD. The 6-PGI) isozymes of zone I I are dimers.

RESULTS

The hematoxylin analysis of PMCs at the diplotenediakinesis stage reveals that the nucleolus is associated with the quadrivalent observed in the structural heterozygotes (HT) obtained in the cross H T X HM (Figure la). In this cross the segregation observed for the quadrivalent at MI was the expected 1:l (1"' + 5ll.7" . mosome ZR was involved in the quadrivalent, but the other pair could not be identified. Three of the bivalents were identified as 2R, 5R and 6R; the remaining two bivalents could not be positively identified, but one of them seems to be 4R (Figures l b and IC).

) (Table 2). The C-banding technique analyses revealed that the chro-


TABLE 2

Single-locus segregation of ten dafferent isozymic loci in the cross HT X HM

181

Loci Distribution of progeny (parental genotypes) (phenotype) x p 1:2:1 x p 3:l x z 1:1

Pgi-I (12) x (12) 54 ( 1 1 ) 47 (12) 22 (22) 23.48*** Pgm-I (12) X (12) 36 ( 1 1 ) 51 (12) 36 (22) 3.58 Per-2 (+-) X (+-) 90 (+) 33 (-)

Got-3 (12) X (11) 57 ( 1 1 ) 66 (12)

Per-3 (+-) X (--) 65 (+) 58 (-) Mdh-1 (13) X ( 1 1 ) 64 ( 1 1 ) 59 (13) Mdh-2b (12) X ( 1 1 ) 59 ( 1 1 ) 64 (12) 6-Pgd-2 (1 2) X ( 1 1 ) 62 ( 1 1 ) 61 (12)

Est-2 (+-) X (+-) 87 (+) 30 (-)

Alk-3 (+-) X (--) 56 (+) 61 (-)

0.22 0.025

0.66 0.21 0.40 0.20 0.20 0.008

Meiotic configuration x s 1:l Quadrivalent 60 (I"' + 5") 63 (7") 0.073

All enzymes with (+) and (-) phenotypes are controlled by loci with one active and one null allele. In the 3:1 segregation the (+) phenotype includes (++) and (+-) genotypes. In the 1 : 1 segregation the (+) phenotype has (+-) genotype.

*** Significant x 2 at 0.001 level.

The isozymic pattern for both the parental and the progeny plants of the same cross (HT X HM) is shown in Figure 2. These progenies segregated for ten different loci (Table 2). The isozymes GOT-3, PGI-1 and MDH-1 are dimers, whereas PGM-1, EST-2, MDH-2, ALK-3, PER-2, PER-3 and PER-4 are monomers (Figure 2). The 6-PGD-2 isozymes are monogenic dimers (Fig- ures Id and 2).

The Got-3, Pgm-1, Pgi-1, Mdh-1, Mdh-2b and 6-Pgd-2 loci showed two active alleles in all of the crosses analyzed. The Mdh-2b locus was located on chromosome 3R (Table 1). In the case of the loci Est-2, Alk-3, Per-2, Per-3 and Per-4 there is one active and one null allele. Therefore, for the last group of loci it is not possible to distinguish the homozygotes for active alleles from the heterozygotes.

Table 2 summarizes the single-locus segregation for the leaf isozymes. Only for the locus Pgi-1 did observed segregation deviate from the expected 1:2:1, whereas the remaining loci segregated as expected.

The linkage relationships between the isozymic loci and the reciprocal translocations are shown in Table 3. In Table 4, two-point linkage tests for the isozymic loci in the H T X HM cross are summarized. It can be seen that Got- 3 and Mdh-2b were linked, as were Mdh-1 and 6-Pgd-2. On the other hand, the Got-3 locus was associated with the translocation and the same results were obtained for the Mdh-2b locus (Table 3).

The zymograms for the crosses (RA X RI)-1 and (RA X RI)-2 (parental and progeny plants) are shown in Figure 3. In progenies from both crosses, the loci Cot-3, Pgm-1, Mdh-2b and Per-4 segregated as expected, as well as loci Alk- 3 and Per-3 in the progenies from the first and second crosses, respectively


+ b

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PER

I 4 0 - - - I V 5 - - - -v

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FIGURE 2.-Diagrammatic representation of leaf isozymes. 1, Structural heterozygote (HT); 2, structural homozygote (HM); 3, the progeny of the cross H T X HM. The different activity zones observed in each isozyme systems are indicated on the right (I, 11, 111, IV, V) and the different alleles shown by the plants are indicated on the left (1, 2, 3, 4, 5) . The Pgm-1, Pgi-I, Got-3, Mdh- I , Mdh-26 and 6-Pgd-2 loci show two active alleles; Alk-3, Est-2, Per-2, Per-3 and Per-4 loci show one active and one null alleles. The Mdh-I locus shows two active alleles (1 and 3) coding for the dark bands diagrammed in the figure. The Mdh-26 locus shows also two active alleles (1 and 2) which coded for the light bands.

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ISOZYMES AND TRANSLOCATIONS IN RYE 185

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2), (3) Riodeva (RI), (4) the progeny of the crosses (RA X RI)-1 and (RA X RI)-2. T h e different activity zones observed in each isozymic system are indicated on the right (I, 11, 111, IV, V), and the different alleles shown by the plants are indicated on the left (1, 2, 3, 4, 5 ) . T h e Pgm-1, Pgi- 1, Got-?, Mdh-I , Mdh-2 and 6-Pgd-2 loci show two active alleles; Alk-3, Est-2, Per-2, Per-3 and Per- 4 loci show one active and one null allele. T h e Mdh-I locus shows two active alleles (1 and 3) coding for the dark bands diagrammed in the figure. The Mdh-26 locus shows also two active alleles (1 and 2) which coded for the light bands.


TABLE 5

Single-locus segregation of different isozymic loci in the crosses (RA X RI)-I and

187

(RA X RI)-2 ~

Distribution of progeny Loci (parental

genotypes) (RA X RI)-I (phenotype) xz 1:l (RA X RI)-2 (phenotype) x z 1:l

Pgm-I (12) X (11) 48 (11) 49 (12) 0.01 49 (11) 51 (12) 0.04

Per-3 (+-) X (--) 56 (+) 41 (-) 2.3 1 53 (+) 47 (-) 0.36 Per-4 (+-) X (--) 52 (+) 45 (-) 0.50 Alk-3 (+-) X (--) 52 (+) 48 (-) 0.16

All enzymes with (+) and (-) phenotypes are controlled by loci with one active and one null allele. In the 1:1 segregation the (+) phenotype has (+-) genotype.

Got-3 (12) x (1 1) 46 (11) 51 (12) 0.25 50 (11) 50 (12) 0.00

Mdh-26 (1 2) X (1 1) 47 (11) 50 (12) 0.09 45 (11) 55 (12) 1 .O

(Table 5) . The linkage analyses for these two crosses revealed that the Got-3 and Mdh-26 loci were linked. Per-3 and Per-4 were also linked in the cross (RA X RI)-1 (Table 6).

DISCUSSION

Our results are in agreement with the previous ones reported on the genetics of isozymes analyzed in the present work. In addition, we found that 6-PGD- 2 isozymes are monogenic dimers in rye (Figures Id, 2 and 3).

Our data from the three crosses reveal that the Got-3 and Mdh-2b loci are linked (Tables 4 and 6). Also Mdh-I and 6-Pgd-2 loci showed linkage in the cross HT X HM (Table 4). The cross (RA X RI)-1 indicates that Per-3 and Per-4 are also linked. The remaining loci analyzed showed independent as- sortment.

These results conform very well to the chromosomal location data summarized in Table 1. There is a discrepancy between the two sources of infor- mation only in the case of loci Per-2 and Per-3. Both loci have been reported before (SALINAS and BENITO 1984b) to be situated in the chromosome arm 2Rp, but they segregated independently in the cross HT X HM. A possible explanation could be that the genetic distance between these two loci is greater than 50 cM.

The distances between the isozymic loci were estimated by means of the maximum likelihood method. The distances obtained between Got-3 and Mdh- 2b from the crosses (RA X RI)-1 and (RA X RI)-2 were 17 f 3 and 25 f 4 cM, respectively. The data from these two progenies can be pooled since the x2 heterogeneity test was not significant at the 5% level. With the pooled data, the estimated distance was 21 f 2 cM (Table 6).

In the cross HT X HM, the estimated distance between Got-3 and Mdh-2b loci was 13 f 3 cM. Exactly the same value was obtained when the linkage relationships between the Got-3 locus and the reciprocal translocation were analyzed. The Mdh-2b locus appears to be totally linked to the translocation as can be inferred by the absence of recombinant plants (Table 3). This locus

188 A. M. FIGUEIRAS E T AL.

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might be located between the centromere and the translocation breakpoint (Figure 4).

Thus, the distance between the Mdh-2b locus and the translocation breakpoint would be about 21 - 13 = 8 cM. This distance was calculated by comparing the distance obtained between the Got-3 and Mdh-2b loci in the crosses (RA X RI)-1 and (RA X RI)-2 with the distance estimated in the cross H T X HM by the same loci.

Previous reports indicate that the cot-3 and Mdh-2b loci are located on the chromosome 3R in rye (TANG and HART 1975; SALINAS and BENITO 1984c) (Table 1). HART (1975) located GOT-3 in wheat on the long arm of homoeologous group 3. It is possible that in rye the location of GOT-3 is also on the long arm of the chromosome 3R, considering the strong homoeology oc- curring between the rye and wheat chromosome arm (HART 1979; SALINAS and BENITO 1983, 1984a,b,c). Accordingly, it is likely that the long arm of the 3R chromosome would be involved in the translocation observed in the structural heterozygote (Figure 4) and the same could be applied to the Mdh- 2b locus (Got-3 and Mdh-26 are closely linked).

On the other hand, the study of the PMCs at diplotene-diakinesis revealed that the nucleolus was associated with the quadrivalent. So, the nucleolar organizer chromosome (I R ) would be implicated in the translocation together with the 3R.

Furthermore, the C-banding technique indicated that the 1 R chromosome is related to the quadrivalent. It was not possible to identify the other chromosome involved in the translocation, but the chromosomes 2R, 5R , 6 R and probably 4R could be excluded (Figure l b and c). The Ail& cv. shows a high polymorphism for heterochromatin bands (Figure IC), and normally it is not possible to identify all of the chromosome complement. However, the chromosomes IR , 2R, 5R and 6 R can usually be clearly recognized (ORELLANA and GIRALDEZ 1981). Therefore, the other chromosome involved in the interchange would either be 7 R or 3R which supports our results obtained using the isozyme markers.

The location of the isozyme structural genes in the Triticeae genomes suggested that the linkage groups have been largely conserved. This has also been found among different rye cultivars (Table 1). Consequently, the isozyme genes can be used as chromosome markers in rye. Therefore, simultaneously using both kinds of markers (cytological and biochemical) it is possible to identify the chromosomes involved in a range of different translocations.

CHOJECKI and GALE (1982) located the Pga-I locus on the 1Rp chromosome arm in rye and on the short arms of the homoeologous group 1 in hexaploid wheat. These authors also suggested the occurrence of a duplication for the Pgi-I locus in hexaploid wheat. In rye, the segregation of this locus in different crosses frequently deviates from that expected (A. M. FIGUEIRAS, unpublished data). Therefore, it is possible that a duplication of the Pgi-I locus is present in both species. This might explain the discrepancy between the observed and expected segregation observed in the H T X HM cross (Table 2).

In the same H T X HM cross the 6-Pgd-2 and Mdh-I loci were found to be


STRUCTURAL HETEROZYGOTE

191

OU A DR I VA L E N T

BREAK MDH POINT GOT

3RS:: 3RL I I

I I

I 1 I '-1 I

I I

I I I

8 13

2 1

DISTANCE FIGURE 4.-Diagrammatic representation of the quadrivalent observed in the structural heter-

ozygote (HT) used in the cross H T X HM. The 3 R and I R chromosomes are involved in the translocation. The isozymic alleles of MDH and GOT that are located on the 3 R chromosome have been named 1 and 2. Also, the genetic distances calculated among the Mdh-Pb, Got-3 loci and the breakpoint on the 3 R chromosome are indicated.


linked by an estimated distance of 16 & 3 cM (Table 4). SALINAS and BENITO (1983) located the 6-Pgd-2 locus on the long arm of the 2R chromosome. Thus, it is possible that Mdh-1 is on the same chromosome.

Similarly, the loci Per-3 and Per-4 were found to be linked in the cross (RA X RI)-1 with an estimated distance of 26 & 4 cM (Table 6). Per-3 is located on the 2Rp chromosome arm (SALINAS and BENITO 1984b). Therefore, our results indicate that both Per-3 and Per-4 are on chromosome 2R.

The rye cultivar Ail& is highly polymorphic for reciprocal translocation. Several different translocations have been observed in this cultivar (CANDELA, FIGUEIRAS and LACADENA 1979). It is interesting to consider the possibility that the cultivars that present polymorphism for reciprocal translocations might also show a heterozygosity greater than that of the cultivars that do not present polymorphism. Sometimes, such a variation in heterozygosity is difficult to observe. Therefore, it is very important to identify the different translocations observed and to have isozyme markers totally and partially linked to the translocations analyzed. For all of these reasons, we are now interested in comparing the polymorphism for translocation to the isozymic polymorphism. Our goal is to identify the chromosomes involved in the different translocations in order to analyze the evolutionary significance of such associations in the plant populations.

The authors are very much in debt to ALICIA DE LA P E ~ A and to EDWARD WILTSHIRE for their inestimable help with the English version of this article.

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