Chromosomal locations of phosphoglucomutase

Chromosomal locations of phosphoglucornutase, phosphoglucose isomerase, and glutamate oxaloacetate transaminase structural genes in different rye cultivars J . SALINAS' A N D C. BENITO' Departamento de GenPtic-a, Fat-ultad de Biologicl, Univer.sid(ld Complutense, Mudrid-3, Spain

Can. J. Genet. Cytol. Downloaded from www.nrcresearchpress.com by Peking University on 06/04/13 For personal use only.

Received May 3 1 , 1984 Revised manuscript received October 30. 1984 SALINAS, J . , and C. BENITO. 1985. Chromosomal locations of phosphoglucomutase. phosphoglucose isomerase. and glutamate oxaloacetate transaminase structural genes in different rye cultivars. Can. J . Genet. Cytol. 27: 105- 1 13. Phosphoglucomutase, phosphoglucose isomerase, and glutamate oxaloacetate transaminase isozymes have been studied in 'Chinese Spring' - 'Imperial', 'Holdfast' - 'King II', and 'Kharkov'-'Dakold' wheat-rye addition lines using starch and polyacrylamide gel electrophoresis. The analyses were conducted with different parts of dry kernels (embryo plus scutellum, and endosperm), leaves, and roots. In all cases the zymogram phenotypes obtained were the same, independent of the tissue analysed. Germination and maturation processes were also studied. One locus involved in the production of phosphoglucomutase isozymes was located on chromosome 4R of 'Dakold' and on 4RS of 'Imperial' and 'King 11' cultivars. Evidence was obtained that one locus controlling the phosphoglucose isomerase isozymes is located on chromosome 1 R of these three rye cultivars analysed, specifically on the short arm of chromosome 1 R in 'King 11'. Also, three loci controlling the glutamate oxaloacetate transaminase isozymes have been placed on chromosomes 7R, 6K. and 3R of the rye cultivars analysed, and more exactly, on 7RL of 'Imperial' and 'King 11' and on 6RL of 'King 11'. Biochemical evidence of homology between chromosome arms of the different cultivars of rye analysed, and homoeology between chromosome arms of wheat and rye is reported. Germination and maturation processes support the hypothesis that the expression of rye isozymes studied in dry kernel, leaf, and root, and during germination and maturation, is controlled by the same chromosome arms. Key words: wheat-rye addition lines, isozyme structural genes, phosphoglucomutase, phosphoglucose isomerase, glutamate oxaloacetate transaminase. SALINAS, J., et C. BENITO.1985. Chromosomal locations of phosphoglucomutase, phosphoglucose isomerase, and glutamate oxaloacetate transaminase structural genes in different rye cultivars. Can. J . Genet. Cytol. 27: 105- 1 1 3. On a etudie les isozymes de la phosphoglucomutase, de la phosphoglucose isomerase et de la glutamate oxaloacetate transaminase chez les lignees d'addition de ble-seigle 'Chinese Spring' - 'Imperial', 'Holdfast' - 'King 11' et 'Kharkov'-'Dakold' au moyen de I'electrophorese sur gel d'amidon et de polyacrylamide. On a effectue les analyses sur differentes parties de grains seches (embryon en plus du scutellum et de I'endosperme), les feuilles et les racines. Dans chaque cas les phenotypes des zymogrammes obtenus etaient identiques quelque soit le tissus analyse. On a aussi itudie les processus de la germination et de la maturation. Un locus implique dans la production d'isozymes de phosphoglucomutase Ctait situe sur le chromosome 4R de 'Dakold' et sur le chromosome 4RS des cultivars 'Imperial' et 'King 11'. On a obtenu des indices rCvilant qu'un locus responsable des isozymes de la phosphoglucose isomerase itait situC sur le chromosome I R des trois cultivars de seigle etudies, soit sur le bras court du chromosome IR de 'King 11'. De plus trois loci responsables des isozymes de la glutamate oxaloacetate transaminase ont ite places sur les chromosomes 7R. 6R et 3R des cultivars de seigle etudies, exactement sur le 7RL d"1mperial' et de 'King 11' et sur le 6RL de 'King 11'. On rapporte des indices biochimiques d'homologie entre les bras chromosomiques des differents cultivars de seigle analyses, et d'homeologie entre les bras chromosomiques du ble et du seigle. Les processus de la germination et de la maturation supportent I'hypothese que I'expression des isozymes de seigle etudies dans les noyaux dches, les feuilles et les racines, de mCme qu'au cours de la germination et de la maturation, est contr6lee par les mCmes bras chromosomiques. Mots cle's: lignees d'addition ble-seigle, genes structuraux d'isozymes, phosphoglucomutase, phosphoglucose isomerase, glutamate oxaloacetate transaminase. [Traduit par le journal]

Introduction zymogram of w h e a t - r ~ e addition lines and parental wheat and rye genes involved in the production of isozymes can be located on specific rye chromosomes and chromosome arms. cultivarsy

'present address: Laboratoire de Genetique moleculaire, lnstitut J. Monod, Universitd de Paris V11, 2, Place Jussieu, 75005 Paris, France. 'Author to whom reprint requests should be addressed.

Chromosomal location of isozyme structural genes can provide information about chromosome homoeology and genetic relationships among related species (Tang and Hart 1975; Hart et 976; Hart a l . 1980; Powling et al. 198 ; Hart and Tuleen 1983). this way, biochemical evidences of chromosome homoe ~ l o gand ~ evolution between in the Triticeae can be obtained by locating isozyme structural genes on the rye chromosomes and chromosome arms (Hart

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( 1979a) and references therein; Chojecki and Gale

1982; Hsam et al. 1982; Koebner and Shepherd 1983). On the other hand, isozyme markers can also be used in studies of chromosome homology and genetic relationships between different cultivars of the same species. Although for the majority of species, aneuploids for this purpose are not available, there are a significant number of wheat lines with added chromosomes of different cultivars of Sec-ule c~recrleL. (for a compendium of wheat - S . c.ereule addition lines, see Lacadena (1977)). To date, only alcohol dehydrogenase structural genes have been assigned to chromosomes of three different rye cultivars ('Imperial', 'King Il', and 'Dakold') (Irani and Bhatia 1972; Tang and Hart 1975); for this reason, the identification of the chromosomes of these cultivars and the correspondence between them and their assignation to the Se~.ulehomoeologous groups has only been possible by means of cytological data, i .e., studies of heterochromatin bands (Gill and Kimber 1974; Darvey and Gustafson 1975; Singh and Robbelen 1975; Gustafson et a!. 1976) or chromosome pairing (Koller and Zeller 1976). In this work, phosphoglucomutase (PGM) E.C. 2.7.5.1 , phosphoglucose isomerase (PGI) E.C. 5.3.1.9, and glutamate oxaloacetate transaminase (GOT) E.C. 2.6.1 . 1 isozymes are analysed. So far, PGI isozymes have only been related to the 1R chromosomes of 'King 11' and 'Imperial' (Chojecki and Gale 1982), and GOT isozymes to the 3R and 6R chromosomes of 'Imperial' (Tang and Hart 1975). Chromosome arm locations of rye PGl and GOT structural genes have not been published. PGM isozymes have yet to be studied in rye. The present paper reports on the chromosomal location of structural genes coding for the PGM, PGl, and GOT isozymes of 'Imperial', 'King ll', and 'Dakold' rye cultivars. Also, the chromosome arm location of 'Imperial' and 'King 11' PGM and GOT structural genes, and 'King 11' PGI structural genes is described. Locations are performed studying different parts of dry kernels, namely embryo plus scutellum, endosperm, leaves, roots, and the germination and maturation processes by means of polyacrylamide and starch gel electrophoresis. On the basis of these results, the genetic control and subunit structure of these isozymes in rye are discussed. In addition to this, biochemical evidence of homology among chromosome arms of the different rye cultivars analysed, and of homoeology among chromosome arms of rye and hexaploid wheat, is presented.

Materials and methods Tritic-um aestivum L. cv. Chinese Spring (CS), S. c.errale L. cv. Imperial (I), CS-I disomic addition lines obtained from E. R. Sears (Missouri), CS-I ditelosomic addition lines

2RL, 4RL, 4RS. 5RS, 7RL. and 7KS supplied by F. J . Zeller (Munchen). T . trt2srivumL. cv. Holdfast (H). S. c-ert)cileL. cv. King 11 (KII). H-K disomic addition lines from J . P. Gustafson (Manitoba). H-KII ditclosomic addition lines excluding 3RL, 3KS. and 7KS obtained from C. N . Law (Cambridge). and T . cirsrivum L. cv. Kharkov (K), S. t~crecilr L. cv. Dakold (D), and K - 0 disomic addition lines supplied by J . P. Gustafson (Manitoba) were analysed. 'I'he rye chromosomes of addition lines were named according to the classification proposed by Kollcr and Zeller ( 1976). The analyses were carried out using different parts of individual kernels, specil'ically enlbryo plus scutellum (E + S) and endosperm (Ed), and 7-day-old seedling leaves and roots. To study germination. dry kernels were placed in petri dishes with moist filter paper at 21 + I0C with a 15-h day. The E + S, Ed, coleoptyle. leaf. and root of germinating kernels were taking 0- 106 h following germination and were studied. Maturation studies were performed on samples taken daily from 5 days after anthesis until the kernel was ripe (30-35 days after anthesis). The E + S, Ed, internal coats (Ci), and external coats (Ce) of maturing kernels of plants grown under greenhouse conditions were anal ysed. In all cases, individual samples were crushed and immersed in 0. I M sodium acetate at pH 7.2. Small pieces of filter paper (Whatman 3MM) were soaked with the crude extracts and placed into the gels. PGM, PGI, and GOT isozymes were revealed using thin-layer polyacrylamide gels 10% w/v (95 : 5 acrylamide - bisacrylamide in weight) and starch gels 12% w/v (Connaught) as electrophoretic supports. Electrophoresis was carried out as previously described by Poulik (1957). After electrophoresis, polyacrylamide and starch (sliced into two slabs) gels were stained following the methods of Brewer and Sing ( 1970). The verification of the addition line chromosomal contents was performed employing the C-banding technique described by Giraldez and Orellana ( 1979).

Results All addition lines showed the 42 wheat chromosomes and the two expected chromosomes or chromosome arms of rye. The rye chromosomes of H - Kll-2R addition line showed a deletion in their short arms; this result is in agreement with Singh and Robbelen (1976). Phosphoglu~.omuruse(Figs. I , 2 , und 3) Embryo plus scutellum, endosperm, and 7-day-old seedling leaves and roots always presented the same PGM pattern, but this pattern was different according to the electrophoretic support used. In starch gels, wheat cultivars exhibited three isozymes designated PGMW I , PGM-W2, and PGM-W3; in polyacrylamide gels only the PGM-W 1 and PGM-W3 isozymes were detected. In all cases, rye cultivars only expressed one isozyme (PGM-R I ). In starch gels, PGM-R 1 isozyme had a stronger intensity than PGM-W I isozyme. When addition lines were electrophoresed using polyacrylamide gel as the electrophoretic support, no different patterns than that of the wheat cultivars were

SALINAS A N D BENITO

PGM AND PGI

PGM AND PGI MATURATION

STARCH e


r;s

n

-

--

5-10 DAYS

era

c:=

0

a=m

IIPGM

UIUI

- -- - - -- E n , ,

I

o

PGIII

I

III III

Ce I KII D

CS-I K-KII K-D

Ed

CI

Ce

LINES LINES LINES

I K11 D

-

n

--

Ed

Ci

CS-I-1R H - KII-1R K D-1R 10-15 DAYS - I 0

-

1

I

I

I

P

G

M

POLYACRY LAMlDE 0

CIZ

E

0

-

m

m

-

m

m

-

-

e m - Ce

Ci CS H K

-- -- -- - - -- - - --c:,

E

E

-

0

c-X

0

0

-

m - -

c=,

0

n

-

m

m

Cc

Ci

m

PGIII

,

1 1 1

Ed

Ce

Ci

I

1 - 1

Ed

Co

CS- I - 4 R H KII-4R K D-4R

-

Ci

-

Ed

CS I -1R H KII-1R K D-1R

Ed

I KII D

FIG. 2. PGM and PGI patterns during the maturation processes of wheat. rye, and I R and 4R addition line kernels using starch gel electrophoresis. FIG. I . Phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI) zymogram phenotypes that wheats. ryes. and critical addition lines present when endosperm (Ed),embryo plus scutellum (E + S). leaf. and root were analysed in starch and polyacrylarr~idegel electrophoresis.

observed. In starch gels, CS- I -4R, CS- I -4RS, H-KII-4R, H-KII-4RS, and K-D-4R addition lines revealed the PGM-W I isozyme having the same intensity that PGM-R I rye isozyme. The other addition lines had the PGM phenotype of wheat. Because the resolution is better in starch than in polyacrylamide gels, germination and maturation studies were only performed using starch gel electrophoresis. During wheat and rye germination no variation in the PGM patterns was observed. During maturation, the PGM zymogram phenotypes of wheat Ed, E + S, and coats showed different isozymes according to their degree of development. Rye kernels presented only quantitative variation in E + S and external coats. In all cases, from the 20th day after anthesis, it was not possible to analyse Ed and Ci separately, and the Ce intensity was very faint. The E + S did not express PGM

activity until 15 days after anthesis. The maturation of addition line kernels was similar to that of wheat kernels; however, the endosperm and internal coats of 4R addition lines exhibited the PGM-W 1 isozyme more intensely than the wheat Ed and Ci. Also, the E + S of these lines showed, from the 15th day of development, the PGM-W I isozyme more intense than the wheat E + S. The kernels of the remaining addition lines had maturation processes equal to that of wheat kernels. Pho.sphogluc~o.srisornerclsr (Figs. I , 2 , urzd 3 ) Embryo plus scutellum, endosperm, leaves, and roots always revealed the same isozymic pattern for PGI. In starch gel, hexaploid wheat exhibited two PGI activity zones: zone 1 (the faster migrating zone) had only one band, named PGI 1 -W I having the same migration as the PGM-W3 isozyme; zone I1 contained six bands (from PG12-W 1 to PG12-W6). In the polyacrylamide gel, zone 1 was the same as in the starch gel; however, zone I1 showed four bands. Rye cultivars displayed the same pattern in the starch and polyacrylamide gel. This pattern also had two activity zones of PGI: zone 1 presented only one band designated PGI I -R 1 and zone 11 was composed of four bands (from

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108

FIG.3. ( a ) Zymograms of PGM and PGI isozymes in polyacrylamide gel (Ed, E + S , leaves. and roots). Lane 9: 1, KII, and D rye cultivars. The rest of the patterns were wheat and addition lines (AL). ( h ) Zymograms of PGM and PGI isozymes in starch gel (Ed, E S. leaves, and roots). Lane 4: CS-I-4RS. H-KII-4RS. and K-D-4R AL. Lane 8: 1. KII, and D rye cultivars. The rest of the patterns were wheat and AL. ( c ) Zymograms of PGM and PGI isozymes during the kernel maturation processes in starch gel (5- 10 days after anthesis). Lane I: wheat external coats (Ce). Lane 2: wheat internal coats (Ci). Lane 3: wheat Ed. Lane 4: Ce of 1 R AL. Lane 5: Ci of 1 R AL. Lane 6: Ed of I R AL. Lane 7: rye Ed. Lane 8: rye Ci. Lane 9: rye Ce.

+

PGI2-R 1 to PGI2-R4). In the starch gel these bands had a slower mobility than PG12-W 1 , PGI2-W2, PGI2-W3, and PG12-W4 wheat bands, respectively, but in the polyacrylamide gel the mobility of these bands was the same. When addition lines were analysed using the polyacrylamide gel as the electrophoretic support, no variation in the PGI phenotypes was observed. In the starch gel, the patterns of CS-I-IR, H-KII-IR, H-KII- IRS, and K-D- IR addition line zone I1 were different to those of wheat. The rest of the addition lines showed the same PGI zymogram as the wheat cultivars. As in the case of the PGM isozymes, germination and maturation studies were carried out utilizing starch gels. No variation in the PGI patterns of wheat and rye was detected during germination. Only quantitative changes in the PGI zymograms were observed during the maturation processes of wheat and rye kernels. After 20 days of development, it was impossible to separate the Ci from the Ed, and external coat patterns were S fainter than endosperm and Ci patterns. The E presented PGI activity from 15 days after anthesis. The maturation processes of addition line kernels were like those of wheat kernels; however, the endosperm and coat zone I1 bands of 1 R addition lines showed a different intensity ratio than that of the bands of wheat Ed and coat zone 11. The same difference was found between 1 R addition line E + S and wheat E S from 15 days after anthesis. The kernels of the other addition lines

+

+

exhibited the same maturation processes as did wheat kernels. Gluturnate oxulouc~etute trunsurninuse (Figs. 4 , 5 , and 6) Embryo plus scutellum, endosperm, and 7-day-old seedling leaves and roots displayed the same GOT isozymatic phenotype; this phenotype was equivalent in starch and polyacrylamide gels. Wheat cultivars expressed three activity zones: the zone 1 (the faster migrating zone) was composed of one isozyme designated G O T l - W l , the zone I1 (the intermediate zone) also contained one isozyme (GOT2-Wl), and the zone 111 included three isozymes named GOT3-W 1 , GOT3-W2, and GOT3-W3. Rye cultivars presented three GOT activity zones too: zone 1 having one isozyme (GOTI-Rl), zone 11 formed by the GOT2-Rl isozyme, and zone 111 containing the isozyme called GOT3-Rl. CS-I-7R, CS-I-7RL, H-KII-7R, H-KII-7RL, and K-D-7R addition lines showed the GOTl-Wl isozyme more intense than the wheat cultivars. CS-I-6R, H-KII-6R, H-KII-6RL, and K-D-6R addition lines displayed the GOT2-W I isozyme more intensely than the wheat cultivars. The zone 111 isozymes of CS-I-3R, H-KII-3R, and K-D-3R addition lines exhibited a different intensity ratio than that of the isozymes of wheat zone 111. The rest of addition lines were like wheat cultivars. During the germination processes of wheat and rye,


GOT

GOT

MATURATION


e

CS H K

7RL 7RL 7R

6R 6RL 6R

3R 3R 3R

I KII D

CS-I H-KII K-D

LINES LINES LINES

FIG.4. Glutamate oxaloacetate transaminase (GOT) phenotypes that wheats, ryes, and critical addition lines present when Ed, E + S, leaves, and roots were analysed in starch and polyacrylaniide gels.

no variation in the GOT patterns was observed. Qualitative (zone I) and quantitative changes (zones 11 and 111) were detected in the GOT zymogram during the maturation processes of wheat and rye kernels. In all cases, the E + S did not show GOT activity until 15 days after anthesis. From 20 days after anthesis, Ed and Ci were analysed together and Ce expressed very faint patterns. The maturation of addition line kernels was similar to that of wheat kernels; however, Ed and Ci of 7R and 6R addition lines presented the GOT I -W I and GOT2-W I isozymes, respectively, more intensely than the wheat Ed and Ci. Besides, the endosperm and internal coat of the 3R addition lines displayed the GOT zone 111 isozymes with a different intensity ratio than that of the isozymes of wheat Ed and Ci zone 111. The E + S of the 7R, 6R, and 3R addition lines showed, after 15 days of development, the same GOT isozymatic phenotypes as those described for endosperm and Ci. The kernels of the remaining addition lines expressed the maturation process of wheat kernels.

Discussion The results acquired in this work indicated that the use of both polyacrylamide and starch gel electrophoresis at the same time, may be useful and makes it possible to obtain results that could not be obtained using only one of them. Phosphoglucomutase isozymes have been described functionally as monomers in Hordeum (Brown et al. 1978), Pinus (Mitton et al. 1979), Zea mavs (Goodman et al. 1980), Carnelia japonica (Wendel and Parks 1982), and S. cereale (Perez de la Vega and Allard 1984). The results obtained suggest that phosphoglucomutase isozymes in rye are controlled by at least one locus (Pgm-1) located on chromosome 4R (specifically on the short arm of I and KlI chromosome 4R). This locus could express at least one allele, the structural gene Pgm-la, which would code the active

5-10 DAYS

Ce

Ci

Ed

CS-I-3R H-KII-3R K- D-3R 10-15

Ce

Ci

Ed

I KII D

DAYS

FIG.5. GOT patterns during the maturation processes of wheat, rye, and 3R addition line kernels using starch and pol yacry lamide gel electrophoresis. Maturation processes of 6R and 7R addition line kernels are not represented.

monomeric subunit N . The subunit a would be the PGM-R I isozyme. In T. aestivurn L. cv. Chinese Spring, the structural genes coding for PGM isozymes have been located on the short arms of group 4 chromosomes (Benito et al. 1984). The location of one rye PGM structural gene on chromosome 4R, and specif icaIl y on 4RS, constitutes biochemical evidence for homoeology between 4RS of rye and the short arms of the chromosomes of homoeologous group 4 of hexaploid wheat. Cytogenetic evidence of this homoeology has been obtained by Koller and Zeller ( 1976). Phosphoglucose isomerase has been described as dimeric in different plants: Adams and Allard ( 1977) in Festuca; Gottlieb (1977) in Clarkia; Hart (1979b) and Chojecki and Gale (1982) in Triticwm; Nielsen (1980) in Loliurn; Powling et al. (1981) in Hordeurn; Wendel and Parks (1982) in Carnelia; Perez de la Vega and A1lard ( 1984) in Secbale. The results obtained when PGI isozymes were studied point out that it was not possible to relate the PGl1 -R I band of zone I to any chromosome of rye because it has the same electrophoretic mobility as did the PGI I-W I wheat band. On chromosome I R, the


C A N . J. GENET. CYTOL. V O L . 27. 1985

FIG.6. ( u ) Zymograms of GOT isozymes in starch and polyacrylarnide gels (Ed, E t S. leaves, and roots). Lanc 6: CS-I-6R, H-KII-6RL, and K-D-6R. Lane 7: CS-I-7RL. H-KII-7RL. and K-D-7R. Lanc 8: I . K11, and D rye cultivars. The rest of the patterns were wheat and addition lines (AL). ( h )Zymograms of GOT lsozynies during the kernel maturation processes in starch and polyacrylamide gels (20th day after anthcsi\). Lane 1: wheat Ce. Lane 2: wheat Ci. Lane 3: wheat Ed. Lane 4: wheat E S. Lane 5: Ce of 3R AL. Lane 6: Ci of 3R AL. Lane 7: Ed of 3R AL. Lane 8: E S of 3R AL. Lane 9: Ed. E S. leaf, and root of wheat cultivars.

+

+

short arm in the case of the K11 cultivar, there must be one PGI locus (Pgi-I) located which would control the PGI isozymes of rye zone 11. This locus, named Gpi-Rl by Hart ( 1 9 7 9 ~and ) Chojecki and Gale ( 1982), could express at least one allele, the structural gene Pgi-la, which would code only the active dimeric form of subunit a. The active form of subunit 11 would be the PG12-R4 isozyme (GPI-R isozyn~e according to Chojecki and Gale ( 1982)). It is difficult to suggest the genetics of the other bands that compose the rye PGl zone I1 pattern (PG12-R I , PG12-R2, and PG12-R3). Multiple band phenotypes for the PGI zone 11 isozymes (also called GPI- I isozymes by Hart (19796) and Chojecki and Gale ( 1982)) have been described in different higher plants (Hart 19796; Jones 1983). Jones ( 1983) proposes that the multiple band phenotype of the ryegrass PGI zone 11 isozymes is due to experimerltal procedures; moreover, Salamini et al. ( 1972) have observed that maize PGI bands show a modification in number and intensity when they are under some chemical treatments. The chromosomal location results obtained agree with those of Hart ( 1 9 7 9 ~ )and Cho-jecki and Gale (1982); these latter authors connected the PGI zone 11 isozymes of 'Imperial' and 'King 11' ryes with chromosome 1R by using the 'Chinese Spring' - 'Imperial' and 'Holdfast' - 'King 11' addition lines. Also, the 1R and I RS addition line patterns support the subunit structure model of the rye PGI zone 11 isozymes proposed by Hart ( 1 9 7 9 ~ )and Cho-jecki and Gale (1982). Hart

+

( 19796) published evidence supporting a dimeric structure model for the wheat PGI zone 11 isozymes; later Chojecki and Gale ( 1982) extended this model to different Triticeae species. In 'Chinese Spring' hexaploid wheat, the PGI isozymes of zone 11 have been defined as dimeric and have been associated with the short arms of chromosomes in ho~noeologousgroup I (Hart 19796). Later, Chojecki and Gale ( 1982) suggested a system of duplicated genes on every short arm of homoeologous group 1 chromosomes. The chromosomal location of one rye PGI structural gene on chromoson~eI R and on I RS is biochemical evidence of the existent homoeology between the IRS rye chromosome arm and the short arms of homoeologous group 1 chron~osomes of hexaploid wheat. This evidence is corroborated by the fact that wheat and rye subunits are able to form active heterodimers in I R and IRS wheat-rye addition lines, as shown by the zymograln phenotypes that these lines represent. Cytological evidence of homoeology between I RS and IAS, and I BS and IDS chromosome arms has been presented by Zeller and Fischbeck (1971). Indications that GOT isozymes exist functionally as dimers have been obtained in different plants and in particular in Gramineae species: MacDonald and Brewbaker ( 1972) in Zeu rnuys, Hart ( 1975) in T. upstivum, Tang and Hart (1975) in S . c~crralu,Hart et al. (1976) in Agropyron, Babbel and Wain (1977) in Hordritm .

TABLEI. Chromosome arms ot 'Ch~neseSpring' wheal and 'Impcrlal'. 'Klng 11'. and 'Dakold' rye cultivars related with the d~fl'crcnti\cvymatlc 4y4tcrii4 4tu~11ed Chromosomal localion

.s.

7.. (if].\t i \,llt?l


Isozymat ic system

PG M PG I GOT GOT GOT GOT

1 1 II Ill

CS

CS

CS

I

K II

11

4AS IAS 7A hAS hAL 3AL

4B1, IBS 7B hBS hBL 3BL

3DS IDS 7D 611s hD1, 30L

3KS IK 7KL

4KS IKS 7KL

4K IK 7K

hK 3K

hKL 3K

hK 3K

When the GOT isozymes of ditelusornic and disomic addition lines were studied, at least three loci controlled the rye GOT isozymes. One locus (Gor-3) located on chromosome 3R controlled the GOT zone I11 iso~ylnes o f rye. Rye GOT zone I 1 isozymes were controlled by one locus ((701-2) located on chroniosome 6K. 6RL, in the case of K I I . The locus Got-I, located on the chromosome 7K. specifically on the long arm of 'Imperial' and 'King 11' chromosome 7R. controlled the GOT zone I isozymes of rye. The results agree with those of Tang and Hart (1975) which indicated that one GOT zone 111 structural gene (Got-R3) and one GOT zone I 1 structural gene (Got-R2) were located on chronioso~iies 3R and 6R, respectively, of 'Imperial' rye. Although our electrophoretic system only permits suggesting the subunit structure of the rye GOT Lone I11 isozymes, i t has made i t possible for the chromosomal location of the locus controlling the rye GOT zone 1 isozymes. The zymograms of 3K addition lines support a dinieric structure for the rye isozynies of zone I l l . The same zyniogranis and, consequently. the same structure has been described by Tang and Hart ( 1975) for the GOT zone I11 isozymes of 'Imperial' rye who studied disomic addition lines. These authors also suggest a dirneric structure for the rye isozymes of zone 11. From the coincidence between our res~~lts and those of Tang and Hart ( 197.5), we can postulate that the GOTZ-RI isozyme. named GOT-2 by 'Tang and Hart (1975), ~ o u l dbe constituted by the dinier tr'tr2. The would be synthesized by thc active dimeric subunit structural gene Got-2tr (Got-RZ), an allele of the Got-2 locus. '['he locus Got-3 could express at least one allele. the structural gene Got-3u (Got-R3). which would code the active dirneric subunit tr'. The dirner t i ' t r ' uould constitute the GOT3-K I isozyme (GOT-3 iso~ynie according to Tang and Hart ( 197.5) ) . On the other hand. one allele of the locus Got-I. the structural gene Gotl a , would code the subunit t r ' which should be related to the GOT-K I isozyme. In hexaploid wheat. the three zones of GOT activity

(I'

~ ~ ~ ~ t ~ C ( i I C

W heat retercncch

Ben1toctaI.(I984) Hart (19791)) Hart (1975) Hart ( 1975) Hart ( 1975) Hart(I975)

have been related to the long arnis ot' group 3 chromosonies (zone Ill. the most cathodal one). to the long arms or group 6 chromosomes (zone 11) and to the short (zone 1). Besides, one arms o f group 6 chro~noso~iies additional zone 1 gene set has been located on the group 7 chromosonies (Hart 1975). Our results constitute biochemical evidence of homoeology between the 3KL, and 6RL rye chromosome arnis and the long arms of honioeologous group 3 and 6 chroniosonies of wheat. Honioeology between the Got-I locus described in this paper and the Got-I set described in wheat by tlart ( 1975) can not be stated. I n consequence. other results are necessary to assert the existence of homoeology between the 7KL rye chromosonie arm and the long arms of honioeologous group 7 chroniosomes of wheat. Cytological evidence of homoeology between 3K. 6K. and 7K rye chromosoriies. and corresponding wheat chroniosomes have been described (Acosta I96 1 ; Riley 1965; Koller and Zeller 1976). Relationships between dit'ferent chromosolnes. chromosome arms, or segments of chromosonies can be studied by the location in each of homologous and homoeologous structural genes. 'The genes that encode isozyrnes are the most suitable t'or this purpose (Hart 1979tr ). For the chroniosomal location ol' structural genes coding for PGM. PGI. and GOT isozynies in the three rye cultivars analysed. the relationships among different chromosomes and chromosome arms of wheat and rye have been pointed out (Table I ). In this paper, we have proved that all the structural genes located are on the same chromosome in the three rye cultivars studied and on the same chroniosorne arms when chromosome arm location has been possible. Also. we have shown that the wheat and rye chroniosomes, and the wheat and rye chromosome arms which include the isozyme structural genes analysed. are homoeologous. 'The results support the relationships established by Koller and Zeller (1976) between the different chroniosomes of 1. K11. and D ryes and their location into the Tri ticineae homoeologous group.

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'Kharkov' wheat by heterochromatin bands and chromosome morphology. Can. J. Genet. Cytol. 18: 339-343. HART,G. E. 1975. Glutamate oxaloacetate transaminase isozymes of Tritic-um,evidence for multiple systems of triplicate structural genes in hexaploid wheat. In Isozymes. Vol. 111. Developmental biology. Edited by C. L. Market. Academic Press. pp. 637 -657. 1979~1.Genetical and chromosomal relationships among the wheats and their relatives. Stadler Genet. Symp. 11: 9-29. 19796. Evidence for a triplicate set of glucoseAcknowledgments phosphate isomerase structural genes in hexaploid wheat. The authors express their sincere appreciation to Biochem. Genet. 17: 585-598. Dr. E. R. Sears, Dr. F. J . Zeller, Dr. J . P. Gustafson, HART,G. E., A. K. M. R . ISLAM, and K. W. SHEPHERD. and Dr. C. N. Law for their generous gifts of the addi1980. Use of isozymes as chromosome markers in the tion lines, and to Mr. E. Sanchez for his technical isolation and characterization of wheat-barley chromoassistance. This work was supported by grants from the some addition lines. Genet. Res. 36: 3 1 1 - 325. Comision Asesora de Investigacion Cientifica y HART,G. E., D. E. MCMILLIN, and E. R. SEARS.1976. Determination of the chromosomal location of a glutamate Tecnica and INAPE (J. Salinas). oxaloacetate transaminase structural gene using Tritic-umAgropyron translocations. Genetics, 83: 49-6 1 . ACOSTA,C. A. 196 1. The transfer of stem rust resistance HART,G. E., and N. A. TULEEN.1983. Chromosomal from rye to wheat. Ph.D. thesis, University of Missouri. locations of eleven E(vrrigia elongcrtcr ( = Agropyron elonADAMS,W. T., and R. W. ALLARD.1977. Effect of polygatum) isozyme structural genes. Genet. Res. 41: ploidy on phosphoglucose isomerase diversity in Festuc-tr 18 1-202. microstac*his. Proc. Natl. Acad. Sci. U.S.A. 74: and W. HUBER. 1982. Genetic HSAM,S. L. K., F. J. ZELLER, 1652- 1656. control of 6-phosphogluconate dehydrogenase (6-PGD) BABBEL, G. R., and R. P. WAIN.1977. Genetic structure of isozymes in cultivated wheat and rye. Theor. Appl. Genet. H. jubatum. I. Outcrossing rates and heterozygosity levels. 62: 317-320. Can. J. Genet. Cytol. 19: 143- 152. 1972. Chromosomal location of BENITO, C., A. M. FIGUEIRAS, and M. T. GONZALEZ-JAEN.[ R A N I , B. N.. and BHATIA. alcohol dehydrogenase gene(s) in rye using wheat-rye 1984. Phosphogluco mutase-a biochemical marker for addition lines. Genetica (The Hague), 43: 195-200. group 4 chromosomes in the Triticinae. Theor. Appl. JONES, T. W. 1983. instability during storage of Genet. 68. In press. phosphogluco-isomerase isoenzymes from ryegrasses BREWER, G. J.. and C. F. SING.1970. An introduction to (Lolium spp.). Physiol. Plant. 58: 136- 140. isozyme techniques. Academic Press, New York. KOEBNER, R. M. D., and K. W. SHEPHERD. 1983. Shikimate and E. ~VEVO. 1978. OutBROWN,A. H. D., D. ZOHARY, dehydrogenase-a biochemical marker for group 5 chrocrossing rates and heterozygosity in natural populations of mosomes in the Triticinae. Genet. Res. 41: 209-2 12. Hordeum spontaneum Koch in Israel. Heredity, 41: KOLLER, 0 . L., and F. J. ZELLER.1976. The homoeologous 49-62. relationships of rye chromosomes 4.R and 7K with wheat CHOJECKI, A. J. S., and M. D. GALE.1982. Genetic control chromosomes. Genet. Res. 28: 177 - 188. of glucose phosphate isomerase in wheat and related speJ. R. 1977. Interspecific gene transfer in plant LACADENA, cies. Heredity, 49: 337-347. breeding. Proceedings of the 8th Congress of Eucarpia, 1975. Identification of DARVEY, N. L., and J. P. GUSTAFSON. Universidad Politecnica de Madrid. pp. 45-62. rye chromosomes in wheat - rye addition lines and triticale MACDONALD, T., and J. L. BREWBAKER. 1972. lsozyme by heterochromatin bands. Crop. Sci. 15: 239 - 243. polymorphism in flowering plants. VII. Genetic control GILL,B. S., and G. KIMBER. 1974. The Giemsa C-banded and dimeric nature of transaminase hybrid maize isokaryotype of rye. Proc. Natl. Acad. Sci. U.S.A. 71: enzymes. J . Hered. 63: I 1 - 14. 1247- 1249. MITTON,J. B., Y. B. LINHART, K. B. STURGEON, and J. L. GIRALDEZ, R., and J. ORELLANA. 1979. Metaphase I bonds, HAMRICK. 1979. Allozyme polymorphism detected in macrossing-over frequency and the genetic length of specific ture needle tissue of ponderosa pine. J. Hered. 70: 86-89. chromosome arms of rye. Chromosoma, 72: 377-385. NIELSEN, G. 1980. Identification of all genotypes in tetraploid GOODMAN, M. M., C. W. STUBER, K. NEWTON, and H. H. ryegrass (Lolium spp.) segregating for four alleles in a WEISSINGER. 1980. Linkage relationships of 19 enzyme Pgi-enzyme locus. Hereditas, 92: 49-52. loci in maize. Genetics, 69: 697-710. PEREZDE LA VEGA,M., and R. W. ALLARD.1984. Mating GOTTLIEB,L. D. 1977. Evidence for duplication and disystem and genetic polymorphism in populations of Seeale vergence of structural gene for phosphoglucoisomerase in ceretrle and S. vavilovii. Can. J. Genet. Cytol. 26: diploid species of Clnrkia. Genetics, 86: 289-307. 308-317. GUSTAFSON, J. P., L. E. EVANS,and K. JOSIFEK.1976. POULIK, M. D. 1957. Starch gel electrophoresis in a disconIdentification of chromosomes in Secale montanum and tinuous system of buffers. Nature (London), 180: 1477. individual S. montanum chromosome additions to


In all cases, no variation of PGM, PGI, and GOT zymogram phenotypes was observed during germination. The chromosomal location results obtained when the maturation of rye, wheat, and addition line kernels was studied, support the hypothesis that the same genes control the PGM, PGI, and GOT isozymes in the different developmental stages of rye kernel and in dry kernel, leaf, and root.



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POWLING, A . , A. K . M. R. ISLAM, and K. W. SHEPHERD. within rye chromosomes in addition lines. Z. Pflanzenzuecht. 76: 11-18. 1981. Isozymes in wheat-barley hybrid derivative lines. TANG,K. S., and G. E. HART. 1975. Use of isozymes as Biochem. Genet. 19: 237-253. chromosome markers in wheat-rye addition lines and in RILEY, R. 1965. Cytogenetics and plant breeding. triticale. Genet. Res. 26: 187-20 1 . Proceedings of the 1 I th International Congress of Genetics. WENDEL, J . F., and C. R. PARKS.1982. Genetic control of Genetics Today, 3: 68 1 -688. isozyme variation in Camelia japonica L. J . Hered. 73: SALAMINI, F., C. Y. TSAI,and 0. E. NELSON.1972. Multiple 197-204. forms of glucosephosphate isomerase in maize. Plant 1971. Cytologische unZELLER.F. J . , and G. FISCHBECK. Physiol. 50: 256- 26 1. tersuchungen zur identifizierung des fremdchromosoms in SINGH,R. J . , and G. ROBBELEN. 1975. Comparison of der Weizensorte Zorba (W 565). Z. Pflanzenzuecht. 66: somatic Giemsa banding pattern in several species of rye. 260 - 265. Z. Pflanzenzuecht. 75: 270-285. 1976. Giemsa banding technique reveals deletions