inheritance and linkage relationships of seed peroxidase loci in hexaploid oat ( avena...

Inheritance and linkage relationships of seed peroxidase loci in hexaploid oat (Avena sativa L.)

N.R. Sharopova, V.A. Portyanko, and A.A. Sozinov

Abstract: Polyacrylamide gel electrophoresis has been used to investigate the inheritance and linkage relationships between anodal (PXA) and cathodal (PXC) seed peroxidases in hexaploid oat (Avena sativa L.). A total of 12 seed peroxidase loci (5 loci of PXA and 7 loci of PXC) were identified in three crosses. Only two Pxc loci (Pxc.5 and Pxc7) were not linked to any peroxidase loci; the others were scored in three linkage groups. The order of the three loci assigned to one of the linkage groups was Pxcl-Pxa.5-Pxc2. The order of loci in the other two linkages were Pxc4-Pxal-Pxa3 and Pxc3-Pxa4-Pxa2. Also, the Pxc6 locus was shown to be linked to the Pxc3 locus. Considering that A. sativa is an allohexaploid, it can be proposed that the three peroxidase linkages represent homoeologous chromosomes.

Key words: seed peroxidase, isozyme inheritance, linkage, Avena sativa.

ResumC : L'Clectrophor&se en gel de polyacrylamide a CtC employCe afin d'Ctudier I'hCrCditC et la liaison entre les peroxydases anodiques (PXA) et cathodiques (PXC) prksentes dans les graines chez l'avoine hexaploi'de (Avena sativa L.). En tout, 12 loci de peroxydases (5 loci de PXA et 7 loci de PXC) ont kt6 identifiks parmi trois croisements. Seuls deux loci Pxc (Pxc5 et Pxc7) n'ktaient pas liCs a d'autres loci de peroxydases tandis que les autres loci ont CtC assignCs a trois groupes de liaison. L'ordre des trois loci assignCs a un des groupes de liaison gCnCtique Ctait Pxcl-Pxa.5-Pxc2. L'ordre des loci chez les deux autres groupes de liaison Ctait Pxc4-Pxal-Pxa3 et Pxa3-Pxa4-Pxa2. De plus, il a kt6 montrC que le locus Pxc6 est lik au locus Pxc3. En tenant compte du fait que A. sativa est un allohexaploi'de, les auteurs suggkrent que les trois groupes de liaison correspondent aux chromosomes homCologues.

Mots cle's : peroxydases de la graine, hCrCditk d'isoenzymes, liaison gknCtique, Avena sativa. [Traduit par la Rkdaction]

lntroduct ion

Peroxidase inheritance has been investigated in various oat species (Marshall and Allard 1969; Clegg and Allard 1973; Berding 1974; Miller 1977; Yen and Sadanaga 1977a, 1977b). But only a few peroxidase loci have been verified through progeny tests in Avena barbata (Marshall and Allard 1969; Miller 1977), A. fatua (Clegg and Allard 1973), and in cultivated hexaploid oats (Yen and Sadanaga 1977~) .

Linkage values between peroxidase and esterase loci were reported by Marshall and Allard (1969) and Berding

(1974). These workers estimated recombination frequencies for two pairs of loci (EstlO-Pxa5 and Pxa5-Acph5) in A. barbata.

The identification of linkage groups is a first step in developing a genetic map of a specific species. But there is little information about isozyme linkage groups in oat available. In the present communication, we report the inheritance of five anodal and seven cathodal seed peroxidase loci in cultivated oat and linkages between them.

Materials and methods

Plant material Corresponding Editor: G. Fedak. The seeds of the five hexaploid oat cultivars were obtained Received August 12, 1994. Accepted January 16, 1994. from several sources. 'Orion 11' (Russian accession num-

N.R. sharopoval and A.A. Sozinov. Institute of Agroecology and Biotechnology, Ukrainian Agrarian Academy of Sciences, Metrologytcheskaya Street, 12, Kiev 252143, Republic of Ukraine. V.A. Portyanko. Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Zabolotnogo Street, 148, 252022, Kiev GSP-22, Republic of Ukraine.

Author to whom all correspondence should be addressed.

ber k-7989) and 'Sovietsky' (k-8257) were obtained from collections maintained in the N.I. Vavilov Institute of Plant Industry, St. Petersburg, Russia. 'Elgin' (k-12296) was received from the South Ural Research Institute of Grain Production, Chelyabinsk, Russia,, and 'Narymsky 943' (k- 1 1 122) was obta ined f rom the Narymskaya Plant Breeding Station, Kolpashevo, Tomskaya province, Russia. 'Selma' (k-11584) was supplied by Dr. Bogachkov, West Siberian Plant Breeding Center, Omsk, Russia.

Genome, 38: 467-471 (1995). Printed in Canada 1 ImprimC au Canada

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Table 1. Two-loci segregation and linkage of the oat seed peroxidase loci in F, of 'Elgin' X 'Selma'."

No. of F, seeds of the

phenotypes: Peroxidase x Recombination

zones AB aB Ab ab (9:3:3:1) frequencies

*Only locus pairings significant at the 99% level of confidence are given.

Fig. 1. Zymograms of cathodal (A) and anodal (B) The running buffer for cathodal peroxidase (PXC) peroxidases in hexaploid oat cultivars: 'Selma' ( I ) , separation was 0.5% (wlv) Tris and 1% (wlv) aluminum 'Elgin' (2), 'Narymsky 943' (3), 'Orion 11' (4), and lactate. This buffer was used both for the stacking gel 'Sovietsky' (5). (pH 3.2) and for the resolving gel (pH 5.2).

3 4 5 Anodal peroxidases (PXA) were resolved in a continuous

A 1 2 gel system containing 8.5% (wlv) acrylamide and 0.17%

Three crosses among the five cultivars were produced in order to study the inheritance of oat anodal (PXA) and cathodal (PXC) seed peroxidases, and to assign them to particular linkage groups. They were 'Elgin' X 'Selma' (Cross I ) , 'Elgin ' X 'Narymsky 943 ' (Cross 2), and 'Orion 11' X 'Sovietsky' (Cross 3).

Extraction a n d isozyme electrophoresis Peroxidases were extracted from single mature oat seeds. Seeds were cut in half and the embryo ends were crushed and placed in 150 FL buffer containing 0.05 M Tris, 20% sucrose, 0.04 M ascorbic acid, and 0.001 M EDTA (pH 7.2) overnight.

The extracts were loaded onto vertical slabs of polyacrylamide gel for electrophoresis. The stacking gel for cathodal peroxidases contained 5% (wlv) acrylamide and the resolving gel contained 8% (wlv) acrylamide and 0.4% (w/v) bis-acrylamide.

iwlv) bis-acrylamide. The gel buffer and running buffer for PXA separation

was 0.01 M Tris and 0.07 M glycine (pH 8.4). Initially, gel slabs were subjected to electrophoresis at a

constant 80 V for 1 h. The cathodal peroxidases were electrophoresed an additional 1.5 h, and the anodal peroxidases were electrophoresed an additional 3 h, both at a constant 250 V.

The staining solution was 0.02% benzidine and 0.09% hydrogen peroxide in 5% acetic acid. The zymogram should be recorded as soon as blue bands become evident, as the bands tend to diffuse and, later, fade away.

Genetic studies Ninety-six to one hundred and fifty seeds obtained from selfed F, plants for each of the three crosses were analyzed. Cathodal peroxidases were scored for all three F, populations, but anodal peroxidases were scored in Crosses 2 and 3 only. Linkage estimates were computed by the max- imum likelihood equations for F, progeny as outlined by Allard (1956). Genetic distances were calculated on the basis of recombination frequencies (Kosambi 1944). In addition, Kosambi's correction (Kosambi 1944; formula 7) was used to calculate adjusted map distances between the extreme positions in three-point tests.

Results and discussion

From one to seven zones of PXC activity were observed in the five oat cultivars under study (Fig. la ) . In the present work we scored all of these isozymes as products of the seven loci. Five zones of anodal peroxidase activity were also analyzed as products of five loci (Fig. lb) .

We have not studied the correspondence of the loci under investigation to any previously published peroxidase loci. The designation for peroxidase loci used was that proposed by Price and Kahler (1983). This style of nomen- clature seems to be convenient for computer search.

Cross 1 ('Elgin' X 'Selma') Only cathodal peroxidases were assayed in this cross. Four segregating zones of PXC activity were observed in the

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Table 2. Two-loci segregation and linkage of the oat seed peroxidase loci in F, of 'Elgin' X 'Narymsky 943'.*



zones AB aB Ab ab (9:3:3:1) frequencies - -

PXC 1 -PXC2 PXC 1-PXA5 PXC2-PXA5 PXC3-PXA2 PXC3-PXA4 PXC5-PXA1 PXC5-PXA3 PXAl-PXA3 PXA2-PXA4


Fig. 2. Zymograms of cathodal (Al, A2) and anodal (B 1, B2) seed peroxidases of two F, populations. (Al, B1) Cross 2: P,, 'Elgin'; P,, 'Narymsky 943'. (A2, B2) Cross 3: P,, 'Orion 11'; P,, 'Sovietsky'.

F, of Cross 1 (Fig. l a ) . Two phenotypes for each zone were found to fit a ratio of 3 "active" : 1 "null". Two-loci segregation data indicated that Pxcl was linked to Pxc2 with a recombination frequency of 0.166 + /- 0.07 and Pxc3 was also linked to Pxc6 with a recombination frequency of 0.155 +/-0.07 (Table 1). Recombination frequencies for two-loci linkages were calculated only when the observed segregation ratios significantly deviated from the expected 9:3:3: 1 at the 1 % level. The recombination frac- tions were equivalent to map distances of 17+/-7.7 and 16+/-7.6 cM, respectively (Fig. 3).

Cross 2 ('Elgin' X 'Narymsky 943') Four polymorphic zones of PXC activity and five of PXA were scored in F, hybrids of 'Elgin' X 'Narymsky 943' (Figs. 2A1 and 2B1). The segregation for each of the zones fitted a 3: 1 ratio. Recombination frequencies for two-loci linkages were calculated only when the observed segregation ratios significantly deviated from the expected 9:3:3: 1 at the 1% level (Table 2). Linkage analyses showed that Pxcl was linked to Pxc2 (recombination frequency 0.083+/-0.05) and to Pxa.5 (recombination frequency 0.048+/-0.04). Pxc2 was also linked to Pxa.5 (recombination

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Table 3. Two-loci segregation and linkage of the oat seed peroxidase loci in F, of 'Orion 11' X 'Sovietsky'.*



zones AB aB Ab ab (9:3:3:1) frequencies


Fig. 3. Genetic linkage between cathodal and anodal Fig. 5. Genetic linkage between cathodal and anodal peroxidase loci Pxcl, Pxa5, and Pxc2 in two F, populations peroxidase loci Pxc4, Pxal, and Pxa3 in two F, populations of cultivated oat. of cultivated oat.

P x c 4 Pxa5

P r a l P r a 3 Pxcl Ppc2 I

I I 23+8.6 cM l4+3.7 CM I cross2 '4.8'4 -0 c 8.0f5.1 cM b

4 * cross2 28+11.0 CM

8.4f5.1 CM -I 27.3 CM (Kosambi correction) e

12.5 cM(Rosambi correction) A- C

Fig. 4. Genetic linkage between cathodal and anodal peroxidase loci Pxc3, Pxa4, and Pxa2 in two F, populations of cultivated oat.

15.7 cM(Kosambi correction)

' 7+4.9 cM ' 10+5.8 cM ' - * 14+6.5 cM

Cross2 4 t 16.8 ~ ~ ( ~ o s a m b i correction)

frequency 0.079+/-0.05). Pxc4 was linked to Pxal and to Pxa3 (recombination frequency 0.218+/-0.07 and 0.257 +/-0.08, respectively). Pxal and Pxa3, in turn, are linked to each other (0.040+/-0.04). Another pair of linked anodal peroxidase loci, Pxa2 and Pxa4 (recombination frequency 0.099+/-0.06), was linked to Pxc3 (0.135+/-0.06 and 0.067+/-0.05, respectively). Map distances in cM for the above mentioned cases are shown in Figs. 3, 4, and 5.

Cross 3 ('Orion 11' X 'Sovietsky') Four polymorphic zones of PXC activity and four of PXA activity occurred in the F, of 'Orion 11' X 'Sovietsky' (Figs. 2Bl and 2B2). It was found on the basis of linkage

tests that Pxc3 was linked to Pxa2 (0.147+/-0.06) and to Pxa4 (0.097+/-0.05) (Table 3). Both of the Pxa loci in turn were linked with each other with the recombination frequency 0.058 + /- 0.04. Apparently, there was no recombination between Pxal and Pxa3. Consequently, Pxc4 was linked to Pxal and Pxa3 with the same recombination frequency of 0.238+/-0.08. Corresponding map distances are displayed in Figs. 4 and 5.

Further to the above, two Pxc loci, Pxc5 and Pxc7, were identified in Cross 3. Two phenotypes for each of the zones were found to fit a ratio of 3 "active" : 1 "null" (85:28, X2 = 0.003 and 92:21, X2 = 2.48, for Pxc5 and Pxc7, respectively). Two-loci segregation data indicated that the two loci segregated independently of the other Pxc and Pxa loci.

Three seed peroxidase loci Pxcl, Pxc2, and Pxa5 were placed in the same linkage group on the basis of the data produced by Crosses 1 and 2. The order of the loci in this linkage was determined to be Pxcl-Pxa5-Pxc2 (Fig. 3). The distances between the members of another linkage group involving the Pxc3, Pxa2, and Pxa4 loci scored for Cross 2 were in good agreement with those scored in Cross 3 ('Orion 11' X 'Sovietsky'). The results favored the gene order Pxc3-Pxa4-Pxa2 (Fig. 4).

Some discrepancies occurred among Pxc4, Pxal , and Pxa3 in Crosses 2 and 3. The Pxal and Pxa3 loci recom- bined in Cross 2 with the frequency 0.0399+/-0.037 (Table 2), but there were no recombinants between them in Cross 3 (Table 3). Thus, Pxa l and Pxa3 are probably tightly linked. If this is true the third linkage also consists

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of three loci linked in the following order: Pxc4-Pxal-Pxa3 (Fig. 5).

So the data obtained for a total of three crosses in this study showed the existence of 12 peroxidase loci for hexaploid oat seed peroxidases. Ten of them were identified in three linkages. The linkage groups involved a minimum of three peroxidase loci each. Considering that Avena sativa is a hexaploid with genome constitution AACCDD, it can be proposed that the three peroxidase linkages represent homoeologous chromosomes.

Because of the large number of loci distributed among several linkage groups and the low cost and low labour of the enzyme assay, oat seed peroxidases seem to be convenient for the construction of a low-saturated oat genome map.

References

Allard, R.W. 1956. Formulas and tables to facilitate the cal- culation of recombination values in heredity. Hilgardia, 24: 235-279.

Berding, N. 1974. Spatial and life cycle distribution of complex allozyme polymorphism in a population of A. fatua L. Ph.D. thesis, University of California, Davis.

Clegg, M.T., and Allard, R.W. 1973. The genetics of elec- trophoretic varients in Avena. 11. The esterase El , E2, E4, E5, E6 and anodal peroxidase APX4 loci in A. fatua. J. Hered. 64: 3-6.

Kosambi, D.D. 1944. The estimation of map distances from recombinant values. Ann. Eugen. 12: 172-175.

Marshall, D.R., and Allard, R.W. 1969. The genetics of elec- trophoretic varients in Avena. I. The esterase E4, E9, E10, phosphatase P5 and anodal peroxidase APX5 loci in A. barbata. J. Hered. 69: 17-19.

Miller, R.D. 1977. Genetic variability in the slender wild oat Avena barbata in California. Ph.D. thesis, University of California, Davis.

Price, S., and Kahler, A.L. 1983. Oats (Avena ssp.). In Isozymes in plant genetics and breeding. Part B. Edited by S.D. Tanksley and T.J. Orton. Elsevier Science Publishers B.V., Amsterdam.

Yen, S.T., and Sadanaga, K. 1977a. Inheritance of leaf peroxidases in oats. Can. J. Genet. Cytol. 19: 303-312.

Yen, S.T., and Sadanaga, K. 1977b. Nullisomic and monosomic analyses of genes controlling leaf peroxidases in oats. Can. J. Genet. Cytol. 19: 395-403.

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inheritance and linkage relationships of seed peroxidase loci in hexaploid oat ( avena...

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