Maize Genetics Cooperation Newsletter vol 84 2010

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of authors.

Maize and Sorghum Research Institute

Pascani, Republic of Moldova

Frequency of reversion of o2-m(r) alleles as a characteristic of specificity of their interaction with regulatory Bg elements ¹

--V. V. Koterniak ²

¹ This note is a shortened version of a full size article which had been submitted for publication, however, due to termination of my work in Maize and Sorghum Research Institute its publication has not been finalized. However I would like to share main result and conclusions of this article with the maize community.

² Present address: Pharmasciance Inc., Montreal, Canada

Reversions of mutable o2-m(r) alleles occur mostly postmeiotically during gametophyte development or later during endosperm development (Montanelli et al., Mol. Gen. Genet. 197: 209-218, 1984; Koterniak; MNL 76: 54; Genetika (Moscow) 39:769-774) and rarely at late stages before meiosis (MNL 76: 54; Genetika (Moscow) 39: 709-712, 2003). Phenotypic expression of o2-m(r) alleles reversion depends on its timing. The earlier it occurs during endosperm development the higher is the portion of the vitreous tissue in variegated endosperm. Reversion occurring before the first mitotic division of the primary endosperm nucleus determines appearance of phenotypically normal kernels or whole endosperm revertants (WER) (Salamini, Molecular and General Genetics 179: 497-507, 1980; Montanelli et al., Molecular and General Genetics 197: 209-218, 1984).

Earlier, the specificity in interaction between regulatory elements Bg-hf and Bg-lf in relation to reversion frequency of o2-hf and o2-lf alleles has been established. Thus, in two or three doses the Bg-lf element determined low reversion frequency of the o2-lf allele but high reversion frequency of the o2-hf allele, whereas the presence in the same doses of Bg-hf determined high reversion frequency of both the o2-hf and o2-lf alleles (Maydica 48: 275-281, 2003).

Rather unexpected therefore was finding of very high reversion frequency of the o2-hf allele in presence of standard Bg-Ref element (this paper). Percentage of phenotypically normal kernels on homozygous o2-hf, Bg-Ref ears was higher than that of the o2-hf, Bg-hf ears obtained under selection for high WER content and often did not differ significantly from the content of normal kernels observed in heterozygous O2 ears.

To reveal plants carrying the wild-type O2 allele (i. e. descendants of embryo revertant kernels) selfing and crossing with the o2-R, +^Bg (rarely with the o2-m(r), +^Bg) testers (i.e. with testers containing recessive o2 alleles and lacking regulatory Bg elements) were made. In contrast to the crosses in which o2-hf, Bg-Ref strains were used as male parent (contained one dose of regulatory element), in reciprocal crosses and selfed ears (containing respectively two and three doses of Bg-Ref) high WER content can significantly change kernel phenotype ratio impeding determination of embryo revertants in this material. Therefore in the analysis of kernel segregation ratios of the latter crosses the ratios characteristic for heterozygous O2 ears, were considered as uncertain, if the testcrosses in which the same plants were used as male parent were not available.

The data on frequency of reversion of o2-hf and o2-lf alleles in presence of Bg-Ref and the earlier published data (Maydica 48: 275-281, 2003) on frequency of reversion of these alleles in presence of the Bg-hf and Bg-lf were obtained in the same experiment carried out in 2002. Determination of embryo revertants was carried out in the progeny of all WER and a part of variegated kernels of two ears of the o2-hf/o2-R, Bg-Ref/+^Bg genotype (ears 02-4574�4742p152 and 02-4573�4742p131) and in the progeny of all kernels of one homozygous o2-hf, Bg-Ref ear (ear 02-4568p131, 253 WER and 168 variegated, i.e. 60.10% of WER).

Evaluation of significance of differences between observed and expected frequencies was carried out by the χ² test.

High WER content is characteristic for the o2-hf, Bg-Ref but not for o2-lf, Bg-Ref genotypes

Lower reversion frequency of the o2-lf allele in comparison to the o2-hf allele in presence of standard Bg-Ref element have been determined on the first and second generation of the crosses of o2-hf, +^Bg and o2-lf, +^Bg strains with the o2-R, Bg-Ref line (Maydica 44: 195-203, 1999). However values of reversion frequency of the o2-hf allele in presence of Bg-Ref which were higher than the reversion frequency of the same allele in presence of the Bg-hf element (i. e. the regulatory element obtained under selection for high WER content) were quite unexpected.

The major part (68%) of homozygous o2-hf, Bg-Ref ears contained more than 50% of phenotypically wild-type kernels (Table 1). The ratio of phenotypically normal to variegated kernels in o2-hf, Bg-Ref genotypes often did not deviate significantly from the ratios of such kernels observed on ears heterozygous for normal O2 allele. Thus on about half of selfed ears this ratio was statistically indistinguishable from 3:1 or higher (Table 1). Most variegated kernels on selfed o2-hf, Bg-Ref ears were characterized by small and very small opaque sectors surrounded by vitreous tissue (Figure 1).

In contrast to the o2-hf, Bg-Ref strains the described phenomenon of high WER content in o2-lf, Bg-Ref genotypes was not observed (Table 1, Figure 2). Among selfed o2-lf, Bg-Ref ears none had the excess of WER kernels over variegated ones (Table 1). Revertant content in o2-lf, Bg-Ref genotypes observed at three Bg-Ref doses was statistically higher than the same parameter observed with two doses of Bg-Ref (Table 1).

Most kernels on the ears with high WER content are not embryo revertants

Out of 67 tested progenies of one of the two studied ears of the o2-hf/o2-R, Bg-Ref/+^Bg genotype (ear 02-4574�4742p152) three were heterozygous O2 plants. No heterozygous O2 plants were found in 59 progenies of the second o2-hf/o2-R, Bg-Ref/+^Bg ear. In the progeny of selfed homozygous o2-hf, Bg-Ref ear (02-4568p131) 18 plants can be considered as embryo revertants (Table 2).

Two heterozygous O2 plants, descendent of o2-hf/o2-R, Bg-Ref/+^Bg ear and 12 of homozygous o2-hf, Bg-Ref ear were found in progenies of 37 and 108 WER kernels, respectively, giving frequency of their formation in progeny of WER kernels equal to 5.41 (2/37�100) and 5.56% (12/(2�108)�100)) respectively. Two plants homozygous for the wild-type O2 allele (originated from WER kernels) were found in the progeny of selfed o2-hf, Bg-Ref ear (Table 2).

It is necessary to mention that presence of variegated kernels in two out of three embryo revertant ears (i.e. in ears heterozygous for wild-type O2 allele and non-mutable o2-R allele) found in the progeny of the o2-hf/o2-R, Bg-Ref/+^Bg ear (Table 2А) is unexpected and needs to be explained (see below).

At 3 out of 16 plants (03-4225p4, 03-4411p4, 03-4432p4 in Table 2B) considered as O2 heterozygotes (i.e. carrying wild-type O2 and o2-hf alleles) in the progeny of selfed o2-hf, Bg-Ref ear, the content of variegated kernels in crosses with the o2-R, +^Bg, tester (used as female parent), was significantly higher than expected. This excess of variegated kernels is apparently conditioned by the same causes as the described above phenomenon of apparition of variegated kernels on heterozygous O2/o2-R, Bg-Ref/+^Bg ears (see below). It is necessary to mention that the content of phenotypically wild-type kernels at these three ears (24.06-42.78%) is much higher than at homozygous o2-hf, Bg-Ref strains (maximum WER content on their ears was equal to 8.07%, Table 3B). Significant deviations from expected ratios due to excess of normal kernels which are mostly observed in the crosses of the o2-hf, Bg-Ref strains with the o2-R, +^Bg tester used as male parent and on selfed ears (Table 2B) are conditioned by high frequency of WER formation especially in presence of two or three doses of regulatory Bg-Ref element (see below).

Revealed embryo revertants did not belong to the same pairs of spikelets, however five embryo revertants on homozygous o2-hf, Bg-Ref ear (Table 2) were in two clusters consisting from two and three contiguous kernels presented in adjacent pairs of spikelets (data not shown).

High WER content in o2-hf, Bg-Ref genotypes needs the presence of two or three doses of the regulatory element

Comparison of WER content on selfed homozygous o2-hf, Bg-Ref ears and in their reciprocal crosses with the o2-R, +^Bg showed that high WER content needs the presence of two or three doses of Bg-Ref. Thus with one dose of this regulatory element, WER percentage (about 2%) is approximately 24 times lower than with three doses of Bg-Ref (Table 3).

With three Bg-Ref doses percentage of phenotypically normal kernels is higher than with its two doses, however the value calculated per one Bg-Ref dose (or as the reversion frequency of o2-hf ) was higher at two doses of Bg-Ref, differences in both cases being insignificant (Table 3).

Possible causes of high frequency of reversion of o2-hf in presence of Bg-Ref

Several causes could be connected with high content of phenotypically wild-type kernels in o2-hf, Bg-Ref genotypes: (i) action of modifiers; (ii) earlier (i. e. before meiosis) developmental stage of o2-hf reversion in presence of Bg-Ref; (iii) specific interaction between o2-hf allele and Bg-Ref element.

An assumption about action of modifiers is unlikely since sharply different on revertant content genotypes o2-hf, Bg-Ref and o2-lf, Bg-Ref have been obtained in the crosses of the same o2-R, Bg-Ref line with closely related strains o2-hf, +^Bg and o2-lf, +^Bg and differences in reversion frequencies of responsive alleles of these strains were not caused by modifiers unlinked to the o2 locus (Maydica 44: 195-203, 1999; MNL 73: 76-79, 1999).

Regulatory element Bg-Ref does not condition earlier reversion of the o2-hf allele in development

Obtained data showed, that frequency of formation of embryo revertants from WER kernels in studied o2-hf, Bg-Ref ears (5.41 and 5.56%) is approximately on the same level with this trait of the o2-hf, Bg-hf strains (1.82-8.18%) (Genetika (Moscow) 39: 709-712; 2003) indicating that reversion of the o2-hf allele in presence of Bg-Ref element (as well as in that of the Bg-hf) occurs mostly at the period from fertilization to the first division of the primary endosperm nucleus. The size of embryo revertant clusters formed from the kernels (of two and three kernels) was also equal to that of observed in homozygous o2-hf, Bg-hf genotypes (Genetika (Moscow) 39: 709-712; 2003). Proceeding from the frequency of formation of heterozygous O2 ears in the progeny of WER kernels obtained from analyzed selfed homozygous o2-hf, Bg-Ref ear (the ear 02-4568p131) and the number of WER kernels on this ear (5.56% and 253, respectively) the apparition of two plants homozygous for wild-type O2 allele found in the progeny of mentioned ear and the apparition of two kernel embryo revertant cluster, as a result of o2-hf reversion in gametes at postmeiotic stages of development is not excluded. However frequency of formation of three kernels embryo revertant cluster (1.7×10^-4 or 0.0556³) on selfed o2-hf, Bg-Ref ear indicates that premeiotic reversion of o2-hf is more probable. Small size of this cluster indicates on both the late stages of premeiotic development at which the reversion of o2-hf can rarely occur and confirms that the early reversion of this allele is not the main cause of high WER content in o2-hf, Bg-Ref genotypes.

Kernel segregation ratios of on certain heterozygous O2 ears and phenotype of o2-hf, Bg-Ref kernels indicate on possibility of intragenic transposition of rbg element in the o2 gene

The o2-hf, Bg-Ref genotypes, besides having high content of phenotypically normal kernels, also have another characteristic feature: their variegated kernels appear as small and very small opaque sectors in vitreous background (Figure 1). Taking into account small size of opaque sectors and their encirclement by vitreous tissue it is possible to conclude that these sectors originate at the late stages of endosperm development as a result of insertion of Bg or rbg elements in wild-type O2 allele leading to inactivation of this allele and therefore to opaque tissue. Similar mechanism, based on intragenic transposition of nonautonomous Ds element has been proposed for the wx1 endosperm variegation observed for the Wx1-m5 allele (Weil et al., Genetics 130: 175-185, 1992).

Since apparition of wild-type O2 allele at the o2-hf, Bg-Ref genotypes is conditioned by excision of the rbg element from the o2-hf allele and if the above described phenotype of o2-hf, Bg-Ref kernels is originated from the rbg insertion in the o2 locus it is possible to refer to such insertion as to the reinsertion of rbg. In case if rbg reinsertions does not lead to exact restoration of the sequence existing before excision of this element (though direct data for rbg is not available, transposons excision are often imprecise and rarely show strong target site preference (see for example reviews of Saedler and Nevers, EMBO J. 4: 585-590,1985; Wessler, Science 242: 399-405, 1988; Walker et al., Genetics 146: 681-693 1997; Kunze and Weil, In: Mobile DNA II. P. 565-610. Edited by N. L. Craig et al. ASM Press, Washington, D.C. P, 2002), these reinsertions are in fact intragenic transpositions of rbg. Earlier (Genetika (Moscow) 39:769-774, 2003a) proceeding from similarity in behavior between o2-hf allele with class 2 mutable an3 petunia alleles (these alleles, which are under control of the dTph1 transposon of the hAT superfamily, have been described by van Houwelingen et al (The Plant Cell. 11: 1319–1336, 1999) an assumption was made that in case of o2-hf allele in or near the o2 locus two receptor rbg elements may be present. In such a case it is possible to expect that presence of two rbg elements increases the probability of their intragenic transpositions. It is interesting to mention that in case of the Wx-m5 allele, a part of new alleles arisen due to intragenic transposition of Ds carries two Ds elements in the wx1 gene (Weil et al., Genetics 130: 175-185, 1992).

Proposed mechanism of Bg or rbg insertion in the wild-type O2 allele explains not only above described phenotype of o2-hf, Bg-Ref kernels but also explains both the apparition of variegated kernels in two out of three embryo revertant O2/o2-R, Bg-Ref/+^Bg ears and higher than expected portion of variegated kernels in 3 out of 16 heterozygous O2/o2-hf, Bg-Ref/+^Bg ears in progeny of selfed o2-hf, Bg-Ref ear (Table 2).

Dosage effects of studied Bg regulatory elements and some inferences about mechanism of action of Bg transposase

Data presented in Table 3B indicate that with two times regulatory element Bg-Ref dose increase (from one to two), reversion frequency of the o2-hf allele increases approximately by 6 times (Table 3B). Considering excision of the rbg element as a biochemical reaction (e.g. Zhang and Peterson, Genetics 153: 1403-1410, 1999) much higher increase of reversion frequency indicates that the order of this reaction is higher than one and that the rbg excision occurs as a result of action of Bg transposase oligomers consisting of several transposase molecules participating in forming of transposition complexes. The same conclusion was made comparing reversion frequency of the o2-hf allele under the dose increase of the Bg regulatory elements from 1 to 2 (Maydica 48: 275-281, 2003).

Though little is known about eukaryotic transposition complexes (transpososomes), participation of several proteins and protein-protein interaction are suggested in their formation (reviewed in Essers et al., The Plant Cell 12: 211–223, 2000). Formation of transposase oligomers has shown to be necessary for activity of the Ac encoded transposase (Kunze et al., Proc. Natl. Acad. Sci. USA 90: 7094-7098; Essers et al., The Plant Cell 12: 211–223, 2000). It is important to note that the C terminus of Ac transposase responsible for dimerization is the most conservative transposase region of the hAT superfamily of transposons (Essers et al., The Plant Cell 12: 211–223, 2000). On the basis of sequence similarity, to this superfamily both the Ac and Bg elements belong that suggests similarity in their transposition mechanisms (Harting et al., Molecular and General Genetics 227: 91-96, 1991; Kunze et al., Proc. Natl. Acad. Sci. USA 90: 7094-7098; Atkinson et al., Proc. Natl. Acad. Sci. USA 90: 9693-9697, 1993).

In this context, insignificant differences in frequency of reversion of o2-hf observed under regulatory elements dose increase from 2 to 3 (Table 3; Genetika (Moscow) 39: 769-774, 2003; Maydica 48: 275-281, 2003) can be explained by aggregation of mentioned transposase oligomers leading to their inactivation. Analogous mechanism of aggregation of the Ac encoded transposase oligomers has been proposed as one of possible mechanisms of the Ds excision inhibition at high transposase concentrations (Scofield et al., The Plant Cell. 4: 573-582, 1992; Kunze et al., Proc. Natl. Acad. Sci. USA 90: 7094-7098; 1993; Heinlein et al., 1994; Heinlein, Plant J. 5: 705–714, 1996; Essers et al., The Plant Cell 12: 211–223, 2000).

Origin of o2-hf and o2-lf alleles and their behavior in presence of regulatory Bg elements can be explained by participation of the rbg element product in rbg excision from these alleles

In contrast to the o2-hf allele, reversion frequency of the o2-lf allele increases under dose increase of all studied regulatory elements from 2 to 3 (Table 1, Figure 2; Genetika (Moscow) 39: 769-774, 2003; Maydica 48: 275-281, 2003). This regularity is observed at different levels of reversion frequency: at low frequency of reversion of the o2-lf in presence with Bg-lf element (Genetika (Moscow) 39: 769-774, 2003; Maydica 48: 275-281, 2003); at medium frequency with Bg-Ref (Table 1) and at high level in combination with Bg-hf (Maydica 48: 275-281, 2003).

Different behavior of o2-hf and o2-lf alleles under regulatory elements dose increase from 2 to 3 can be explained assuming interaction of Bg transposase oligomers with differing products of the rbg elements inserted in these alleles, the interaction resulting in changed properties of transpososomes. The possibility of such interaction is indicated by sequence similarity between the Bg and rbg elements: rbg differs from Bg by small deletion and insertion events and the two elements share more than 75% homology based on sequence data (Hartings et al., Molecular and General Genetics 227: 91-96, 1991). If the product of the rbg element inserted in o2-lf allele in its interaction with Bg transposase oligomers determine higher level of inactivation of transposase oligomers than the level of inactivation of the oligomers containing the product the rbg element present in the o2-hf allele, this can explaine different behavior of studied alleles with the regulatory elements dose increase from 2 to 3.

For other transposable element systems there are data indicating on rather complicated character of interaction between autonomous and non-autonomous elements. Thus, Kunze et al. ( Proc. Natl. Acad. Sci. USA 90: 7094-7098, 1993) have established that some transpositionally inactive transposase plasmids lead to an increase of Ds excision frequency when they are co-expressed with the active truncated transposase. In work of Cuypers et al. (EMBO J. 7: 2953-2960, 1988) a product of the defective En-I102 element was suggested to be responsible (acting as competitive inhibitor) for the reduced ability of the autonomous En element to induce excisions of the receptor element. Interestingly, for the En/Spm system of maize transposons it was shown that interaction between two proteins TnpA and TnpD is essential for forming transposition complexes (Raina et al., Proc. Natl. Acad. Sci. USA 95: 8526-8531, 1998). These proteins sharing significant degree of homology originate from a single Spm transcript by alternative splicing (Masson et al., Cell 58: 755-765, 1989)

Accepting above presented hypothesis it is possible to conclude that apparition of the sharply different o2-hf and o2-lf alleles as a result of change in state of the initial o2-m(r):3449 allele under disruptive selection for WER content (Maydica 44: 195-203, 1999) is conditioned by changes in the rbg elements affecting in opposite directions the ability of rbg products for interaction with transposition complexes responsible for rbg excision. Accordingly, changes in the initial Bg-3449 element, which conditioned apparition of Bg-hf and Bg-lf elements under the same disruptive selection (Maydica 44: 195-203, 1999), could lead to the changes in their encoded transposases that affected affinity of these transposases toward rbg element products.

Another indication of effect of selection on Bg-hf elements (and hence on its encoded transposase) can be the higher frequency of reversion of o2-hf in presence of the Bg-Ref element than in presence of Bg-hf. The changes in the Bg-hf conditioning certain upper level of the rbg excisions could be determined by used method of disruptive selection for high WER content (in which the Bg-hf element was obtained): the ears containing significantly more than 50% of WER were not selected for next cycle of selection (Maydica 44: 195-203, 1999) since in this case it were more difficult to distinguish the ears with high WER content from the ears heterozygous for normal O2 allele.

Table 1 - Distribution of ears obtained on homozygous o2-hf, Bg-Ref and o2-lf, Bg-Ref plants and in their crosses with o2-R, +^Bg tester (male parent) based on the ratio of phenotypically normal (n) to variegated (v) kernels

Plant genotype	Number of ears					% WER�	RF, %�
	total studi-ed	with the n/v ratio
		=1�	>1	=3�	>3
Selfed plants
o2-hf, Bg-Ref	38	12	26	5	13	47.91a��	15.97a
o2-lf, Bg-Ref	13	1	0	0	0	17.24b	5.75b
Crosses with o2-R, +^Bg used as male parent
o2-hf, Bg-Ref	30	4	6	2	1	26.08c	13.04c
o2-lf, Bg-Ref	19	1	1	0	0	4.31d	2.16d

� Significant by χ² test.

�� A common letter at the means indicates on insignificance of differences (P=0.05).

� Here and in Table 3, reversion frequency (RF), calculated as WER percentage per one dose of responsive allele.

� Calculated without considering ears with the ratio of WER to variegated kernels characteristic for heterozygous O2 plants.

Table 2 - Kernel segregation in embryo revertants found in progeny of two ears of o2-hf/o2-R, Bg-Ref/+^Bg and homozygous o2-hf, Bg-Ref genotypes

Plant number	Crossing with o2-R, +^Bg or with o2-m(r), +^Bg (^b) used as female parent				Selfing (^a) or crossing with o2-R, +^Bg or with o2-m(r), +^Bg (^b) used as male parent
	Number of kernels			χ^21:1 ^c	Number of kernels			χ^21:1	χ^23:1
	n^d	v	o		n	v	o
1	2	3	4	5	6	7	8	9	10
A. Progeny of the ear 02-4574�4742p152 of o2-hf/o2-R, Bg-Ref/+^Bg genotype
03-4210p1^e	-	-	-	-	148	0	148	0.00	-
03-4210p13	-	-	-	-	166	8	175	0.00	-
03-4446p7	-	-	-	-	84	33	118	0.00	-
B. Progeny of the ear 02-4568p131 of homozygous o2-hf, Bg-Ref genotype
03-4211p19	74	59	0	1.69	197	83	0	46.41***	-
03-4212p7^f	26	12	0	5.16*	220^b	77^b	0^b	68.85***	-
03-4212p19	78	61	0	2.08	210	50	0	98.46***	-
03-4219p3	88	75	0	1.04	174	70	0	44.33***	-
03-4225p4^e	70	221	0	78.35***	147	40	0	61.22***	-
03-4226p16	71	0	0	-	158	0	0	-	-
03-4404p13^g	97	97	0	0.00	147	67	0	29.91***	-
03-4404p16^g	28	20	0	1.33	177	104	0	18.96***	-

Table 2 (continued)

1	2	3	4	5	6	7	8	9	10
03-4406p7^e	66	62	0	0.13	122	45	0	35.50***	-
03-4411p4	80	107	0	3.90*	171	69	0	43.35***	-
03-4415p19	103	109	0	0.17	326^a	39^a	0^a	-	39.89***
03-4418p13	28	41	0	2.45	171	95	0	21.71***	-
03-4420p4^e	82	23	0	33.15***	219	181	0	3.61	-
03-4421p16	-	-	-	-	310	0	0	-	-
03-4428p3	70	76	0	0.25	112	59	0	16.43***	-
03-4432p3^ef	128^b	153^b	0^b	2.22	228^a	32^a	0^a	-	22.33***
03-4432p4^f	65	120	0	16.35***	137^a	35^a	0^a	-	1.98
03-4433p13	59	50	0	0.74	117	67	0	12.78***	-

^c For progenies of the ear 02-4574x4742p152 (part A of the table) the 1:1 ratio was the ratio of the sum of normal and variegated kernels to opaque ones; for the progenies of the ear 02-4568p131 (part B of the table) the ratios 1:1 and 3:1 were the ratios of phenotypically normal kernels to variegated.

^d Here and in Table 3, n, v, o indicate phenotypically normal (normal or WER), variegated and opaque kernels respectively.

^e Plant originated from variegated kernel (not marked are plants from phenotypically normal kernels).

^f, ^g Plants from clusters of three (^f) and two (^g) embryo revertants consisting from contiguous kernels presented in adjacent pairs of spikelets (data not shown).

*, *** Significance of deviation from expected at P=0.05 and P=0.001, respectively.

- Indicates not applicable (for the χ² test) or data not available

Table 3 - Comparison of whole endosperm revertants (WER) content in o2-hf, Bg-Ref genotypes observed at one, two and three doses of regulatory element Bg-Ref

Plant number	Crossing with o2-R, +^Bg used as female parent (1 Bg-Ref dose)				Selfing (A) or crossing (B) with the o2-R, +^Bg used as male parent(3 or 2 Bg-Ref doses, respectively)
	Number of kernels			WER, %	Number of kernels			WER, %	RF, %
	n	v	o		n	v	o
1	2	3	4	5	6	7	8	9	10
A. Comparison between 1 and 3 doses of the Bg-Ref
03-4425p16	0	12	0	0	141	201	0	41.23	13.74
03-4427p4	1	28	0	3.45	68	164	0	29.31	9.77
03-4432p19	2	104	0	1.89	78	123	0	38.81	12.94
03-4433p3	3	107	0	2.73	179	53	0	77.16	25.72
03-4433p4	4	84	0	4.55	108	129	0	45.57	15.19
03-4433p19	2	131	0	1.50	180	131	0	57.88	19.29
03-4435p3	1	182	0	0.55	140	164	0	46.05	15.35
Total	13	648	0	1.97a�	894	965	0	48.09b	16.03c
B. Comparison between 1 and 2doses of the Bg-Ref
03-4218p7	2	145	0	1.38	114	145	0	44.02	22.01
03-4218p19	2	172	0	1.16	74	158	0	31.90	15.95
03-4222p4	0	154	0	0.00	99	126	1	44.00	22.00
03-4224p7	18	223	0	8.07	144	128	0	52.94	26.47
03-4226p10	1	84	0	1.19	91	207	0	30.54	15.27
03-4226p19	4	50	0	8.00	89	169	0	34.50	17.25
03-4229p19	7	136	0	5.15	128	112	0	53.33	26.67
Total�	34	964	0	3.53a	739	1045	1	41.42b	20.71c

� A common letter at the means indicates on insignificance of differences (P=0.05).

� Only 7 out of 34 studied reciprocal crosses are presented (analogous data on both samples).

Figure 1 - Kernel phenotypes observed at o2-m(r) alleles. Upper row (from left to right): phenotypically wild-type (whole endosperm revertant, WER) kernel formed as a result of o2-m(r) excision before the first division of primary endosperm nucleus; variegated kernel (vitreous sectors in opaque background) arisen due to o2-m(r) reversion in presence of a Bg element during endosperm development, commonly observed at o2-m(r) alleles; opaque kernel phenotype conditioned by the o2-m(r) allele in absence of the regulatory Bg elements. Two variegated kernels in the lower row which are characterized by small and very small opaque sectors in vitreous background are usually observed in o2-hf, Bg-Ref genotypes.