Using isozymes as markers, data indicative of polymorphisms in the progeny produced from crossing two beet plants, homozygous on contrastive alleles of enzyme loci, were obtained

Evgenii Levites

English

Nature Precedings

Limitation of F1 hybrids uniformity law
E.V. Levites
Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of  
Sciences,10 Lavrentiev Ave. 630090 Novosibirsk, Russia
Using isozymes as markers, the data indicative of polymorphism in the progeny 
produced from crossing two beet plants, homozygous on contrastive alleles of 
enzyme loci, were obtained.
Keywords:  sexual  plant  reproduction,  agamospermy,  malic-enzyme,  gluc-
osephosphate isomerase, sugar beet. 
Introduction
The  origin  and  development  of  genetics  is  based  on  the  studies  of 
polymorphism  emerging  in  sexual  progenies  and  conditioned  by  two 
independent  probable  processes:  chromosome  divergence  in  meiosis  and 
chromosome integration under gametes merging. Polymorphism in the progenies 
that  form  even  without  meiotic  genome  transformation  of  a  cell  entering 
embryogenesis  (mitotic  agamospermy)  indicates  the  existence  of  one  more 
number of mechanisms that provide variability under seed reproduction (Levites 
et  al.,  1998).  To  explain  the  polymorphism in  agamospermous  progenies,  a 
hypothesis suggesting chromosome sites differential polyteny in a cell before its 
entering embryogenesis and a loss of excessive chromatide regions under the 
first embryogenetic division (Levites, 2005, 2007) was set forth. According to 
the suggested hypothesis,  only two allelic copies that attached to the nuclear 
membrane or nuclear matrix are left. The data on induction of inherited changes 
in wheat and sugar beet by means of affecting nuclear membranes assisted with 
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detergent Triton X-100 (Makhmudova et al., 2009, Kirikovich, Levites, 2009) 
are  in  favor  of  this  supposition.  Considerable  arguments  in  favor  of  this 
hypothesis were obtained in the analysis of agamospermous progenies of triploid 
sugar beet plant (Levites, Kirikovich, 2010, 2011). Marker enzyme phenotypes 
and their ratios revealed in agamospermous progenies are well explained by a 
high polyteny level of chromosome sites coding marker enzymes in mother plant 
cells entering embryogenesis. And, finally, a decrease of heterozygotes share in 
an agamospermous sugar beet progeny (Levites, Kirikovich, 2012) is a weighty 
argument  in  favor  of  polyteny.  This  decrease  is  explained herein  by  a  post-
meiotic  apozygotic  combinatorial  process  that  proceeds  in  heteroallelic 
(heterozygous) egg cells carrying a doubled chromosome set. This combinatorial 
process  is  a  randomized  equiprobable  loss  (diminution)  of  excessive 
chromatides by a diploid egg cell before entering embryogenesis. As a result of 
such a  combinatorial  process,  three types  of  cells  are  formed,  part  of  which 
replenishes  homozygous  classes  and  only  the  left  part  is  the  base  for 
heterozygous progeny formation.
Thus,  studies  of  agamospermous sugar  beet  progenies  give more and more 
proofs  for  the  hypothesis  about  the  effect  of  chromosome  polyteny  for 
agamospermous progenies variability. As agamospermy is only a part of existing 
ways  of  seed  reproduction,  the  search  for  an  answer  to  the  question  —  if 
polytenization  of  chromosomes,  or  their  separate  sites,  takes  place  in  plant 
sexual reproduction — is undoubtedly interesting. In this connection, the aim of 
the present contribution was studying variability in hybrid beet progenies.
Materials and methods
Seeds of F1 reciprocal hybrids produced from crossing only two plants: a green 
plant of sugar beet inbred line KWS-9c and red table beet plant No 19. These 
plants  were  differed  in  alleles  of  gene  Gpi2 controlling  the  marker  enzyme 
glucosephosphateisomerase  (GPI2,  E.C.  5.3.1.9.)  and  gene  Me1 controlling 
malic-enzyme (ME1, E.C. 1.1.1.40.). The sugar beet plant had genotype Gpi2-S/
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Gpi2-S (short -  SS) and Me1-S/Me1-S (short — SS) and, accordingly, the slow 
migrating GPI2 and ME1 isozymes; the red table beet plant had genotype Gpi2-
F/Gpi2-F  (short  -  FF)  and  Me1-F/Me1-F (short  -  FF)  and,  accordingly,  fast 
migrating GPI2 and ME1 isozymes. These two plants hybridization process was 
realized on an isolated plot distant from any flowering beet plants at not less 
than  two  kilometers.  GPI2  and  ME1 were  analyzed  at  the  seed  stage  in  F1 
progeny set on the green and red plants.
Isozymes  electrophoretic  separation  in  starch  gel  and  their  histochemical 
staining  were  carried  out  on  the  earlier-described  methods  (Vallejos,  1983; 
Levites, 1986). Scanning electrophoregrams was made using Biodoc device.
Results and Discussion
Isozyme spectrum GPI2 of homozygous  beet plants is presented with a one-
band spectrum having fast (FF) and slow (SS) electrophoretic mobility (Fig. 1). 
In heterozygous hybrid plants, spectrum GPI2 has parent homodimeric isozymes 
FF and SS and one heterodimeric isozyme FS with intermediary electrophoretic 
mobility (Fig. 1).
+
     1    2     3     4      5     6
Fig. 1. Isozyme patterns of GPI2 in beet seeds. Phenotypes: FF (1, 6), SS (4), FS 
(2, 3, 5). Migration is toward the anode.
Homozygous spectrum of beet  ME1 is presented with a one-band spectrum 
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with  fast  (FF)  or  slow  (SS)  electrophoretic  mobility  (Fig.  2).  Heterozygous 
hybrid spectrum of beet ME1 has parent homotetrameric isozymes FFFF, SSSS 
and three heterotetrameric isozymes FFFS, FFSS and FSSS with intermediary 
electrophoretic  mobility  (Fig.  2).  Electrophoretic  distinctions among 5 allelic 
ME1 isozymes are weak; therefore heterozygous ME1 spectrum is revealed as a 
regular enzyme activity zone (Fig. 2).
+
      1     2    3     4      5    6
Fig. 2. Isozyme patterns of ME1 in beet seeds. Phenotypes: FS (1, 4, 5), SS (2, 
3), FF (6). Migration is toward the anode.
F1 progenies of hybrids  sugar beet x red table beet (short  SB x RT) and red 
table beet x sugar beet (short  RT x SB) were analyzed at seed stage. All the 
seeds  SB x RT studied on isozyme spectra GPI2 and ME1 were classified as 
heterozygous. Heterozygousity of hybrids is conditioned by the thing that they 
were produced from hybridization of plants homozygous on contrastive alleles 
of genes controlling the present marker enzymes.
The basic part of seed progeny  SB x RT had a standard three-band isozyme 
spectrum GPI2  typical  of  diploid  heterozygotes,  in  which  the  heterodimeric 
enzyme manifests itself most intensively. Among 200 analysed F1 seeds, three 
seeds  were  found  to  have  a  five-band  spectrum  GPI2  (Fig.  3)  in  which, 
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alongside with isozymes typical of a common heterozygous spectrum, there are 
two isozymes with a lower electrophoretic mobility.
+
      1    2    3     4     5
Fig. 3. Isozyme patterns of GPI2 in beet seeds. Phenotypes: FS (1, 2, 3, 5), FSSM 
(4). Migration is toward the anode.
It  is  impossible  to  explain  the  appearance  of  these  phenotypes  with  the 
pollution of outside pollen as parent plants were growing on a well-isolated plot. 
It is also impossible to explain by classical mutation as abnormal phenotypic 
frequency (0.015) is several orders higher than that of spontaneous mutations. 
On  the  whole,  emerging  of  abnormal  phenotypes  in  F1 progeny  can  be 
considered as a limitation of F1 hybrids uniformity law.
As abnormal GPI2 spectra have not only FF, FS and SS isozymes, and also two 
more isozymes with electrophoretic mobility lower than that in homodimers SS, 
it  is  possible  to  conclude  that  these  abnormalities  are  conditioned  by  the 
capability of allele Gpi2-S (short — S) to transfer into the new Gpi2-SM (short - 
SM)  state.  The  same  phenotypes  were  found  by  us  when  analyzing 
agamospermous  sugar  beet  progenies  (Levites,  Kirikovich,  2011).  Thus,  the 
presence  of  five  isozymes  in  spectrum  GPI2  points  out  the  thing  that  the 
products of three alleles F, S and SM at once are present in the cell. Symmetricity 
of  enzyme intensity  distribution  in  the  five-band spectrum indicates  the  two 
relevant  moments:  1)  the  process  of  monomeres  (subunits)  integration  into 
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dimeric  enzyme  molecules  proceeds  randomly  and  equiprobably;  2)  enzyme 
subunits synthesized by normal alleles (Gpi2-F and Gpi2-S) and changed allele 
Gpi2-SM develop in each cell at equal quantities, i.e. activities of each of two 
initial and one changed allele are equal.
The five-band abnormal spectrum of GPI2 is a unique phenotype in whose 
appearance  two  events  happened  at  once:  doubling  of  allelic  dosage  and 
transition of one of double-doses into a new state.  The frequency of each of 
these events is not high, but their simultaneity points that the abnormality may 
emerge in case when an embryogenesis-entering cell has not only allele Gpi2-F 
but  also  two  copies  of  allele  Gpi2-S, one  of  which  undergoes  changes  and 
transforms into state Gpi2-SM.
The electrophoresis technique we use allows us to determine the phenotype of 
each seed right on several enzymes by means of getting some horizontal slices 
(sections)  of starch gel  and their  further  histochemical  staining.  The analysis 
showed that  seeds  having a  changed phenotype on GPI2 have  no change of 
malic-enzyme  (ME1),  which  corresponds  to  the  standard  spectrum  ME1 
revealed in diploids having no abnormalities on GPI2. These data indicate the 
thing that the Gpi2 dosage increase is  not connected with an increase of the 
whole genome and/or gametes non-reduction. Appearance of an additional dose 
of allele  Gpi2-S in cells of plant generative organs may be a consequence of 
polytenization  of  chromosome  site  carrying  this  allele.  Independence  in  loci 
Gpi2 and Me1 expression, with the presence of dosal changes in one of them, is 
in favor of the hypothesis on differential chromosome polyteny in cells of plant 
generative  organs.  The  obtained  result  point  out  the  thing  that  differential 
polyteny may also manifest itself in gametes. It is this thing that is the reason for 
the limitation of F1 hybrids uniformity law.
And, finally, the data obtained when analyzing RT x SB hybrid progeny is the 
most drastic expression of such variability. Among the 37 analyzed  RT x SB 
hybrid seeds, a seed with malic-enzyme spectrum which was presented only by 
mother  isozymes  —  namely  FFFF  homotetrameres  were  revealed  (Fig.  4). 
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However, the GPI2 spectrum in this seed was heterozygous (Fig.5).
+
     1    2       3     4              5    6     7
Fig. 4. Isozyme patterns of ME1 in RT x SB hybrid beet seeds. Phenotypes: FS 
(1, 3, 4, 5 - 7), FF (2). Migration is toward the anode.
+
     1     2      3     4             5     6   7
Fig. 5. Isozyme patterns of GPI2 in RT x SB hybrid beet seeds. Phenotypes: FS 
(1 - 7). Migration is toward the anode.
This event may be explained  as the egg cell introduced a high allelic  Me1-F 
polyteny level into the zygote. This polyteny led to the combinatorial process 
proceeding in the zygote before its entering embryogenesis. As a result of this 
process, paternal allele Me1-S was lost.
The polyteny-connected  changes we observed well  accord with the data  of 
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variability  conditioned  by  gene  amplification.  For  instant,  researchers  that 
studied  the  appearance  of  gigantic  flax  plants  (genotrophs)  characterised  by 
increased habitus came to the conclusion that such changes are conditioned by a 
9-10% of DNA content increase that proceeds not by an increase of a certain 
chromosome  number,  but  by  all  chromosomes  increase  simultaneously,  their 
separate sites being proliferated (Evans et al., 1966; Evans, 1968; Durrant, 1971; 
Durrant,  Jones,  1971;  Timmis,  Ingle,  1974;  Cullis,  1973).  Cytogenetic 
amplification is expressed in an increase of certain chromosome parts having 
homogeneously stained sites. It is known about high mutability of loci that were 
subjected to amplification (spontaneous or induced) (Ignatova, 1982). This fact 
well accords with the appearance of abnormal  GPI2 spectrum revealed in this 
experiment.
Amplification is usually understood as an increase of DNA sequences copies 
by  means  of  repeats  formations.  But  a  different  way  is  also  possible.  It  is 
polytenization  of  separate  chromosome  sites.  The  presence  of  polytene 
chromosomes in cells surrounding the embryosac and in the cells of embryosac 
proper was shown in a number of plant objects  (Nagle, 1976; D’Amato, 1984; 
Carvalheiro, 2000; Morozova, 2002). It is these facts that served as base for the 
hypothesis explaining sugar beet progenies polymorphism forming by means of 
agamospermy  (Levites,  2005,  2007).  Elimination  of  excessive  allelic  copies 
proceeding according to  probability  laws leads  to  outlined  precised  ratios  of 
phenotypic  classes  in  progenies  indicating  the  existence  of  a  combinatorial 
process underlying variability under agamospermy. It is combinatorial process 
that allows us to dwell on the thing that polymorphism in an agamospermous 
progeny is determined not by the emergence of allelic genes linear repeats, but 
their endoreduplication that leads to separate chromosome sites polytenization.
It is characteristic that the type of abnormal phenotypic classes GPI2 coincides 
in  sexual  and  agamospermous  progenies  (Levites,  Kirikovich,  2011).  For 
example, in both cases phenotypes FSSM are revealed. The affinity of abnormal 
phenotypes  and their  connection  with the  appearance of  an additional  allelic 
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dose allows us to hypothesize on the thing that the emergence of abnormalities is 
conditioned by chromosome polytenization in  both agamospermy and sexual 
reproduction.
It is worth noting that Gpi2 changes were added to F1 by the egg cell. This fact 
well  accords  with  the  known  data  on  the  thing  that,  e.g.  Allium tuberosum 
polytenization frequency is 80 % in female generative cells, male being 3.9 % 
(Kojima, Nagato, 1992). This is an additional argument in favor of the thing that 
the revealed variability is conditioned herein by chromosome polytenization.
Summing up the  results  obtained earlier  and those  in  this  contribution,  it  is 
possible  to  say  that  polytenization  is  a  powerful  variability  factor  –  in  both 
sexual  and  agamospermous  plant  progenies.  Considering  the  dependence  of 
DNA synthesis from an external (exogenic) factor (Cullis, 1973), one can assert 
the thing that polytenization is one of the ways to record the information about 
acquired traits.
Acknowledgement 
The research was financed by Grant No 99 on the SB RAS Integrative Project 
for the period of 2009–2011. The author would like to express his gratitude for 
the help on electrophoretic analysis to Genina E.S. and for the English version of 
this article to Alexander V. Zhuravlev, SB RAS Executive Interpreter.
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Triton X-100 Induces Heritable Changes of Morphological Characters in Triticum aestivum L.

Limitation of F1 hybrids uniformity law

http://precedings.nature.com/documents/6734/version/1

Limitation of F1 hybrids uniformity law

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