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The lines of Chinese Spring marked with the alleles
cn-A1 (7A) and cn-D1 (7D) wore kindly provided
by Dr. O. P. Mitrofanova (Mitrofanova 199.1), Institute of Plant
Industry, St., Petersburg, Russia. ANK-32A received the marker trait
(cn-A1) from chlorina-1 line; the mutant (AH-215 x
BC6 Chinese Spring) was the donor of the marker trait
(cn-D1) for ANK-32B. The genes were relocated employing
monosomics, mono7A and mono7D from the monosomic series Milturum 553
(Tsilke and Zharkov 1981). The aneuploids were kindly provided by
Prof. R.A. Tsilke, Agrarian University, Novosibirsk, Russia.
The F1 hybrids of aneuploids with the carriers of chlorina
markers in a hemizygous state failed to differ from the plants
carrying the normal allele. All the plants displaying chlorina
phenotype that appeared m the progeny of monosomic F1
hybrids were disomics. Therefore, the segregation in the progeny of
an F1 monosomic was assessed according to the progenies Of
F2 plants. Occurrence of chlorina plants among the
F3 plants of a given family indicated that the initial
F2 plant carried the marker gene. Such plants were united
into one class with the plants exhibiting chlorina phenotype in
F2. The absence of plants with chlorina phenotype m
F3 indicated the. wild type allele in the initial
F2 plant.
To study the chloroplast structure with electron microscope, the
basal part of the first leaf plate was fixed with 2. 5% glutaric
aldehyde in 0. 1% phosphate buffer (pH 7.4) over 2 h, post-fixed in
1% osmium tetroxide for 1 h, and embedded in Araldite resin.
Ultrathin sections were produced using an Ultracut ultramicrotome
(Leica, Switzerland), stained with uranyl acetate and lead citrate,
and examined under a JEM 100S electron 'Microscope (JEOL, Japan).
Morphometric analysis was carried out in 50 chloroplast negatives of
each line; using test grid, the numbers of dots within the entire
chloroplast and only within its membrane structures (granum and
stroma thylakoids) were calculated. The relative volume of the
chloroplast membrane structure was calculated by the formula Vv =
P1/Pcl, where Vv is the relative volume of the
membrane structures, P1 number of dots within the
chloroplast membrane structures, and Pcl the number of
dots within the entire chloroplast. The values obtained were
processed using the Statistica software package.
Results and discussion
The F1 hybrids of the NILs with their recurrent parent were green,
While a 3 : 1 ( green : chlorina) segregation was observed in F2
(Table 1). Both monosomic and disomic
F1 plants were green in the crosses, mono 7A x ANK-32A and
mono 7D x ANK-32B. Assessing of the F2 plants according to
their F3 progenies has demonstrated the lack of
segregation in these crossing combinations. The inefficiency of
marker alleles of the both loci in heterozygous as well as in
hemizygous states indicates the dose effect. This allowed us to
create morphologically marked monosomics 7A and 7D carrying the
alleles cn-A1 and cn-D1 in the critical chromosomes.
The phenotype of hemizygotes in these lines are marked with green
color, while euploids display chlorina phenotype.
Disagreement of our observations on the inefficiency of one dose of
these markers with the previously obtained results (Pettigrew et al.
1969; Pettigrew and Driscoll 1970; Washington and Sears 1970) may be
connected with the employment of Chinese Spring mutants that,
possibly, have different, genetic background, including, in
particular, additional mutations able to enhance the marker's effect.
The genotype of Chinese Spring itself could enhance the phenotypic
manifestation of one dose of genes cn-A1 and cn-D1.
Sufficient number of backcrosses in our lines (BC9)
guaranteed the absence of modifier genes, while involvement of
different cultivars (Novosibirskaya 67, Milturum 553) in the genetic
analysis excluded the mistakes connected with the peculiarities of
the tester's genetic environment.
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