- Obtention of a second and a third allele for X\a\/x\a.gene.
A second allele (x\a2) was obtained in Castelar by means of X-rays treatments applied to seeds of a translocated genotype T (6-7)d (Prina and Favret, 1988). In comparison to OUM 215, this mutant showed a lower difference in colour between heterozygotes and normal homozygotes. They look alike at heading, but they are easy to distinguish from each other at early stages.
A third allele (x\a3) is herein reported. This was obtained through sodium azide treatments 1x10^(-3) M, pH 6, applied in aquous solution to seeds of MC 20 genotype (mutant Castelar accession number). Diallelic crosses among chlorina plants of the three above- mentioned origins segregated xantha seedlings on the F1 generation, indicating the three mutants belong to the same gene. This last material showed marked differences among the three segregated genotypes, similarly to what was observed for OUM 215 or even more. We think the different expressions are not necessarily due to functional differences among the three alleles; otherwise, they could be produced by the influence of the different genetic backgrounds.
The phenothypic frequencies in the M5 generation originated from heterozygous plants ( X\a\x\a3) are presented in table 1. They statistically did not differ from a. 1:2:1 ratio, as was expected for a single nuclear gene with semidominant expression.
-Use of the third X\a\/x\a mutant as a genetic marker for locating newly induced mutants in the long arm of chromosome 1.
One thousand seeds harvested on heterozygotes (chlorina) plants of MC 20 origin were treated with EMS ( 0,25% v/v ), for 20 hours, and sowed in the field nursery. In M1 generation, xantha, chlorina and normal seedlings were segregated. At maturity only chlorina plants were harvested, since xantha seedlings died at second leaf stage and normal plants were cut off before heading. The progenies of 708 spikes (corresponding to 177 M1 plants) were analyzed at the greenhouse and 58 of them segregated new chlorophyll mutants.
-Two chlorophyll mutants associated with X\a\/x\a segregation:
Only those chlorophyll mutants clearly deviating from random segregations were considered for further linkage analysis. This was the case of two newly induced mutants, one yellow, lighter than the xantha (X\a\x\a), that we named clear xantha (xc) and one virido-albina, with green top and albina bottom (va). Homozygous xcxc seedlings were lethal at second leaf stage, while vava plants were weaker than normal ones, occasionally surviving at field conditions. Both mutant alleles had recessive expression.
A third case producing segregation distortion of X\a\/x\a gene, but showing no visible change, is being analyzed at present and will be later on reported.
The phenotypic frequencies observed in the selfed progenies of double heterozygous plants X\a\x\a3-Xcxc and X\a\x\a3-Vava, both in repulsion , are shown in tables 2 and 3, respectively. According to these results, both genes are closely linked to X\a\/x\a. In table 4 the phenotypic frequencies of the F2 generation coming from double heterozygous plants (Xcxc-Vava) indicated they are located at both sides of X\a\/x\a locus.
-Conclusions:
Mutagenic treatment of heterozygotes for easily recognized markers is an appropiate methodology for mapping new genes in the corresponding chromosome region and also for artificially building balanced lethal systems ( Favret and Ryan, 1966 ) for a given chromosome segment. With slight effort, two new chlorophyll markers were located in the long arm of chromosome 1, having one of them an even more distal position than X\a\/x\a gene.
References :
Allard, R. W., 1956. Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24 (10). Univ. of California.pp 235-279
Favret, E. A. and Ryan, G. S., 1966. New useful mutants in Plant Breeding.In: Mutations in Plant Breeding. IAEA, Viena. pp.49-61.
Konishi, T., 1972. An incomplete dominant chlorophyll mutation on chromosome 1. Barley Genetics Newsletter 2: 43-45.
Prina, A. R. and Favret, E. A., 1988. Influence of marker genes on the expression of somatic mutations in barley. J. Heredity 79: 371-376.
Takahashi, R., Hayashi, J., Konishi, T., Moriya, I., 1972. Inheritance and linkage studies in barley. Ber. Ohara Inst. .Biol.Okayama Univ. 15: 147-168.
Table 1. Phenotypic frequencies in selfed progenies of heterozygous plants X\a\x\a3.
Normal Chlorina Xantha Total P for
1:2:1 ratio
453 906 459 1818 >0.95
Normal: X\a\x\a; Chlorina: X\a\x\a3; Xantha: x\a3\x\a3.
Table 2. Phenotypic frequencies in selfed progenies of double heterozygous
plants X\a\x\a3-Xcxc (in repulsion).
N C X cX P for expected ratio % Recombination
Xc X\a Indep. (1)
3:1 3:1
40 1043 542 542 a)>0.99 >0.99 <0.001** 3.69 ± 0.41
b)>0.99 - <0.001** 3.76 ± 0.41
a)x\a3\x\a3\xcxc = 0
b)x\a3\x\a3/xcxc= X\a\x\a\xcxc= X\a\x\a3\xcxc.
N:Normal: X\a\x\a-XcXc/Xcxc;
C:Chlorina: X\a\x\a3-XcXc/Xcxc;
X:Xantha:x\a3\x\a3-XcXc/Xcxc;
cX:Clear Xantha: X\a\X\a\/x\a/x\a3 x\a3\x\a3-xcxc.
(1): The recombination was estimated based on acumulation of
expected recombinants.
Table 3. Phenotypic frequencies in selfed progenies of double heterozygous
plants X\a x\a3-Va va (in repulsion).
N C X VA XA P for expected ratio % Recombination
Va X\a Indep. (1)
3:1 3:1
30 421 202 218 2 >0.8 >0.2 <0.001** 10 ± 3.34
N:Normal: VaVa/Vava-X\a\x\a;
C:Chlorina: VaVa/Vava-X\a\x\a3;
X:Xantha: VaVa/Vava-x\a3\x\a3;
VA:virido-albina: vava-X\a\X\a\/x\a\x\a3;
XA:Xantha-alba: vava-x\a3\x\a3.
(1): For maximum likelihood (Allard, 1956)
Table 4. Phenotypic frequencies in selfed progenies of double heterozygous
plants Xc xc-Va va. (In repulsion)
N VA cX cXA P for expected ratio % Recombination
Xc Va Indep. (1)
3:1 3:1
47 23 24 2 >0.6 >0.8 <0.05** 26.72 ± 9.35
N:Normal: XcXc/XcXc-VaVa;
VA:virido-albina: XcXc/Xcxc-vava;
cX: Clear xantha: xcxc-VaVa/Vava;
.cX-A: Clear xantha-alba: xcxc-vava.
(1): For maximum likelihood (Allard, 1956)