The materials consisted of F/2/ individuals and their F/3/ progenies derived from three crosses, each of which was made by a combination between a strain carrying the variant isozyme allele and a linkage stock containing multiple markers on the corresponding chromosome. Isozyme genotypes of the F/2/ individuals were investigated by starch-gel electrophoresis using the first leaf of each 10-day-old seedling grown under a 12-h illumination cycle in a growth cabinet kept at 18°C. Furthermore, segregation at the Aat2 locus and that of marker alleles were examined in F/3/ progenies.

As shown in Table 1, Est5 and Pgd1 are linked with a recombination value of 6.5%, and both of them are loosely linked to fc for chlorina on the short arm of chromosome 1. One of the parents, Pgd1-fc, was developed from a cross between 'K 12' (Okayama University Accession Number, OUI602)(Konishi and Yoshimi, 1993a) and Colsess V (fc) (OUL003)(Konishi and Yoshimi, 1993b). Meanwhile, Aat2 is assigned on chromosome 6 by the addition line analysis (Hart et al., 1980). But, no linkage study of Aat2 has been conducted, because any variant allele could not be detected at the locus. Recently, we found the null allele at the Aat2 locus in a strain of H. spontaneum, Spont. MRC 5 (OUH779), collected in Morocco by a Spanish team (Molina-Cano and Conde, 1980). Linkage analysis revealed that Aat2 is located between o for orange lemma and l9 for dense spike on the long arm of chromosome 6, with recombination values of 32.6% between Aat2 and o and 26.9% between Aat2 and l9, respectively.

Hvid and Nielsen (1977) demonstrated that two alleles, Fl and ne, at the Est9 locus control the presence and absence of the Est9 band appeared on the cathodal side of the starch gel, respectively. As the staining intensity of the band was strongly influenced by genetic backgrounds and growing condition of the seedlings, some varieties and segregants showed very weak bands which were difficult to determine their genotypes. We also found a variant allele in a strain of H. spontaneum, Spont. 6908 (OUH614), showing the slower migrating band than the Fl one. Using the strain as one of the parents, linkage analysis was carried on. Est9 is located on the long arm of chromosome 7, arranging fs, s, r and Est9 from the centromere to the telomere, as illustrated in Fig. 3.

These isozyme loci are scattered on three of the seven barley chromosomes, so the isozyme markers may be linked with some agronomically important loci such as those controlling disease resistance.

References:

Hart, G. E., Islam, A. K. M. R., and Shepherd, K. W. 1980. Use of isozymes as chromosome markers in the isolation and characterization of wheat-barley chromosome addition lines. Genet. Res., Camb. 36:311-325.

Hvid, S., and Nielsen, G. 1977. Esterase isoenzyme variants in barley. Hereditas 87:155-162.

Konishi, T., and Yoshimi, R. 1993a. Geogaphical distribution of isozyme alleles for phosphogluconate dehydrogenase in barley. Genome 36:512-516.

Konishi, T., and Yoshimi, R. 1993b. Linkage analysis of Pgd1 for phosphogluconate dehydrogenase in barley. Barley Genet. Newsl. 22:70-72.

Molina-Cano, J. L., and Conde, J. 1980. Hordeum spontaneum C. Koch em. Bacht, collected in Southem Morocco. Barley Genet. Newsl. 10:44-47.

Table 1. Segregation of isozyme and linkage markers in F/3/ progenies of three corsses

__________________________________________________________________________
                                                                Recombi-
Gene pair Cross¹ Phase² Segregation³             Total   X²L    nation
  a    b                                                        value (%)
__________________________________________________________________________
 fc  Est5   A       R   4:10:53/19:95:13/47:13:1   255  225.8   13.7± 1.65
 fc  Pgd1   A       C   53:12:2/11:102:14/1:6:54   255  282.0   10.1± 1.41
Est5 Pgd1   A       R   1:5:61/10:106:2/59:9:2     255  377.5    6.5± 1.13

Aat2  o     B       R   14:21:31/26:65:25/30:16:6  234   43.6   32.6± 2.78
Aat2 l9     B       R   9:26:31/21:75:20/31:19:2   234   61.2   26.9± 2.49
 o   l9     B       C   26:26:9/32:53:35/12:23:18  234   10.8   independent

Est9 fs     C       R   13:39:12/25:74:36/22:34:19 274    5.1   independent
Est9  r     C       R   4:19::41/18:90:27/42:18:15 274  105.2   25.3± 2.22
Est9  s     C       R   8:39:17/30:67:38/22:36:17  274    5.6   independent
 fs   r     C       C   21:33:16/52:69:28/12:27:21 279    8.5   independent
 fs   s     C       C   34:27:9/36:92:21/4:24:32   279   56.9   28.6± 2.36
  s   r     C       C   37:32:5/44:72:27/4:25:33   279   56.7   30.3± 2.44
___________________________________________________________________________

Fig. 1. Linkage map of fc, Pgd1 and Est5 on the short arm of chromosome 1.

Fig. 2. Linkage map of o, Aat2 and l9 on the long arm of chromosome 6.

Fig. 3. Linkage map of fs, s, r and Est9 on the long arm of chromosome 7.