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Materials and Methods
Chinese Spring ditelosomic lines of homoeologous group of 6 were
used for identifying (alpha-amylase isozyme bands and also for
telocentric mapping. Aka, a Japanese local variety, and Jones Fife,
each having the specific zymogram pattern, were also used.
Triticum turgidum (L.) Thell.ssp turgidum conv.
durum (Desf.) MK cv. LD 222 and ssp. dicoccum (Schrank)
Thell. var. georgicum Dek. et Man. were AABB tetraploid used
in this study. Ditelo 6BL-monotelo 6BS of LD 222 used for telocentric
mapping in Emmer wheat carries telocentric chromosomes introduced
from the corresponding Chinese Spring telosomic lines into LD 222
genetic background (Nishikawa unpublished).
Alpha-amylase was extracted by homogenizing a germinating grain in 1
mI of 0.05M tris-HCl buffer (pH 7.0, containing 0.002 M
CaCl2E2H20), followed by centrifugation at
25,000 x g for 10 minutes. Alpha-amylase isozymes in the extract were
separated by thin layer (19 x 14 x 0.5 cm) polyacrylamide (0.5%) gel
isoelectrofocu sing. Carrier ampholites (Pharmalyte: Pharmacia,
Sweden) and electorode solutions used were shown in Table
1.
Electrophoresis was performed by applying constant current
(2.1w/cm3Egel) from the constant power supply (SJ-1065,
Atto Ltd., Tokyo) for three hours. The gel was soaked in 3% starch
sol for 10 minutes, incubated at 38oC for 2 minutes and
stained with Lugol's solution.
Results and
Discussion
It is well known that alpha-amylase isozymes in germinating grain
are grouped into two types, Malt type and Green type, the former are
encoded by the genes on the long arm of chromosomes of homoeologous
group 6. This was reconfirmed in this study as shown in
Fig.1.
Among some twenty bands detected, however, Bands 3, 10 and 14
remained unknown about the chromosome arms, on which the genes
concerned are located. In addition, three added bands, 9, 13 and 23
occurred in ditelo 6DS, which was not detected in disomic. There is
no definite explanation how these three bands appeared, but there are
two possibilities; the long arm of 6D carries the inhibiting effect
to these bands, and the other is that missing the major bands nearby,
Bands 8, 12, and 24 enables to discover Bands 9, 13 and 23 which were
masked by them. Out of 17 common wheat cultivars tested, Aka, a
Japanese local variety, lacked Bands 20, 21 and 22, and had Bands 15
and 15', and Jones Fife lacked Bands 16 and 18 and had Bands 1, 1',
15 and 15', in comparison with Chinese Spring, respectively
(Fig.2-a,-b).
1) Band 20 (former Band 2, cf: Nishikawa et al. 1981)
Aka lacked Band 20 (Fig.2-a),
which was known to be encoded by the gene on the long arm of
chromosome 6A (Nishikawa et al.1981), the allele for Band 20 being
designated as alpha-Amy-A1a, and the one for missing Band 20
as alpha-Amy-A1b. Though not shown in Table
2, unexpected
chromosome complements such as 40+t and 41 occurred among
BC1 plants. Because all the PMC's in F1
monotelodisomic for 6AL formed a heteromorphic bivalent, and
the missing chromosome is considered as one other than 6A, those
plants with the aberrant chromosome number were treated as 41+t or 42
plants. With 8 crossover type in total 216 plant, the recombination
value between alpha-Amy-A1 and centromere was estimated as 3.8
plus or minus 0.01 (%), in other word, alpha-Amy-A is located
on the Iong arm of 6A apart from the centromere with the distance of
3.8 plus or minus 0.01 (%) (Fig.3-a).
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