A Database for Triticeae and Avena
II.20. High lysine barley - a summary of the present research development
Lars Munck. The Swedish Seed Association, S-268 00 Svalöf, Sweden.
Hiproly barley (CI 3947) was found in the world barley collection (Hagberg
and Karlsson 1969, Munck et al. 1969a) with the dyebinding (DBC) screening
techniques (Mossberg 1969). Hiproly barley although high in protein content
(about 16%) showed a protein and amino acid composition similar to low
protein (about 10%) commercial lines (Munck et al. 1969b). This means increased
content in % of Kjeldahl protein (Nx6.25) of albumines, lysine, asparatic
acid and methionine and decreased content of prolamines, glutamic acid,
cysteine and proline. It was found that these effects were due to a major
change restricted to the endosperm part of the seed (Munck 1970) depending
on a major gene segregating 1:3 (Munck et al. 1970). Significant deviations
for 1:3 were found in some crosses pointing out the possibility of genetic
modifiers. The gene was by linkage or as a pleiotropic effect associated
with a morphological trait resulting in a strong starch protein adherence
of meal preparations as seen in the light microscope (Munck et al. 1970).
Analysis of segregating F2 populations in a phytotron experiment with two
daylengths (Hagberg et al. 1970) revealed no difference between high lysine
and normal segregants regarding daylength response, plant height, number
of spikes per plant, numbers of seeds per plant. The 1000-kernel weight
was, however, about 5 absolute grams lower as a mean in the high lysine
part of the material. A significant amount of positive deviates was found
among the high lysine lines. The shriveled Hiproly seed could be considerably
improved in seed quality by crossing without affecting amino acid composition.
Endosperms of different developmental stages from Hiproly and CI 4362 ("isogenic
normal sisterline") were homoenized with an Ultraturrax at 2°C and
extracted twice with water followed by 1.2% NaCl and 70% EtOH. Centrifugations
were made in a high speed centrifuge and extracts were analysed with micro-Kjeldahl.
The water extract was diluted with EtOH to a strength of 70% to determine
the EtOH precipitable and EtOH soluble Kjeldahl protein. Hiproly showed
increased ability to produce EtOH precipitable albumines especially at
rather late stages in the endosperm development (30-40 days after anthesis)
compared to CI 4362. At yellow ripening stage (about 40% water content)
Hiproly total protein contained 11.3% of this fraction compared with 5.9%
in CI 4362. This difference was 30% less after drying similar endosperms
indicating denaturation effects on the proteins. The prolamines were strongly
reduced in Hiproly protein - 26.5% compared with 38.3% in the sisterline.
Electrophoresis studies of the EtOH soluble H20 fraction and
the EtOh soluble proteins (prolamines) in polyacrylamid (7.5%) gels using
beta-alanine buffer at pH 5.0 indicates differences in extractability
of the catodic component between the two barley lines. A characteristic
pattern of four bands soluble in EtOH and in 0.1 M formic acid appeared
in the alcohol soluble subfraction of the water extract in Hiproly and
in the EtOH (prolamine) fraction in CI 4362. Further studies regarding
the anodic component of the albumines with electrophoresis using 15% gels
in TRIS buffer at pH 8.6 confirm the great quantitative increase of this
fraction introduced by the Hily gene (see also Munck 1972). These studies
were made on single endosperm level from segregating populations, always
with yellow ripe non dried frozen seeds (water content about 40%) to avoid
denaturation effects. No certain qualitative differences could be ascertained.
An extra band in the globulines of the Hiproly and Hily seeds could be
found in the albumines of CI 4362 and normal segregants. Extracts from
single endosperms (table 1) analysed with electrophoresis were confirmed
with amino acid analysis, It was possible by analysing several seeds per
plant to detect normal and high lysine homozygotes as well as heterozygotic
plants (extracts on seed base). A segregation not significant: deviating
from 1:3 was found in the F2 generation from the cross Hiproly x Kristina.
The relative strength between different bands varied considerably between
homozygotic high lysine plants indicating the presence of modifiers. It
was also possible to isolate such lines with the DBC method (see table
2). Other amino acid levels changed in the Hiproly barley were also modified
either towards the direction of the normal or Hiproly. These differences
have been confirmed two years with several analyses on single plant and
The DBC screening analysis was tested on 235 F4 lines emerging from
F2 plants homozygotic for the gene according to amino acid analysis. Twelve
low DBC lines were found which were all found to be high in lysine according
to amino acid controls in F4. Starch protein relationship showed the adherance
type. These lines gave more coarse meal in milling with a hammer mill.
Rechecking the lines in F5 another year did not confirm the low DBC values
Morphological studies on sections reveal that both Hiproly and CI 4362
have a dense protein subaleuron layer, comparable to that of hard wheats.
This is not the case with the variety Kristina which more resembles the
structure of soft wheats. These facts seem to affect milling characteristics,
Hiproly and CI 4362 giving more coarse meal than Kristina.
Another morphological character separated from the above described is
the starch protein adherence character, Kristina and CI 4362 giving good
separation between starch granules and protein in meal preparations in
contrast to Hiproly. Out of 235 high lysine lines in F4 seventeen lines
gave low starch protein adherence as Kristina and CI 4362, Amino acid composition
was not different from 15 control plants and the standard deviation of
protein and amino acids g/16 g N were of the same magnitudes. Nevertheless,
the usual negative correlation between e.g, lysine g/16 g N and crude protein
as well as the positive correlation between e.g. glutamic acid g/16 g N
and crude protein were only significant for the seventeen lines with deviating
starch protein adherence, while there were no signs of such correlation
for the control material. Tentatively the adherence trait could affect
the timing of the protein synthesis so that low lysine prolaimines are
starting and finishing earlier at higher water contents in the endosperm,
independently of the amount of nitrogen translocated to the seed. Such
a changed pattern as well as the adherence trait itself could also be related
to the apparent smaller cell size of the Hiproly endosperm. The starch
protein adherence character is difficult to use as an indicator for the
high lysine gene in backcrosses which could indicate that it is inherited
separately as a gene, linked with the high lysine gene.
Similar effects could be obtained with morphological modifiers which
do not affect the overall amino acid composition, but which neutralize
a tentative pleiotropic morphological side effect of the high lysine gene.
A marked starch protein adherence effect was found in otherwise normal
barley varieties (e.g. Monte Christo). Further integrated genetical-ultrastructural-biochemical
work is needed to grasp this complex.
Nutritional experiments with mice and rats confirm the improved nutritional
value of the new barley lines obtained from the crosses. An increase (at
9.4% protein level in diet) of net protein utilization (NPU) from 59.8
to 68.7% and biological value from 71.2 to 80.6% could be obtained for
restrictively fed rats when lysine g/16 g N increased from 3.25 to 4.13.
Protein content of the seeds were 12.6 and 12.4%. Neither the particle
size of the barley meals nor the starch protein adherence character interacted
significantly with the feeding results.
Mapping studies show that the high lysine gene is located in the 7th
chromosome (K.-E. Karlsson, Barley Genetics Newsletter No. 2, 1972). The
symbol lys is suggested for the gene referring to the practically
important increase of the amino acid lysine which is limiting for the use
of barley protein in e.g. pig feeds. It should be remembered that several
other amino acids are affected by the gene. The symbol lys should
be revised when the basic action of the gene is better understood
Contributions from the following persons and institutions are involved
in this note: The Swedish Seed Association, Svalöf: A. Hagberg and
G. Persson (plant breeding), K.-E. Karlsson (DBC screening, crossing, gene
mapping), L. Munck (coordination, biochemistry, nutrition). Institute of
Genetics, Lund: P. Malnoe, A. L. Tallberg and P. Knutsson (electrophoresis),
K. Hazell (morphology). Institute of Biochemistry, Uppsala: D. Eaker and
R. Thorzelius (amino acid analyses). National Institute of Animal Sciences,
Copenhagen, Denmark: B. 0. Eggum (rat tests).
Table 1. Amino acid analyses of extracts from single
endosperms with and without the Hily gene.
Table 2. Amino acid content* g/16 g N of Hiproly,
normal segregant, high lysine segregant and modified high lysine segregant.
Hagberg, A. and Karlsson, K.-E. 1969. IAEA (Vienna) Symp. Proceed. STI/PUB/212:17-21.
Hagberg, A., Karlsson, K.-E. and Munck, L. 1970. IAEA (Vienna) Symp.
Mossberg, R. 1969. IAEA (Vienna) Symp. Proceed. STI/PUB/212:151-160.
Munck, L. 1970. IAEA (Vienna) Symp. Proceed. STI/PUB/258:319-329.
Munck, L. 1972. American Chemical Society Symp. on seed proteins. Los
Angeles, March 30, 1971 (In press)
Munck, L., Karlsson, K.-E. and Hagberg, Aw 1969a. J. Swed. Seed Ass.
Munck, L., Karlsson, K.-E. and Hagberg, A. 1969b. Bartey Genetics II
Munck, L., Karlsson, K.-E., Hagberg, A. and Eggum, B. 0. 1970. Science
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