66                              PNL Volume 21 1989 RESEARCH REPORTS
TRYPSIN INHIBITOR GENES ARE LINKED TO R AND Tl
Smirnova, O.G., S. M. Rozov, and V. A.Berdnikov
Institute of Cytology and Genetics, Academy of Sciences
Siberian Division, Novosibirsk, USSR
Pea seed trypsin inhibitors are poorly studied, possibly due to their amount being less than in other legumes. Using chromogenic substrates for inhibitor identification, inhibitors were proved to be heterogenous in any single genotype of pea (1,7). A wide range of variation in inhibitor activity was shown among cultivars (4,6). After comparison of different inhibitor extraction methods (2), we have chosen a 5% perchloric acid ex­traction of seed flour followed by acetic acid-urea gel electrophoresis (Fig. 1). Trypsin inhibitor bands were determined by the gelatine replica method (3). A single extraction was shown to be enough for almost com­plete removal of perchloric acid extracted proteins.
An analysis of 100 samples from the collection of the N. I. Vavilov Institute of Plant Industry (USSR) enabled us to observe more than ten types of inhibitor electrophoretic patterns. They differ in the number of fractions and in their relative amounts (Fig. 2). It should be noted that some of the bands with trypsin inhibitor activity also possess chimotryp-sin inhibitor activity.
To study the inheritance of the trypsin inhibitor genes, we crossed line WIR 2524 (ssp. elatius) with the tester-line NGB 1238. The former lias five electrophoretic bands with trypsin inhibitor activity while the latter possesses only two subtle ones (Fig. 3). The segregation of band patterns observed in F2 progeny is in agreement with the joint inheri­tance of all trypsin inhibitor components (Table 1). The corresponding gene complex was designated IP (Inhibitor of Proteases). The IP complex is shown to be located in the same linkage group as R and Tl (Table 2A), 21.3 ± 2.7 map units from gene Sa-K9 (5) and at 34.8 ± 3.1 map units from gene tl. Location of the IP genes was also studied in the cross of our laboratory line 'Sprint' (alleles R, Tl, His-lS, Sa-K9F, IPF) with the tester-line NGB 1018 (alleles r, tl, His-1F, Sa-K9S, IPS). The results of the segregation are shown in Table 2B. The genetic distance between the IP and Sa-K9 genes appeared to be different in the two crosses examined: 21.3 ±2.7 and 15.3 ±2.5 map units, respectively. This dif­ference may be accounted for by the differences in genetic background be­tween the subspecies being used. The genetic map of the r-tl chromosome segment based on data presented in this and the previous paper (5) is as tollows:
1.   Ghavan, J. K., and J. Hejgaard. 1981. J. Sci. Food Agric. 32:857-862.
2.   Gofman, J. J., and I. M. Vaisblay. 1975. Prikladnaja Biochimija i Mikrobiologija. 11:777-783 (in Russian).
PNL Volume 21 1989 RESEARCH REPORTS                      67
3.   Konarev, A. V. 1986. Biochimija. 51:195-201 (in Russian).
4.   Mukhsinov, V. X., and V. V. Hanghildin. 1978. Selskochozjajstvennaja Biologija. 8:190-195 (in Russian).
5.   Smirnova, 0. G., S. M. Rozov, and V. A. Berdnikov. 1989. PNL 21:63.
6.   Vaisblay, I. M. 1978. Izvestija Akademii Nauk SSSR. 6:840-848.
7.   Vaisblay, I. M. 1979. Izvestija Akademii Nauk SSSR 1:133-137 (in Russian) .
Table 1. Segregation of trypsin inhibitor phenotypes in F2 progeny of two crosses .
Cross
Homozygote 1
Heterozygote
Homozygote 2
Total
Chi-square
WIR 2524 x NGB 1238 Sprint x NGB 1018
25 26
60 46
30 26
115 98
0.65 0.37
Table 2. Distribution of F2 plants upon phenotypic classes.
A. Cross
WIR
2524 x NGB
1238
Gene pair
Chi-
Recomb.
X
Y
XY XYy
xy
XxY
XxYy
Xxy
xY
xYy
xy
square
fract.
S.E.
Tl
IP
11(1) 14
4
13
34(7)
11
1
12
15(2)
19.9
34.8
3.1
Sa-K9
-----
IP
14 13
2
11
37
11
0
10
17
36.2
21.3
2.7
Expected
7 14
7
15
29
15
7
14
7
-
-
-
B. Cross Sp
r i n t x
NGB 101?
Gene pair X Y
XY
XYv
Xy
XxY
XxYy
Xxy
xY
xYy
xy
Chi-square
Recomb. fract.
S.E.
R
IP
12
1 3
3
12
2 5(4)
10
2
8
13
19.7
31 .1
3.3
Tl
IP.
12
11
3
12
28 (3)
10
2
7
13
21 .0
28.1
3.2
His-l
IP
IP
15
18 6
9
6
12
2 1
6
9 6
12
31
34 25
9
6
12
2 2 6
6
6
12
15
19
6
38.8-49.9
20.9 15.3
2.9
Sa-K9
2.5
Expected
-
For genes Sa-K9, His-1, and IP capital letter stood for mother phenotypes. Observed double crossovers are given in parentheses.
oo
Fig. 2. Trypsin inhibitor variants in different pea genotypes staining with Coomassie R-250.
Fig. 1. Pea WIR 2263 seed proteins.
A - Coomassie R-250 Staining
B - Identification of trypsin inhibitor bands
with gelatin-replica method a) perchloric acid extraction, b) hydrochloric acid extraction (pH of extract 4.2); c) water extraction.
Fig. 3. Trypsin inhibitor and K9 electrophoretic variants of pea lines
used in genetic analysis. a) WIR 2524; b) F1 WIR 2524 x NGB 1238; c) MGB 1238; d) Sprint; e) F Sprint x NGB 1018; f) NGB 1018