GEM Report: Mark Campbell, Avinash Karn, Diana Acevedo (Truman
State University), Jay-lin Jane, Hongxin Jiang (Iowa State University)
and Yusheng Wu (South Dakota State University).
Chicago ASTA meeting– December 11th 2008.
Introduction
As a GEM public cooperator, our objective has been toward the development of
Amylomaize VII germplasm using GEM breeding material as the source of the high
amylose genes and as the source genes for superior agronomic traits such as enhanced
yield and plant health. We feel that development of high amylose germplasm is a
valuable pursuit since there continues to be great interest in its application, especially in
biodegradable plastics and as a nutraceutical. GEM germplasm has been an excellent
source of genetic diversity, which is highly beneficial due to the limited number of
research and development programs with this specialty corn area.
Genetic studies to examine high amylose modifier gene(s)
One specific area of our research has been toward the understanding of the
genetic basis and inheritance of the high amylose modifier genes that elevate starch
amylose to 70% in the presence of the recessive amylose-extendet (ae) allele. Yusheng
Wu of South Dakota State University has conducted the mapping of high amylose
modifiers from the population derived from the cross between H99ae x GEMS-0067ae.
For this study, H99ae served as the low amylose content parent, while GEM0067
functioned as the high amylose content parent. Also included are F1: H99xGEM0067
and F2 mapping population. The data was collected through polyacrylamide and super
fine resolution agarose gel electrophoresis. MapManger and QTL cartographer were used
for the QTL analysis.
Table 1. Statistical tests and QTL information
χ2 test, Duncan test and QTL information for the SSR primers used in the study
Oberved no.
Genotypic mean
Primer
χ2
H99 F1
G
H99
F1
G
umc1397
26
80
39
3.88
70.0 a
68.2 a
69.9
umc1298
41
78
32
1.24
67.8 a
69.0 a
69.2
phi265454 40
86
25
5.90
69.6 a
68.5 a
68.2
phi96100
30
84
37
2.56
67.7 a
69.3 a
68.4
umc2363
37
57
49
7.90
69.2 a
68.3 a
68.6
phi083
3
116 27
58.55
65.4 a
68.5 a
69.6
umc2118
41
32
58
38.68
69.1 a
69.4 a
67.9
umc1780
33
82
32
1.98
68.4 a
69.2 a
68.2
umc2256
36
60
31
0.78
68.4 a
68.8 a
67.9
umc2117
31
82
33
2.27
67.0 a
69.1 a
69.2
umc1655
32
74
37
0.52
67.1 b
69.0 ab 70.0
bnlg1350a 33
75
31
0.93
67.8 b
67.9 b
70.6
umc2027
25
80
37
4.31
69.7 a
67.7 a
70.0
a
a
a
a
a
a
a
a
a
a
a
a
a
Chr
cM1
cM2
LRS
Add
Dom
1.03
1.09
1.11
2.01
2.02
2.04
3.00
3.01
3.01
3.04
3.04
3.08
4.06
226
847
973
28
49
285
0
11
23
190
191
445
349
0
62
95
0
0
61
0
28
37
103
120
150
0
2.8
1.4
1.1
1.1
1.1
0.7
1.7
0.7
1.0
2.9
3.8
4.5
4.8
0.95
-1.65
0.45
0.58
-0.49
-0.54
-0.30
-0.54
-0.30
-0.54
1.03
-0.89
-0.79
1.19
-0.59
0.76
-0.34
0.96
0.51
0.96
1.23
0.34
1.90
-1.39
1.46
-2.15
1
umc1086
30
74
42
2.00
69.9 a
67.8 a
69.1 a 4.08
499
74
umc1365
52
64
29
9.29
67.5 b
68.6 ab 70.7 a 5.01
125
0
umc1784
27
95
24
13.38
67.1 b
68.6 ab 70.5 a 5.03
279
0
umc2161
32
79
32
1.57
63.8 c
68.9 b
72.6 a 5.03
295
28
umc1941
30
75
38
1.24
67.5 b
68.6 b
71.0 a 5.06
493
86
umc1524
35
74
37
0.08
68.9 ab 67.3 b
70.9 a 5.06
493
103
umc2313
32
72
41
1.12
66.4 b
69.1 a
70.0 a 6.01
92
0
bnlg249
37
78
31
1.18
66.5 b
69.1 ab 70.2 a 6.01
98
26
umc1006
36
75
35
0.12
66.3 b
69.1 a
70.1 a 6.02
125
39
umc1105
41
77
28
2.75
67.5 b
68.6 ab 70.4 a 6.04
190
64
umc1136
30
76
39
1.46
68.3 a
68.1 a
69.5 a 6.04
211
64
umc1314
24
88
32
8.00
67.8 a
68.7 a
69.2 a 6.05
285
160
bnlg1792
26
90
30
8.14
69.5 a
69.6 a
65.7 b 7.02
190
N/A
umc1997
34
75
37
0.23
69.6 a
68.4 a
68.2 a 8.06
436
0
umc1724
40
70
35
0.52
69.3 a
68.1 a
68.9 a 8.06
475
41
bnlg1031
30
74
42
2.00
68.1 a
68.4 a
69.4 a 8.06
475
125
phi033
36
52
26
2.63
68.2 a
68.9 a
68.8 a 9.01
32
0
phi028
38
68
29
1.21
68.2 a
68.6 a
69.3 a 9.01
32
6
umc1170
37
82
32
1.45
68.5 a
68.5 a
69.6 a 9.02
101
10
phi061
46
69
31
3.52
67.6 a
69.0 a
69.4 a 9.03
177
27
umc1196a 28
71
41
2.44
69.9 a
68.4 a
68.1 a 10.07 445
N/A
Notation: Obs: observed number; H99: low amylose parent; F1: H99xGEM00667; G: GEM0067, high
amylose parently; Chr: chromosome and bin; cM1: genetic distance on IBM; cM2: genetic distance
on current mapping population;LRS: likelyhood ratio statistic, the threshold 11.5; Add: additive effect;
Dom: dominance effect. For χ2 test, df=2, a=0.05, χ2=6.20. For Duncan test, the means with the same
Letter are not significantly different, otherwise, significantly different. N/A: not available.
3.2
4.4
4.4
42.0
9.6
9.9
7.6
8.2
9.0
1.5
1.5
0.8
N/A
1.4
1.1
1.0
0.6
0.8
0.8
2.1
N/A
0.53
-1.73
1.93
-0.21
1.93
-0.21
4.06
0.65
2.55
-2.45
2.46
-2.76
1.32
0.73
1.61
0.77
1.56
0.84
1.04
-0.74
1.04
-0.74
0.61
0.07
N/A
N/A
-0.49
-0.46
0.27
-0.99
0.82
-0.49
0.46
0.24
0.75
-0.20
0.81
-0.45
0.76
0.60
N/A
N/A
In Table 1, the chi-square test is shown in order to determine whether the
observed number of marker genotypes in the mapping population fit into a 1:2:1 ratio.
The Duncan’s test was used to establish whether the means of those genotypes were
significantly different. Based on the results of those two tests, potential QTLs might be
on chromosome 5 and 6, respectively. The primer umc2161 on chromosome 5 bin 3 had
the highest LRS, indicating that the QTL is potentially nearby. The fine mapping on the
region will be performed sequentially. The primers bnlg249, on chromosome 6 bin1, and
umc1006, on chromosome 6 bin 2, had the LRS values closest to threshold value, 11.5. It
is justifiable to continue looking for the QTL on those two bins.
Four primers, phi033, phi028, umc1997 and phi061, were gene markers for Sh1,
SbeIIa and Wx, which are involved in amylose formation in the starch synthetic pathway.
None of these showed that the means of three genotypes were significantly different.
However, it is worth doing the analysis of gene interaction between two genes mentioned
above to rule out their involvement. The data of the rest of the primers did not provide
any apparent useful information on detecting the QTL on other chromosomes.
2
Figure 1. Starch branching enzyme isoforms and sites of activity and
chromosomal locations (aG – granule associated, S – Soluble) (From
Tetlow et al 2004, and maizeGDB).
Figure 2. QTL LOD scores on eight chromosomes included for analysis
Chr 01
Chr 02
3
Chr 03
Chr 04
Chr 05
Chr 06
Chr 08
Chr 09
In Figure 2, the QTL mapping LOD scores are shown on the eight chromosomes.
From the data, the potential QTL on chromosome 5 and 6 are shown on the upper graph
with the threshold of LOD on the y-axis being 2.5 and indicated by a horizontal line. The
lower graph indicates the additive effect on each chromosome.
During the summer of 2006, genetic stocks of H99ae and GEMS-0067 were
kindly grown in the genetics nursery of Drs. Tracie Bierwagen, Alan Meyers and Martha
James at Iowa State University for starch branching enzyme isoform analysis. Three
isoforms of interest are SBEIIb, SBEIIa (ae) and SBE1 and are displayed in Figure 1
where chromosomal location and primary tissue of expression are shown. Endosperm
samples were obtained 18 days after pollination and used to determine the presence of the
isoforms in a zymogram (Figure 3). Of greatest interest was the apparent altered mobility
4
of the starch branching enzyme 1 (SBE1) isoform. It is interesting to note that GEMS0067 does not appear to be a null mutant for sbe1, but rather a dysfunctional protein
product that has resulted from selection for high amylose within GEM germplasm.
Figure 3. Zymogram of H99ae and GEMS-0067 showing three isoforms
of starch branching enzymes (Sbe1, Sbe2a and Sbe2b).
Yield and Agronomic Data
A yield trial was conducted in two locations in 2008. One normal check, two
amylomaize VII checks from an undisclosed source, and 37 experimental amylomaize
5
VII GEM x GEM hybrids were investigated. Twenty nine of the experimental hybrids
were new and were produced by crossing 12 new SS lines to a common tester
SCR01:N1310-358-1-B-B///GEMS-0067 and 17 new NS lines to a common tester
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-0067 (Figure 4). Although
this assumption was based on limited data, these lines were chosen as testers because a
previous yield analysis suggested that these lines had good general combining abilities.
The remaining 8 experimental hybrids were repeats from previous years and were chosen
because of their high yield. The yield trial from the Kirksville location has not yet been
harvested. Northeast Missouri experienced a wet summer and standing water put a great
deal of stress on this yield trial (Figure 5). Data from this location, unfortunately, may
not prove useful.
Figure 4. Breeding scheme for development of amylomaize VII hybrids
using a common tester.
Summer Rain (Kirksville, 2008)
20
15
inches
Figure 5. Total monthly rainfall
reported at KTVO television
station in Kirksville, Missouri for
the summer of 2008. A wet season!
10
5
0
May
June
July
Aug
Month
Yield data from the Ames, IA location is provided in this report and summarized
in Table 2. Significant effects due to genotype were seen for all traits except stalk
6
lodging. The two Amylomaize VII checks yielded similarly, at approximately 123 bu./A.
This data revealed that our use of the testers, SCR01:N1310-358-1-B-B///GEMS-67 and 201101_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67, was possibly not a good choice as they
generally did not yield well relative to the amylomaize checks. In fact, only one
exceeded them, that being FS8A(S):S09-362-1-B///GEMS-67 x SCR01:N1310-358-1-B-B///GEMS67. Hybrids repeated from previous yield trials did well since they exceeded the checks.
TWT
STAND
RT LDG
MOIST
Pedigree
SKLDG
Table 2. Yield and agronomic traits for experimental GEM x GEM
amylomaize VII hybrids in Ames, IA in 2007
66.3
24.8
49.4
43.5
2.5
0.0
66.4
29.8
44.3
50.0
3.0
0.0
77.1
21.2
45.1
59.0
4.5
0.0
80.9
27.3
49.7
50.0
8.0
1.0
81.2
28.3
46.9
49.5
2.0
0.5
81.6
27.4
48.5
52.5
9.5
0.5
84.5
25.3
47.5
37.0
2.5
0.5
84.7
23.2
50.3
43.0
2.0
0.0
86.3
26.0
50.4
47.0
3.0
0.0
89.1
24.0
48.9
57.5
2.5
0.0
90.9
28.1
48.4
57.0
12.0
0.0
94.4
95.2
26.5
28.4
50.5
50.5
52.0
54.0
4.5
6.0
1.0
2.5
Yield
bu/ac
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
AR16035:S02-615-1-B-B///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
UR13085:N0215-14-1-B///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
AR03056:N09-182-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
SCR01:N1310-358-1-B-B///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
CUBA164:S2012-444-1-B///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
UR10001:S1813-257-1///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
CH05015:N15-3-1-B-B///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
UR13085:N0215-14-1-B///GEMS-67 x
7
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
98.7
25.3
48.7
44.0
3.0
0.0
98.8
25.1
50.2
52.5
4.0
0.0
99.2
27.5
50.6
51.0
4.5
0.0
100.5
27.2
48.5
56.5
3.5
2.0
103.1
25.5
46.9
46.0
2.5
0.0
107.8
28.6
50.5
39.5
4.0
0.0
109.2
20.4
47.0
53.0
2.0
0.5
109.6
29.1
47.8
53.0
3.5
1.5
110.6
27.5
48.4
51.5
5.0
0.0
112.5
26.6
46.8
47.0
7.0
0.5
113.8
28.2
47.8
46.5
2.0
0.5
119.6
26.5
49.3
57.0
1.5
1.0
120.9
25.7
49.6
52.0
9.5
0.0
121.8
122.5
26.9
21.9
49.4
50.0
56.5
57.5
5.0
2.0
0.0
0.0
122.8
123.6
26.3
21.7
49.9
50.1
43.5
58.5
2.0
5.5
0.0
0.0
124.0
22.9
49.4
52.0
2.5
0.0
125.8
26.6
49.6
38.0
2.0
1.0
CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
CH05015:N1502-86-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
AR16035:S02-615-1-B-B///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
UR11003:S0302-1011-1-B-B////(CUBA164:S2012-444-1-B///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67 x
DKXL370:N11a20-36-2-B///GEMS-67
AR03056:N09-191-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
AR03056:N09-182-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
DREP150:N2011d-624-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
AR03056:N09-191-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67 x
SCR01:N1310-358-1-B-B///GEMS-67
FS8B(T):N11a-110-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
amylomaize VII check 1
BARBGP2:N08a18-332-1-B-B////DKXL370:N11a20-31-1-B-B-B///GEMS-67 x
CUBA164:S2012-444-1-B-B-sib////AR16035:S02-615-1-B-B///GEMS-67
amylomaize VII check 2
FS8A(S):S09-362-1-B///GEMS-67 x
(SCR01:N1310-358-1-B-B///GEMS-67
DK212T:N11a12-191-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
8
DXKL370:N11a20-322-1-B-B////DKXL370:N11a20-31-1-B-B-S///GEMS-67 x
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
125.9
26.1
50.4
56.5
5.5
0.0
129.7
26.2
51.3
54.0
1.5
1.0
130.8
28.6
51.3
58.5
6.0
0.0
133.1
27.0
49.3
55.5
6.5
0.0
143.0
25.9
48.9
59.0
8.5
0.0
144.6
25.9
51.7
55.5
4.0
1.0
149.3
200.4
25.4
22.9
51.3
54.8
54.0
53.0
2.0
1.0
0.0
0.0
20.4
29.8
25.9
26.4
44.3
54.8
49.2
2.7
37.0
59.0
51.3
2.2
1.0
12.0
4.2
7.5
DXKL370:N11a20-322-1-B-B////DKXL370:N11a20-31-1-B-B-SB///GEMS-67
x CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
DKXL370:N11a20-199-2-B-B-B////DKXL370:N11a20-31-1-SIB///GEMS-67 x
CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
DXKL370:N11a20-322-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
x CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
DKXL370:N11a20-199-2-B-B-B////DKXL370:N11a20-31-1-B-B-///GEMS-67 x
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
CH05015:N1502-86-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
FS8B(T):N11a-87-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67 x
CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
normal hybrid check (Agrigold 6479 , 112D)
Min
Max
Mean
LSD
Source
Genotype
Block
ANOVA – yield trial Ames, Iowa
Plot
Root
Stalk
TWT YLD
Wt.
lodge
lodge
**
**
**
*
ns
ns
ns
ns
ns
*
Stand
**
ns
Moist
**
ns
Lines were evaluated for general combining ability by determining the mean
value for traits over all hybrids made using a specific line (Table 3). Since the yield trial
predominantly used the two testers, the comparison of lines is not very balanced.
However, certain lines did show especially promising yield potential as a result of their
good representation in the field. Among NS lines, DKXL370:N11a20-36-2-B///GEMS-67
performed well in nine hybrids. Also, FS8B(T):N11a-87-1-B-B////DKXL370:N11a20-31-1-B-BSIB///GEMS-67 performed exceptionally well, although this was from the average of only
two hybrids. Among SS lines, two performed well: CHIS775:S1911b-120-1-B-BB////AR16035:S02-615-1-B-B///GEMS-67 and CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-BB///GEMS-67, both of which occurred in 5 different hybrids. For all the above mentioned
lines that appeared to have good combining ability, their moisture contents were
substantially higher than the checks.
9
0.0
2.5
0.4
5.7
0.0
0.0
0.0
1.2
To conclude, two lines of interest, DKXL370:N11a20-199-2-B-B-B////DKXL370:N11a2031-1-B-B-///GEMS-67 and CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67,
should be considered for use as testers in the future.
10
Table 3. General combining ability for lines used in 2007 yield trial in Ames, IA
SS
n
Yield
(bu/ac)
MOIST
TWT
STAND
SKLDG
RTLDG
AR16035:S02-615-1-B-B///GEMS-67
UR10001:S1813-257-1///GEMS-67
CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEMS-67
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
UR11003:S0302-1011-1-B-B////(CUBA164:S2012-444-1-B///GEMS-67
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
(FS8A(S):S09-362-1-B///GEMS-67
2
1
1
17
4
1
5
5
1
84.7
86.3
98.8
99.4
108.3
109.2
121.1
123.4
124.0
27.7
26.0
25.1
27.0
25.6
20.4
25.8
25.9
22.9
45.6
50.4
50.2
49.0
47.4
47.0
50.0
50.8
49.4
48.0
47.0
52.5
50.9
53.3
53.0
50.5
50.6
52.0
2.8
3.0
4.0
4.9
5.8
2.0
4.5
2.7
2.5
0.0
0.0
0.0
0.6
0.1
0.5
0.4
0.2
0.0
NS
n
Yield
(bu/ac)
MOIST
TWT
STAND
SKLDG
RTLDG
(UR13085:N0215-14-1-B///GEMS-67
(SCR01:N1310-358-1-B-B///GEMS-67
CH05015:N15-3-1-B-B///GEMS-67
AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DREP150:N2011d-624-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DKXL370:N11a20-36-2-B///GEMS-67
BARBGP2:N08a18-332-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DK212T:N11a12-191-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
FS8B(T):N11a-87-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
2
12
4
5
1
9
1
1
2
88.1
93.0
108.2
106.4
113.8
115.9
122.8
125.8
135.5
27.8
24.7
25.6
27.3
28.2
27.4
26.3
26.6
26.2
50.1
48.4
49.4
48.7
47.8
49.4
49.9
49.6
50.3
52.0
48.9
53.4
49.5
46.5
54.9
43.5
38.0
55.3
7.0
3.5
3.3
5.4
2.0
5.6
2.0
2.0
3.5
1.8
0.2
0.8
0.4
0.5
0.3
0.0
1.0
0.0
amylomaize VII check 1
amylomaize VII check 2
normal hybrid (asgrow) check
1
1
1
122.5
123.6
200.4
21.9
21.7
22.9
50.0
50.1
54.8
57.5
58.5
53.0
2.0
5.5
1.0
0.0
0.0
0.0
11
Generation Means Analysis
We have obtained additional information
from this study that we intend to submit
a manuscript of this coming winter.
Again, the study was done in order to
better understand the inheritance of the
high amylose modifier gene(s) using a
generation means analysis (GMA)
model, which takes advantage of the fact
that starch is under triploid genetic
control. Genetic material for this study
was made using the two parents H99ae
and GEMS-0067, which have amylose
content of around 55% and 70%
respectively. The material was
generated as shown in Figure 6.
Figure 6. Self and reciprocal
crossings between H99ae (H) and
GEMS-0067 (G) and the F1 were
made in order to develop genetic
material for the generation means
analysis study.
Data regarding additional grain characteristics from the material and values of
these are shown in Table 4. Total starch content values were variable and no linear trend
with the presence of the modifier(s) was observed. However, the selfed GEMS-0067 did
have the lowest value, suggesting that the modifiers raise amylose levels at the expense
of total starch. Resistant starch levels were also somewhat variable; however, a linear
trend definitely occurred upon increasing the presence of the modifier(s) with the greatest
resistant starch value found to be associated with GEMS-0067. The modifier(s) have a
profound influence on this trait nearly doubling the value compared to genotypes with the
ae allele alone.
Light microscopy images were taken of granules from each of the nine
generations, four of which are shown in Figure 7, which include the two selfs and two
reciprocal crosses. The images are of phase contrast and of polarizing light to observe
the birefringence (Figure 8). Birefringence refers to the light bending property of the
crystalline structure within the granule. It is determined by placing polarizing films
above and below the granules, which are offset from each other by 90o. Normally light
will not pass the two filers when positioned in this fashion. If light is rotated by a
substance, such as the crystalline granule, it will be able to pass both filters and when it
does it will typically form the classic ‘maltese cross’ within each granule. Birefringence,
12
therefore, is a way to compare starches for crystallinity which may influence other
properties. Table 4 shows the proportion of granules with weak or no birefringence as a
percent of the total. Weakest birefringence occurred GEM-0067 and GEMS-0067 when
crossed by the F1, suggesting that modifiers play a role in reducing crystallinity within
the granule. The reduced birefringence might be due to the irregularly shaped or
filamentous granules. Images were analyzed using the program Image-J and evaluated
for granule area, perimeter and circularity. In all cases, the lowest value occurred with
GEM-0067 with a trend toward increasing values as the presence of modifiers was
reduced.
Table 4. Total starch, resistant starch and proportion of granules with
weak birefringence for nine generations of a Generation Means
Analysis (G-GEMS-67, H – H99ae, F1 – GEMS-67/H99ae).
Generation
Total starch content (%)+
s.e. (n = 3 ears)
Resistant starch
content (%)
Proportion of granules
with weak birefringence
(%)
G/G
59.3+ 1.5
35.0  0.5
27.2
G/F1
63.4+0.6
29.5  0.8
26.9
G/H
65.1+0.7
32.0  1.4
8
F1/G
61.4+1.3
28.1  0.8
13.5
F1/F1
62.5+1.0
28.2  0.7
10.7
F1/H
68.8+2.2
12.9  0.2
6.1
H/G
62.8+1.0
29.4  0.7
9.7
64.1+1.3
64.7_0.1
18.4  1.3
8
6
H/F1
H/H
15.7  0.2
13
Figure 7. Perimeter, circularity and area values determined from the
nine generations using Image-J (G-GEMS-67, H – H99ae, F1 – GEMS67/H99ae).
0.85
0.05
0.80
pixals
0.04
0.75
0.03
pe ri mete r
0.70
0.02
ci rcul a ri ty
0.65
4  (area/perimeter 2)
0.06
0.01
a re a
H/H
H/F1
H/G
F1/H
F1/F1
F1/G
G/H
G/F1
0
G/G
0.60
generation
Figure 8. Phase contrast and polarized light images of self and reciprocals of H99ae
and GEMS-0067
G/G
G/G
14
G/H
G/H
H/G
H/G
H/H
H/H
15
16
Total Starch Analysis of GEM lines
A study was conducted to evaluate a method for determining starch content of
corn and to apply it to new GEM amylomaize VII lines (Figure 9). The method
generally involved pre-treatment with DMSO, reaction with amylase enzyme and
determination of glucose using a YSI instrument based on immobilized glucose-oxidase
enzyme technology. We tested the method on pure starch samples in order to establish if
results would be influenced by amylose content. Due to the resistant nature of the high
amylose genotypes, incomplete hydrolysis resulted in only a slight decrease in total
starch compared to normal starch. More importantly, no difference was observed
between starches possessing 55% amylose compared to 70% amylose. We therefore can
conclude that our results with grain samples should not be influenced by variation in
amylose content among our ae genotypes.
Total Starch %
Figure 9. Total starch analysis for standard starch samples obtain from Cargill
(Hammond,IN).
110
100
90
80
70
60
50
40
25%
55%
Starch Standard
70%
Another experiment was conducted to investigate the amount of variation within a
small population of F4 ears derived from the cross between H99ae and GEMS-0067
(Figure 10). The objective was primarily to determine repeatability of our analysis using
three sub-samples from single ground samples derived from one ear. We did not measure
genetic diversity within this population since we tested unreplicated ears. There was
found to be only a 6% spread (max-min) in total starch values. However, significant
difference was found suggesting that our method was robust. Total starch values from
this study were then compared to amylose values in order to determine if increasing
amylose was done at the expense of total starch (Figure 11). Lowest total starch values
were found generally with samples of the highest amylose values, although one sample of
relatively high amylose deviated greatly from this trend.
17
Figure 10. Variation among F4 progeny ears from the cross between
H99 and GEMS-0067.
Total Starch % of grain
68
66
64
62
60
58
56
54
52
F4/306
F4/603
F4/183
F4/46
F4/84
F4/43
F4/51
F4/329
F4/100
F4/36
F4/507
F4/608
F4/324
F4/201
F4/80
F4/J6
F4/27
F4/228
50
F4 ear source derived from H99 x GEMS-0067
Total starch % of grain
Figure 11. Scatter plot comparing amylose values and total starch
values from 18 F4 progeny ears from H99ae and GEMS-0067.
66
65
R2 = 0.0109, ns
y = -0.0155x + 62.942
64
63
62
61
60
59
58
0
20
40
60
80
100
Amylose % of starch
18
Table 5. Total starch values of thirty-one individual ears determined from three subsample for GEM lines
having amylose values in the 60 and 70 percent range.
group
Mean total
starch value of
triplicate
determinations
S.D.
AR03056:N09-191-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
AR03056:N09-250-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
ns
ns
58.4
59.6
0.06
0.15
AR03056:N09-24-1-B-B-B////(CH05015:N15-3-1-B-B///GEMS-67
AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
AR03056:N09-182-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
ns
ns
ns
59.9
61.3
63.0
0.21
0.00
0.06
AR03056:N09-182-1-B-B-B////(CH05015:N15-3-1-B-B///GEMS-67
CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
ns
ns
ns
64.7
56.1
60.7
0.06
0.10
0.21
CH05015:N1204-57-1-B-B////(CH05015:N15-3-1-B-B///GEMS-67
ns
65.2
0.06
DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DKXL370:N11a20-234-2-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
ns
ns
ns
55.9
61.4
67.1
0.20
0.26
0.06
DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DREP150:N2011d-624-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
FS8B(T):N11a-322-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
(H99 ae /// GEMS-67
ns
ns
ns
ns
70.7
63.9
54.3
62.6
0.10
0.10
0.10
0.06
(H99 ae //// H99 ae /// GEMS-67
(SCR01:N1310-358-1-B-B///GEMS-67
(UR13085:N0215-14-1-B///GEMS-67
(UR13085:N0215-14-1-B///GEMS-67
ns
ns
ns
ns
70.6
67.5
64.8
66.3
0.00
0.10
0.06
0.06
Ear sample (F4)
Heterotic
19
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-671-2-1) - 1X
ss
72.3
0.06
AR16035:S02-615-1-B-B///GEMS-67
AR16035:S02-615-1-B-B///GEMS-67
CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEMS-67
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
ss
ss
ss
ss
ss
59.0
69.4
60.0
61.0
55.4
0.06
0.12
0.12
0.11
0.06
CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
ss
ss
57.2
62.2
0.06
0.06
DKB844:S1601-73-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
ss
61.2
0.15
(FS8A(S):S09-362-1-B///GEMS-67 2-1-1 ) - 1
UR10001:S1813-257-1///GEMS-677-2-2-1) - 1
ss
ss
64.8
68.6
54.3
72.3
62.7
0.11
0.12
Min
Max
mean
Source
DF
SS
MS
F
P
Inbred F5 Ears (E)
ss vs. ns
Subsample within ear
Error
30
1
62
186
6197.32
1.38
846.28
19.98
206.57
1.38
13.65
0.107
15.13
0.39
128.1
0.0000
0.5300
0.0000
20
Total starch of grain (%)
Figure 12. Scatter plot comparing total starch and amylose of 25 GEM
lines.
80
70
60
50
Spearman's rank correlation ( rs ) = -0.04
40
30
20
0
20
40
60
80
Starch amylose (%)
Total starch values for a group of 31 ears from GEM lines that are in the process
of being selected for high amylose modifiers are shown in Table 5. The spread in the
values was found to be much larger (max-min=18%) compared to the intra-population
variation in the H99ae x GEMS-0067set, possibly because of the diverse genetic
background. Sub-sample values were very repeatable and the data suggests that our
method will be very useful in screening for total starch in new lines and possibly for
NIRS calibration development. Figure 12 revealed that little correlation occurred
between amylose and total starch suggesting that the effect of background could be useful
in minimizing the total starch reduction effect of modifier(s).
21