2011 GEM Report: Truman State University,
Annual GEM Cooperator meeting, ASTA, Dec. 7, Chicago, IL
Development and evaluation of specialty starch germplasm utilizing GEM
biodiversity to optimize grain quality, composition, and yield.
Duration 2011 – 2015
Mark Campbell, Avinash Karn, Marianne Emery, Akriti Panthi, Jessica Issleib, Elizabeth Vanover,
Jillian Burke and Emily Mauch
General Objectives: In our project, genetic materials from the GEM program have been used to
demonstrate the value of exotic maize germplasm by developing parent lines and hybrids with altered starch
traits that target current trends in industry and consumer interests. Until now, all of our germplasm
developed at Truman has relied exclusively on materials from GEM which include breeding crosses composed
of either 25% or 50% exotic germplasm, of primarily of Latin American origin, with proprietary lines from
private cooperators (1). Using this material, our efforts have focused on developing two distinct hybrid types
with unique starch properties. The first includes high-amylose corn which has a number of niche
applications, one of which includes its use as a source of resistant starch (RS) having prebiotic properties with
well documented health benefits and functional attributes making it well suited as a dietary supplement in
food fortification (2). The second starch type involves developing a commercially viable source of a slowly
digested starch (SDS) from native (unmodified) starch which has been studied for its potential applications in
controlling blood sugar (3). Although a number of applications exist, our emphasis is towards an SDS starch
having a combination of a lowered glycemic-index (GI) and RS content for a slowed release of sugar into the
blood that could be used therapeutically for diabetic and glycogen disorder patients suffering from nocturnal
hypoglycemia. Already, GEM materials have provided a source of unique alleles altering starch for these
applications. The diversity of materials also provides an ideal opportunity to identify genetic backgrounds to
overcome decreased yield and grain quality associated with mutations altering starch structure. In addition
to several classic starch mutations including waxy (wx) and amylose-extender (ae) a novel allele at the starch
branching enzyme 1 (sbe1) exists in our material we refer to as (sbe1::gems67) originating from GEMS-0067
are in our lines. Current grain prices and premiums on specialty grains require these improvements so that
production, milling and marketing can be economically viable. Specific objectives include:
1. Develop and examine the chemical and physical properties of inbreds and hybrids for the RS starches
including Amylomaize class V (ae), class VII (ae sbe1::gems67) for future release.
2. Identify other genetic factors that influence starch properties and grain quality of ae or the combination
of ae and sbe1::gems67
a. Improve marker assisted selection for recovery of our major high-amylose modifier (sbe1::gems67) and
confirm other candidate modifiers whose existence, role and importance requires further study
including sbe1b and sbe2a
b. Evaluate variations in endosperm protein quantity, quality and their spatial distribution which may
influence grain texture and expression of ae sbe1::gems67 and other genotypes under investigation.
3. Examine properties of SDS starches from lines derived from GEM germplasm possessing the double- and
triple- mutant combinations of both ae wx and ae wx sbe1::gems67 genotypes. Specifically amylopectin
fine structure and rate of hydrolysis will be studied using a number of methods including apparent
amylose from the amylopectin-iodine complex, chromatographic separation of de-branched starch and
rate of hydrolysis determined both in vitro and in vivo.
4. Investigate allelic variation at the sbe2a locus (Bin 8.06) within GEM and other exotic maize germplasm
using a proposed gene-centered approach designed for screening allelic diversity within germplasm
called MAGIC (mutant-assisted gene identification and characterization) (4).
Line and Hybrid Development: Table 1 lists candidate parent lines for future release currently in
our program. Lines, having been originate from GEMS-0067, have been developed overtime by
crossing onto either newly released stiff-stalk (SS) or Non-Stiff Stalk (NS) GEM release to preserve
their heterotic identify. Development of this large set derived from the common ancestor was
required for subsequent line development and yield testing of inbreds having common modifying
alleles for full expression of the high amylose trait. Also, an absence any Amylomaize lines in the
public domain limited hybrid evaluation of our materials and usefulness to the public. The
pedigrees do not indicate the number of self-, sib- or bulking for each generation following a
cross. In general, however, F1 plants were selfed, segregating ae F2 kernels were visually
identified in the F1 ear. These ae F2 kernels resulted in plants that were selfed to produced ears
having only ae F3 kernels. This seed was planted ear-to-row and F3 plants were again selfed to
produce F3 ears. Approximately 10 kernels per ear were sampled for a single starch extraction
and assayed using a standard amylose-iodine colorimetric technique described by Williams et al
(5). Several private GEM cooperators generously assisted in providing winter and summer
nursery space to expedite inbreeding. Amylomaize inbreds were also selected during inbreeding
for traits including visually acceptable kernel quality, lack of ear rots and vigor of plant.
A group of public inbreds including H99ae and
OH43ae were grown and selfed out each
season to provide a reference ae genotype
lacking any modifier that would results in
amylomaize V (~55% amylose) starch. Many
of these public lines, incidentally, performed
poorly in seasons especially with above
average precipitation these produced weak
plants with little or no seed. These inbreds
were the result of littlef adaptation, perhaps,
to the environment in Northern Missouri
where silt/clay-rich soils having poor drainage
tend to dominate (Fig. 1). The GEM materials
developed in this region might also be of
importance for breeders considering
adaptability to water logged soil. Additionally
it should be noted that this breeding material
is never subjected to routine
fungicide/insecticide seed treatments and, by
its very nature, possesses endosperm
mutations.
Figure 1. Missouri Major Land Resource
Areas. http://soils.missouri.edu/maps/mlra.asp#109
Table 1. Amylomaize lines of both SS and NSS heterotic groups existing in varying degrees of
inbreeding (>F3) grown in summer of 2011 selected, but not all confirmed, for ae and sbe1::gems67 .
Additionally, chromatographic amylose determination size exclusion HPLC, In need of GEMS or GEMSN
identifiers.
GEMS-0067 = GUAT209:S13 X (OH43ae X H99ae) = GUAT209:S13//(OH43ae/ H99ae)
Chronological sequence of crossing: /// - first crossing from GEMS-0067, //// second crossing…etc
SS Amylomaize Inbreds No.
AR16035:S02-615-1-B-B///GEMS-67
2011-01_SE32_S17_F2S4_9148/////CHIS775:S1911b-120-1-B-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
2011-01_SE32_S17_F2S4_9148-Blk22/00/////CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
DKB844:S1601-073-001-B-B-B-B-B/////CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS-67
2011-01_SE32_S17_F2S4_9148-Blk22/00/////DKB844:S1601-73-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
BR105:S1612-008-001-B-B/////DKB844:S1601-73-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
DKB844:S1601-73-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
2088-01_DK212T_S11_F2S4_9157-Blk29/00-sib-B-B-B/////DKB844:S1601-73-1-B-B-B////AR16035:S02-615-1-B-B///GEMS-67
2011-01_SE32_S17_F2S4_9148-Blk22/00////AR16035:S02-615-1-B-B///GEMS-67
CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEMS-67
CHIS775:S1911b-120-001-B-B-B-B/////CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEMS-67
DKB844:S1601-073-001-B-B-B-B-B/////CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEMS-67
GUAT209:S13 08a-120-001-B-B////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
CHIS775:S1911b-120-001-B-B-B-B////2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
2011-01_SE32_S17_F2S4_9148-Blk22/00////2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
CUBA164:S2012-444-001-B-B-Sib////2011-01_SE32_S17_F2S4_9148-Blk22/00-sib/// GEMS-67
CHIS740:S11411a-783-002-b-b-b////2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
CUBA164:S2012-444-1-B///GEMS-67
CHRIS775:S1911b-120-1-B-B-B////CUBA164:S2012-444-1-B///GEMS-67
CUBA164:S1511b-325-001-B-B-B-B-B-Sib/////CHRIS775:S1911b-120-1-B-B-B//// CUBA164:S2012-444-1-B///GEMS-67
2011-01_SE32_S17_F2S4_9148-Blk22/00////CUBA164:S2012-444-1-B///GEMS-67
FS8A(S):S09-362-1-B///GEMS-67
2011-01_SE32_S17_F2S4_9148-Blk22/00////FS8A(S):S09-362-1-B///GEMS-67
CHIS740:S11411a-783-002-b-b-b/////CUBA164:S1511b-325-1-B-B////FS8A(S):S09-362-1-B///GEMS-67
BVIR155:S2012-029-001-B-B////UR10001:S1813-257-1///GEMS-67
CHIS740:S11411a-783-002-b-b-b////UR10001:S1813-257-1///GEMS-67
DKB844:S1601-073-001-B-B-B-B-B////UR10001:S1813-257-1///GEMS-67
UR10001:S1813-257-1///GEMS-67
2011-01_SE32_S17_F2S4_9148-Blk22/00////UR10001:S1813-257-1///GEMS-67
UR11003:S0302-1011-001-b-b-b////UR10001:S1813-257-1///GEMS-67
BR51675:N0620-033-001////UR10001:S1813-257-1///GEMS-67
BR52060:S0210-143-001-b-b-b////UR10001:S1813-257-1///GEMS-67
GUAT209:S13 08a-120-001-B-B////UR10001:S1813-257-1///GEMS-67
CUBA164:S2008c-289-001-B-B/////AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
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NS Amylomaize Inbreds No.
DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
9353-01/97_DK888N11F2S3_7451-17-b-b/////CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
BARBGP2:N08a18-332-001-B-B-B/////CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DK212T:N11a12-191-001-B-B-B/////CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
FS8B(T):N11a-087-001-b-b-sib-b-b/////CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
CH05015:N1502-086-001-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DKXL370:N11a20-199-002-B-B-B-Sib/////CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
AR03056:N09-191-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
AR03056:N09-24-1-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
AR03056:N09-182-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
CHO5015:N1204-057-001-b-b-b/////AR03056:N09-182-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
CHO5015:N1204-057-001-b-b-b/////AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
CHO5015:N1204-057-001-b-b-b/////AR03056:N09-250-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DKXL370:N11a20-199-002-B-B-B-Sib/////AR03056:N09-182-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
BR51675:N0620-033-001/////AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
AR03056:N09-191-001-B-B-B-Sib/////DKXL370:N11a20-234-2-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
CL-G1607(CML420):N11-008-001-007/////DKXL370:N11a20-234-2-B-B-B////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
MDI022:N21-B-002-003///// DKXL370:N11a20-234-2-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DK212T:N11a12-191-001-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
CH0515:N1502-086-001-b-b-b/////UR13085:N0215-14-1-B-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
DK888:N11-B-027-001-B-001/////DREP150:N2011d-624-1-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
FS8B T):N11a-322-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
UR13085:N0215-14-1-B///GEMS-67
CHO5015:N1204-057-001-b-b-b/////UR13085:N0215-14-1-B///GEMS-67
9353-01/97_DK888N11F2S3_7451-17-b-b////UR13085:N0215-14-1-B///GEMS-67
BARBGP2:N08a18-332-001-b-b-b/////UR13085:N0215-14-1-B///GEMS-67
BR51675:N0620-033-001////UR13085:N0215-14-1-B///GEMS-67
CL-G1607(CML420):N11-008-001-007////UR13085:N0215-14-1-B///GEMS-67
CH0515:N1502-086-001-b-b-b////UR10001:S1813-257-1///GEMS-67
CH05015:N15-3-1-B-B///GEMS-67
CH05015:N1204-57-1-B-B////CH05015:N15-3-1-B-B///GEMS-67
AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS-67
AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEMS-67
BR51403(PE001):N16-B-044-004-001-002G-003/////AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEMS-67
CH05015:N1502-086-001-B-B-B/////AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS-67
CH05015:N1502-086-001-B-B-B/////AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEMS-67
DK212T:N11a12-191-001-B-B-B/////AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS-67
DK212T:N11a12-191-001-B-B-B/////AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEMS-67
DKXL370:N11a20-199-002-B-B-B-Sib/////AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEMS-67
AR03056:N09-191-001-B-B-B-Sib/////AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS-67
BR51403(PE001):N16-B-044-004-001-001C-001////CH05015:N15-3-1-B-B///GEMS-67
BR51721(RN07):N20-B-017-002////CH05015:N15-3-1-B-B///GEMS-67
CH05015:N1502-086-001-B-B-B////FS8A(S):S09-362-1-B///GEMS-67
DKXL370:N11a20-199-002-B-B-B-Sib////FS8A(S):S09-362-1-B///GEMS-67
DKXL380:N11-B-007-010-B-002/////CHRIS775:S1911b-120-1-B-B-B////CUBA164:S2012-444-1-B///GEMS-67
SCR01:N1310-358-1-B-B///GEMS-67
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Yield evaluations of amylomaize GEMN by GEMS entries that were planted in the summer of 2010 in Ames, IA
were again evaluated in the summer of 2011.Ames. Seed was produced in a winter nursery and most entries
included either AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67 as a NS tester or
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS67 as a SS tester because of their superior
performance in previous yield trials. Yields were much greater overall in 2011 (128.1 Bu/Ac ) compared to 2010
(98.9 Bu/Ac). The top yielding Amylomaize hybrid in both 2010 (150.65 Bu/Ac) and 2011 (170.2 Bu/Ac) was
observed for the hybrid CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS67 X AR03056:N09-191001-B-B-B-Sib/////AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67.
The proprietary Amylomiaze check hybrids displayed
the best dry down with moisture percentages near
13%, while the top GEM hybrid was at 19% which
was slightly below relative to other GEMN x GEMS
amylomaize hybrid. Although not recorded, the
GEMS x GEMN hybrids may likely be later in maturity
resulting from their tropical background. Although the
genetic mechanism of the elevated amylose of the
checks is not known, another consideration might be
differences in carbohydrates metabolism in the plant
influencing the quantity of free sugars and thus, water
potential.
Figure 2. Design of a partial diallel experiment grown near Kirksville, MO for which data is currently
is being collected and processed.
A yield trial including entries made up in a partial diallel of GEMS x GEMN amylomaize line was grown
near Kirksville during the summer of 2011 (Fig. 2). Partial flooding in the spring resulted in a portion of
the experiment being severely stunted. Yield data collection is nearly complete since the experiment
has been hand harvested and. As mentioned earlier data from this study and future studies may
increase prioritizing performance in Missouri and may provide useful insight into the environmental
challenges in the heavy loess soil and clay pan region. Additionally, the origin of germplasm
collections are currently being used in studying the diversity of soils in Latin America and its role in
microclimate adaptation.
Fig. 3 Distribution of silt deposits in the US and
collection locations of accessions used in GEM
breeding crosses used in our progra. Predicted silt
(%) content in the whole soil profile.
http://spatial-analyst.net/wiki/index.php?title=Interpolation_of_ISRICWISE_international_soil_profile_data
Table 2. Pooled yield and agronomic performance of GEMN x GEMS amylomaize hybrids grown in Ames, IA
2011 and 2012.
NS GEM amylomaize VII Tester
AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
CHRIS775:S1911b-120-1-B-B-B////CUBA164:S2012-444-1-B///GEMS67
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS67
UR10001:S1813-257-1///GEMS67
CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEMS67
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS67
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS67
CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEMS67
DKB844:S1601-73-1-B-B-B////AR16035:S02-615-1-B-B///GEM67
AR16035:S02-615-1-B-B/GEMS67
CUBA164:S2012-444-1-B///GEMS67
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS67
CUBA164:S2012-444-001-B-B-Sib////2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS67
CHIS775:S1911b-120-001-B-B-B-B////2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS67
GUAT209:S1308a-120-001-B-B////UR10001:S1813-257-1///GEMS67
Wt
Lb.
Yield
(bu/ac)
MOIST
Y/M
%
TWT
lb/bu
Stand
Skldg Rtldg
%
%
%
13.1
103.5
17.7
6.1
57.3
64.3
8.3
8.0
12.9
99.4
20.2
5.0
55.4
81.2
9.6
3.6
15.1
115.6
20.3
5.9
54.7
83.6
3.8
1.9
14.5
113.5
19.2
6.0
56.3
71.6
9.2
8.7
13.3
102.6
20.4
5.1
53.7
68.5
9.8
4.5
10.4
80.3
19.5
4.3
53.6
58.1
19.4
2.0
6.0
49.0
19.0
2.5
38.7
61.3
21.2
1.9
16.1
123.6
20.1
6.4
55.2
76.4
11.9
7.2
14.7
110.0
22.4
5.0
54.2
78.0
5.0
2.9
14.2
107.2
21.6
5.1
56.7
74.3
5.7
2.2
16.8
131.0
19.5
6.8
56.0
71.6
3.6
5.0
15.6
121.3
19.8
6.2
55.5
80.9
11.2
1.4
14.9
117.5
18.5
6.5
57.1
79.3
17.0
5.0
17.4
134.8
20.2
6.7
55.0
76.3
4.5
4.3
110.8
19.7
5.7
55.3
68.4
13.3
1.7
109.2
20.9
5.4
55.4
78.8
11.5
5.4
105.2
16.7
6.4
55.8
82.6
18.5
1.8
DKB844:S1601-073-001-B-B-B-B-B/////CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B14.3
B///GEMS67
2088-01_DK212T_S11_F2S4_9157-Blk29/00-sib-B-B-B/////DKB844:S1601-73-1-B-B-B////AR16035:S0214.3
615-1-B-B///GEMS67
GUAT209:S1308a-047-001-B-B/////CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEMS67
13.2
SS GEM amylomaize VII Tester
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS67
Wt
Lb.
Yield
(bu/ac)
MOIST
Y/M
%
TWT
lb/bu
Stand
Skldg Rtldg
%
%
%
AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
11.5
90.9
18.6
4.9
53.9
53.5
6.4
6.6
AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
15.6
118.8
21.0
5.8
54.3
82.3
10.3
12.1
CH05015:N1204-57-1-B-B////CH05015:N15-3-1-B-B///GEMS67
10.8
86.2
18.4
4.7
56.3
57.0
3.2
2.5
UR13085:N0215-14-1-B///GEMS67
12.9
97.7
21.5
4.7
56.0
74.1
9.9
9.3
AR03056:N09-182-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
14.9
113.8
21.0
5.7
54.9
65.1
7.0
11.1
AR03056:N09-191-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
13.1
98.8
21.8
4.6
53.0
58.4
5.7
4.6
DKXL370:N11a20-234-2-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
18.0
137.7
21.1
6.7
55.2
76.4
7.8
11.2
FS8BT:N11a-322-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
13.0
98.0
22.0
4.6
54.9
61.6
7.6
35.0
CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
12.5
97.1
20.4
4.8
55.0
58.5
8.9
3.0
DREP150:N2011d-624-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
17.7
136.3
20.7
6.8
56.3
60.6
7.2
4.5
DKXL370:N11a20-31-1-B-B-SIB///GEMS67
14.4
109.1
21.4
5.3
55.9
74.2
5.3
5.4
UR13085:N0215-14-1-B///GEMS67
15.8
121.0
20.7
6.0
55.8
72.2
6.6
2.7
DKXL370:N11a20-31-1-B-B-SIB///GEMS67
16.0
123.1
20.5
6.1
56.2
65.5
5.0
4.2
DK212T:N11a12-191-001-B-B-B/////CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEM67
16.6
126.2
21.2
6.1
54.6
64.2
4.0
2.9
DK212T:N11a12-191-001-B-B-B/////AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEM67
19.4
151.3
19.7
7.8
55.8
75.4
7.7
4.4
AR03056:N09-191-001-B-B-B-Sib/////AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
20.6
160.4
19.6
8.4
57.1
78.3
4.4
4.4
SS GEM amylomaize VII Tester
Wt
Yield
MOIST
Y/M
TWT
Stand
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS67
Lb.
(bu/ac)
%
lb/bu
Skldg Rtldg
%
%
%
AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
13.8
106.7
19.7
5.5
54.5
78.9
8.4
7.0
CH05015:N1204-57-1-B-B////CH05015:N15-3-1-B-B///GEMS67
15.7
122.0
19.3
6.5
54.5
76.9
19.8
3.5
FS8BT:N11a-322-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
17.4
134.4
20.3
6.8
55.9
78.0
6.2
3.0
DKXL370:N11a20-31-1-B-B-SIB///GEMS67
15.7
120.2
21.0
5.9
56.7
77.7
2.7
4.6
AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
10.6
81.3
21.1
3.9
54.5
61.2
6.8
6.5
Amylomaize/normal check hybrid
Figure 4. Ears of hybrid
AMY VII Check #1
12.7
103.5
14.7
7.0
55.0
80.5
8.0
0.9
AMY VII Check #2
13.8
112.3
15.5
7.3
54.9
71.2
3.9
3.2
Asgrow normal
21.8
173.0
17.9
9.9
58.4
76.8
3.6
1.5
2010 ave
12.3
98.9
21.4
4.7
53.5
74.4
15.5
10.1
2011 ave
17.0
128.1
18.3
7.1
56.5
68.7
1.6
0.7
Entry (p-value)
0.000
0.000
0.000
0.000
0.000
0.000
0.552
0.006
Year (p-value)
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS67 X AR03056:N09-191-001-B-B-B-Sib/////AR03056:N09-182-1-B-B-
B////CH05015:N15-3-1-B-B///GEMS67 displaying good ear quality in addition to being the top yielding amylomaize hybrid in both 2010 and 2011 in Ames, IA.
Table 3. Grain physical properties from two hybrid ears taken per plot in Ames, IA 2010 yield trial.
Density
(g/mL)
Ear
wt. (g)
HKW
(g)
displacement vol
(mL)
Grit #200
separation
%
CHRIS775:S1911b-120-1-B-B-B//// CUBA164:S2012-444-1-B///GEMS67
1.3
185.3
30.6
5.7
6.2
2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS67
1.3
143.2
22.3
4.4
4.9
UR10001:S1813-257-1///GEMS67
1.4
156.2
28.9
5.1
5.0
CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEM67
1.9
181.3
29.2
3.9
5.9
CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEMS67
1.4
197.6
30.1
5.3
5.7
CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEM67
1.2
219.4
27.7
5.6
5.7
CUBA164:S2012-966-1-B-B////AR16035:S02-615-1-B-B///GEMS67
1.3
166.1
23.5
4.4
6.6
DKB844:S1601-73-1-B-B-B////AR16035:S02-615-1-B-B///GEM67
1.4
131.3
27.0
4.8
6.9
AR16035:S02-615-1-B-B/GEMS67
1.4
161.2
28.4
5.2
6.4
FS8AS:S09-362-1-B///GEMS-67
DKB844:S1601-073-001-B-B-B-B-B/////CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B///GEMS67
0.9
1.5
1.3
1.3
1.4
1.4
1.3
179.0
129.4
172.1
182.8
193.5
166.7
191.9
24.0
36.5
28.3
24.8
25.9
31.4
26.8
7.0
6.2
5.5
4.8
4.6
5.5
5.2
5.9
7.2
4.2
8.0
5.7
7.1
5.1
2088-01_DK212T_S11_F2S4_9157-Blk29/00-sib-B-B-B/////DKB844:S1601-73-1-B-B-B////AR16035:S02-615-1-BB///GEMS67
1.3
201.1
27.0
5.1
5.8
1.4
1.3
1.4
233.4
178.4
177.3
27.4
21.6
27.4
4.9
4.1
5.1
6.5
6.1
6.0
Ear wt.
156.7
HKW
27.9
displacement
5.4
Grit #200
separation
7.7
141.9
23.8
4.8
6.8
NS GEM amylomaize VII Tester
AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
CUBA164:S2012-444-1-B///GEMS67
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS67
CUBA164:S2012-444-001-B-B-Sib////2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS-67
CHIS775:S1911b-120-001-B-B-B-B////2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GEMS67
GUAT209:S1308a-120-001-B-B////UR10001:S1813-257-1///GEMS67
GUAT209:S1308a-047-001-B-B/////CHIS740:S1411a-783-2-B-B////AR16035:S02-615-1-B-B///GEMS67
BVIR155:S2012-029-001-B-B////UR10001:S1813-257-1///GEMS67
ave.
SS GEM amylomaize VII Tester
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS67
Density
1.3
AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
1.2
AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
CH05015:N1204-57-1-B-B////CH05015:N15-3-1-B-B///GEMS67
1.3
120.7
26.3
4.9
7.0
UR13085:N0215-14-1-B///GEMS67
1.4
114.8
21.2
3.7
7.6
AR03056:N09-182-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.4
158.1
27.3
5.0
6.5
DK212T:N11a12-191-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.4
150.5
26.4
4.8
7.3
DKXL370:N11a20-234-2-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS-67
1.4
171.7
30.9
5.7
6.5
FS8BT:N11a-322-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.3
185.5
27.1
5.2
7.1
CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.4
142.3
25.2
4.5
5.1
DREP150:N2011d-624-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.3
209.3
24.3
4.6
7.5
DKXL370:N11a20-31-1-B-B-SIB///GEMS67
H99ae///GEMS67
UR13085:N0215-14-1-B///GEMS67
DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.3
1.4
1.3
1.1
1.5
1.4
168.1
125.0
159.5
173.7
203.6
152.5
23.2
23.5
23.7
23.5
25.1
22.8
4.4
4.2
4.5
5.1
4.3
4.1
11.5
6.4
8.7
6.2
5.8
6.5
1.4
1.3
1.3
186.4
145.8
159.2
24.4
26.8
25.2
4.4
5.0
4.7
5.0
5.9
7.0
DK212T:N11a12-191-001-B-B-B/////CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
DK212T:N11a12-191-001-B-B-B/////AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEM67
AR03056:N09-191-001-B-B-B-Sib/////AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
AR03056:N09-182-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
ave.
Density
Ear wt.
HKW
100 K
displacem
ent
CH05015:N1204-57-1-B-B////CH05015:N15-3-1-B-B///GEM67
1.3
155.3
26.6
5.1
6.6
AR03056:N09-250-1-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.3
151.1
27.9
5.3
6.2
FS8BT:N11a-322-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.3
170.2
29.0
5.7
5.8
DREP150:N2011d-624-1-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.4
210.4
27.2
5.0
4.9
DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.4
173.6
29.2
5.1
7.4
UR13085:N0215-14-1-B///GEMS67
1.3
176.0
26.1
4.9
6.6
DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.4
161.6
25.6
4.5
6.1
AR03056:N09-24-1-B-B-B////CH05015:N15-3-1-B-B///GEMS67
1.2
174.1
27.4
5.6
4.8
UR13085:N0215-14-1-B///GEMS67
1.4
225.3
25.6
4.6
7.1
DKXL370:N11a20-234-2-B-B-B////DKXL370:N11a20-31-1-B-B-SIB///GEMS67
1.4
160.7
24.7
4.4
6.6
ave.
1.4
175.8
26.9
6.2
Density
Ear wt.
HKW
5.0
100 K
displacem
ent
Grit #200
separation
AMY VII Check #1
1.3
124.9
23.2
4.3
7.2
AMY VII Check #2
1.4
195.5
26.4
4.9
11.1
Normal Check
1.3
223.8
26.2
5.1
12.6
ave.
1.3
181.4
25.3
4.8
10.3
overall ave.
1.4
170.8
26.4
4.9
6.6
0.000
0.002
0.002
0.031
0.695
0.491
0.437
0.363
SS GEM amylomaize VII Tester
CUBA164:S1511b-325-1-B-B////AR16035:S02-615-1-B-B/// GEMS67
Amylomaize/normal check hybrid
Entry (p-value) 0.028
Year (p-value) 0.971
Grit #200
separation
Grain quality and physical properties: In Table 5, measurements of grain physical were recorded in
order to determine variation which may influence wet and or dry milling. Density was measure by determining
dry wt. of 25 kernels and placing them into a graduated cylinder. The cylinder was placed on a laboratory scale
and filled with 90% ethanol until the total volume reached 40 mL. Seed density was determined and expressed
in g/mL. A method modified from that described by Noble et al (2000) was used to determine kernel breakage.
Ten kernels were placed in an electric coffee mill for approximately 5 sec. (6). The flour was weighed and
passed through a #200 grade sieve. Material passing through was weighed and expressed as a percentage of
the original flour volume. An analysis of variance demonstrated that these various physical properties varied
significantly. The normal check displayed the greatest percent of grit separated from the flour but for all other
traits did not appear unqiue. Densities, ranged from 0.9 g/mL AR03056:N09-24-1-B-B-B////DKXL370:N11a20-31-1B-B-SIB///GEMS67
1-B-B///GEMS67
SIB///GEMS67.
x
x
FS8AS:S09-362-1-B///GEMS-67 to 1.5 g/mL CUBA164:S1511b-325-1-B-B////AR16035:S02-615-
DK212T:N11a12-191-001-B-B-B/////CH05015:N1204-57-1-B-B////DKXL370:N11a20-31-1-B-B-
Grain density is often a good indicator of dry milling and suitability for food corn. In Amylomaize,
fractionation of starches can be difficult due to small granule size making sedimentation and/or centrifugation
more challenging. Dry milling may provide an ideal alternative and these measures need to be examined in the
future along with application testing.
Introgression of GEM amylomaize into highland and lowland Nepali maize germplasm
Recent studies supported by the World Health
Organization (WHO) suggest that high amylose maize
starch used in conjunction with traditional Oral
Rehydration Treatments (ORT) may improve recovery of
diarrhea outbreaks from cholera and non-cholera
sources (Ramakrishna et al, 2008). In order to develop
materials for future application that may require
affordable source of resistant starch, GEM amylomaize
VII materials are being introgressed into locally adapted
lowland and highland material using the USDA
germplasm Nepali maize collection. The collection is
also being studied in further detail in order to collect
special soil and environmental data of collection sites by
students as a class exercise in the application of soil taxonomy
and soil physical properties for soil science courses at Truman.
Fig 5B. Collection sites of Nepali maize
accessions, world regions of cholera
vulnerability and strains documented
throughout the country (Hendriksen et al, 2011).
http://mbio.asm.org/content/2/4/e00157-11.full.pdf+html
Fig 5A. Collection sites of Nepali maize accessions
and soil taxonomic maps like that showing soil
great-groups of suborders in this geographically
diverse region.
8. Zea mays subsp. mays Ames 10300 -- Ames 10300
7. Zea mays subsp. mays (TUSA RANI) -- PI 175334
Zea mays subsp. mays No. 1 -- PI 208468
4. Zea mays subsp. mays (NGAWAL) -- PI 208469
6. Zea mays subsp. mays (SANKHU) -- PI 208470
3. Zea mays subsp. mays (MAJERIPATAN) -- PI 208471
5. Zea mays subsp. mays (POKHARA) -- PI 208472
2. Zea mays subsp. mays (KATHMMANDV VALLEY) -- PI 208473
1. Zea mays subsp. mays -- PI 286440
9. Zea mays subsp. mays Nepal 1310 -- PI 511577
10. Zea mays subsp. mays Nepal 1701 -- PI 511578
11. Zea mays subsp. mays Nepal 1901 -- PI 511580
12. Zea mays subsp. mays Nepal 2402 -- PI 511581
13. Zea mays subsp. mays Nepal 2901 -- PI 511583
Table 4. Nepali recipient populations for GEM
amylomaize VII in 2011 Kirksville nursery
14. Zea mays subsp. mays Nepal 2903 -- PI 511585
15. Zea mays subsp. mays Nepal 3101 -- PI 511586
16. Zea mays subsp. mays Nepal 3403 -- PI 511587
17. Zea mays subsp. mays Nepal 3903B -- PI 511589
28. Zea mays subsp. mays Nepal 6201 -- PI 511603
18. Zea mays subsp. mays Nepal 3904B -- PI 511590
29. Zea mays subsp. mays Nepal 6202 -- PI 511604
19. Zea mays subsp. mays Nepal 3905A -- PI 511591
30. Zea mays subsp. mays Nepal 6401 -- PI 511605
20. Zea mays subsp. mays Nepal 3908 -- PI 511593
31. Zea mays subsp. mays Nepal 7102 -- PI 511606
21. Zea mays subsp. mays Nepal 4104 -- PI 511595
32. Zea mays subsp. mays Nepal 7505 -- PI 511607
23. Zea mays subsp. mays Nepal 4109 -- PI 511597
25. Zea mays subsp. mays Nepal 5501 -- PI 511600
24. Zea mays subsp. mays Nepal 4509 -- PI 511599
22. Zea mays subsp. mays Nepal 4108 -- PI 511596
26. Zea mays subsp. mays Nepal 5503 -- PI 511601
27. Zea mays subsp. mays Nepal 5603 -- PI 511602
Amylose screening of newly develop lines from GEM releases:
Although many lines have constantly expressed the amylomaize VII starch type. More recent lines and
several less recent lines have required large grow-outs of segregating families in order to verify the high
amylose trait. A number of factors can complicate this process including laboratory error,
environmental and micro-environmental effects, non-representative sampling of early lines. Situations
where recovery is difficult could also reflect novel genetic factors including additional copies of starch
branching enzymes that compensate for the presenece of sbe::gems67 . We currently are utilizing a
colorimetric method utilizing multichannel pipettes and absorbance reading with a microtiter reader. A
protocol is being refined and simplified which will help the volume of screening especially where data is
generated by undergraduates having little laboratory experience but learn quickly and suggest
improvements regularly. Table 6 is a list of inbred families grown in 2010 and evaluated for amylose. It
is exceedingly lengthy but is an important record of lines from which commercial amylomaize V, VI and
VII could be derived. Samples sent to Indiana were difficult to grow in MO with little success in
recovering modifiers in the past.
Table 5. Recovery of amylomaize VII
genotypes grown in the summer of 2010
KV 2010 :230
KV 2010 :229
KV 2010 :219
KV 2010 :134
KV 2010 :224
KV 2010 :219
KV 2010 :228
KV 2010 :231
KV 2010 :226
KV 2010 :227
KV 2010 :437
KV 2010 :116
KV 2010 :121
KV 2010 :474
KV 2010 :465
KV 2010 :468
KV 2010 :470
KV 2010 :473
KV 2010 :472
KV 2010 :471
72.4
71.7
66.0
65.5
60.5
57.5
57.5
50.6
46.5
43.6
81.4
78.8
70.3
64.5
58.0
57.2
56.3
51.6
49.7
45.5
2011-01_SE32_S17_F2S4
_9148-Blk22/00-si
///GEMS67
2088-01_DK212T_S11_
F2S4_9157-Blk29/00-sib-B-BBGEMS-0006/////
DKB844:S1601-73-1-B-B-B////
AR16035:S02-615-1-B-B///
GEMS67
AR03056:N09-182-1-B-BB////(CH05015:N15-3-1-BB///GEMS67
AR03056:N09-182-1-B-B-B
////DKXL370:N11a20-31-1-B-BSIB///GEMS67
KV 2010 :466
43.6
KV 2010 :474
42.9
KV 2010 :467
38.1
KV 2010 :464
30.3
KV 2010 :470
21.5
KV 2010 :446
59.0
KV 2010 :447
56.8
KV 2010 :445
80.7
KV 2010 :448
45.3
KV 2010 :449
36.3
KV 2010 :444
76.5
KV 2010 :380
85.8
KV 2010 :122
83.7
KV 2010 :375
78.8
KV 2010 :380
78.6
KV 2010 :375
78.6
KV 2010 :379
56.3
KV 2010 :377
22.4
KV 2010 :378
21.8
KV 2010 :376
15.3
KV 2010 :115
67.2
B////DKXL370:N11a20-31-1-B-
AR03056:N09-191-001-B-B-BSibGEMN0089/////AR03056:N09-182-1B-B-B////CH05015:N15-3-1-BB///GEMS67
AR03056:N09-191-001-B-B-BSibGEMN0089/////DKXL370:N11a20234-2-B-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
AR03056:N09-191-001-B-B-BSibGEMN0089////DKXL370:N11a20-311-B-B-SIB///GEMS67
AR03056:N09-191-1-B-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
AR03056:N09-24-1-B-B-
KV 2010 :410
71.2
KV 2010 :484
73.4
KV 2010 :120
71.2
KV 2010 :483
64.2
KV 2010 :486
63.8
KV 2010 :487
60.0
KV 2010 :485
45.3
KV 2010 :123
69.0
KV 2010 :456
48.3
KV 2010 :457
42.3
KV 2010 :442
76.2
KV 2010 :441
66.8
KV 2010 :442
58.9
KV 2010 :117
47.5
B////(CH05015:N15-3-1-BB///GEMS67
AR03056:N09-24-1-B-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
AR03056:N09-250-1-B-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
BARBGP2:N08a18-332-001-B-BBGEMN0110/////CH05015:N1204-571-B-B////DKXL370:N11a20-311-B-B-SIB///GEMS67
BVIR155:S2012-029-001-BBGEMS0175////UR10001:S1813-2571///GEMS67
CH05015:N1204-57-1-BB////(CH05015:N15-3-1-BB///GEMS67
CH05015:N1204-57-1-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
KV 2010 :190
83.6
KV 2010 :201
79.1
KV 2010 :196
76.5
KV 2010 :211
74.8
KV 2010 :189
74.3
KV 2010 :198
71.8
KV 2010 :207
70.5
KV 2010 :216
67.9
KV 2010 :199
67.8
KV 2010 :200
65.6
KV 2010 :210
64.3
KV 2010 :215
64.2
KV 2010 :205
63.3
KV 2010 :202
63.1
KV 2010 :214
62.3
KV 2010 :195
56.9
KV 2010 :203
54.5
KV 2010 :209
21.1
KV 2010 :208
15.2
KV 2010 :209
7.6
KV 2010 :304
72.0
KV 2010 :136
70.2
KV 2010 :304
60.1
KV 2010 :174
90.5
KV 2010 :178
84.5
KV 2010 :181
82.1
KV 2010 :170
77.8
KV 2010 :299
59.9
KV 2010 :180
74.9
KV 2010 :300
21.4
KV 2010 :171
73.6
KV 2010 :301
10.6
KV 2010 :172
73.3
KV 2010 :302
2.1
KV 2010 :175
72.6
KV 2010 :303
-1.0
KV 2010 :126
68.5
KV 2010 :427
71.0
KV 2010 :176
62.0
KV 2010 :426
52.5
KV 2010 :169
61.9
KV 2010 :177
60.9
KV 2010 :460
33.9
KV 2010 :429
70.9
KV 2010 :461
43.2
KV 2010 :428
54.4
KV 2010 :428
54.1
KV 2010 :459
23.9
KV 2010 :251
71.2
KV 2010 :245
67.3
KV 2010 :247
58.0
KV 2010 :244
54.2
KV 2010 :242
46.2
KV 2010 :250
22.7
KV 2010 :246
21.8
KV 2010 :193
87.9
KV 2010 :206
85.8
KV 2010 :197
83.9
CH05015:N1502-086-001-B-BBGEMN0112/////AR03056:N09-24-1-BB-B////CH05015:N15-3-1-BB///GEMS67
CH05015:N1502-086-001-B-BBGEMN0112////DKXL370:N11a20-311-B-B-SIB///GEMS67
CH05015:N15-3-1-BB///GEMS67
CHIS740:S1411a-783-2-BB////AR16035:S02-615-1-BB///GEMS67
CHIS775:S1911b-120-001-B-BB-BGEMS-0113//// 201101_SE32_S17_F2S4_9148Blk22/00-sib///GEMS67
CHIS775:S1911b-120-001-B-BB-BGEMS0113/////CHIS740:S1411a-7832-B-B////AR16035:S02-615-1B-B///GEMS67
CHIS775:S1911b-120-1-B-BB////AR16035:S02-615-1-BB///GEMS67
KV 2010 :243
15.7
KV 2010 :221
67.3
KV 2010 :133
66.0
KV 2010 :217
62.5
KV 2010 :218
62.1
KV 2010 :220
54.0
KV 2010 :221
44.3
KV 2010 :423
46.3
KV 2010 :422
57.0
KV 2010 :287
79.6
KV 2010 :292
69.3
KV 2010 :291
67.8
KV 2010 :298
66.7
KV 2010 :294
63.0
KV 2010 :293
62.4
KV 2010 :295
60.5
KV 2010 :137
55.6
KV 2010 :296
44.8
KV 2010 :138
43.7
KV 2010 :286
27.9
KV 2010 :290
16.1
KV 2010 :425
23.7
KV 2010 :424
44.9
KV 2010 :252
78.5
KV 2010 :252
73.6
KV 2010 :253
73.4
KV 2010 :253
61.7
KV 2010 :139
71.9
KV 2010 :279
61.3
KV 2010 :282
45.7
KV 2010 :281
39.7
KV 2010 :284
32.5
KV 2010 :283
25.1
KV 2010 :462
39.6
KV 2010 :433
70.1
CHRIS775:S1911b-120-1-B-BB////(CUBA164:S2012-444-1B///GEMS67
CUBA164:S1511b-325-001-B-BB-B-B-SibGEMS0063/////CHRIS775:S1911b120-1-B-BB////CUBA164:S2012-444-1B///GEMS67
CUBA164:S1511b-325-1-BB////AR16035:S02-615-1-BB///GEMS67
CUBA164:S2012-444-001-B-BSibGEMS-0092//// 201101_SE32_S17_F2S4_9148Blk22/00-sib///GEMS67
CUBA164:S2012-444-1B///GEMS67
CUBA164:S2012-966-1-BB////AR16035:S02-615-1-BB///GEMS67
DK212T:N11a12-191-001-B-BBGEMN0114/////AR03056:N09-182-1B-B-B////CH05015:N15-3-1-BB///GEMS67
DKB844:S1601-073-001-B-B-B-
KV 2010 :431
66.8
KV 2010 :430
23.6
KV 2010 :435
47.9
KV 2010 :434
43.2
KV 2010 :272
82.7
KV 2010 :271
81.9
KV 2010 :270
71.3
KV 2010 :267
66.3
KV 2010 :275
44.1
KV 2010 :269
31.2
KV 2010 :273
26.4
KV 2010 :266
24.9
KV 2010 :453
56.5
KV 2010 :452
41.2
KV 2010 :451
53.2
KV 2010 :451
35.4
KV 2010 :362
74.4
KV 2010 :165
74.0
KV 2010 :124
65.0
KV 2010 :367
49.9
KV 2010 :369
6.5
KV 2010 :129
81.0
KV 2010 :132
72.5
KV 2010 :421
70.7
KV 2010 :419
30.6
KV 2010 :185
90.5
KV 2010 :182
80.2
KV 2010 :184
68.8
KV 2010 :128
68.2
KV 2010 :183
56.0
KV 2010 :256
55.2
KV 2010 :261
55.1
KV 2010 :264
40.2
KV 2010 :260
37.6
KV 2010 :262
23.4
KV 2010 :168
76.5
KV 2010 :125
73.2
KV 2010 :439
54.1
B-BGEMS0115/////CUBA164:S1511b325-1-B-B////AR16035:S02615-1-B-B///GEMS67
DKB844:S1601-073-001-B-B-BB-BGEMS0115////UR10001:S1813-2571///GEMS67
DKB844:S1601-73-1-B-BB////AR16035:S02-615-1-BB///GEMS67
DKXL370:N11a20-199-002-B-BB-SibGEMN0094/////AR03056:N09-182-1B-B-B////DKXL370:N11a20-311-B-B-SIB///GEMS67
DKXL370:N11a20-234-2-B-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
DKXL370:N11a20-31-1-B-BSIB///GEMS67
DREP150:N2011d-624-1-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
FS8A(S):S09-362-1-B///GEMS67
FS8B(T):N11a-322-1-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
GUAT209:S13 08a-120-001-BBGEMS0184////CHIS740:S1411a-783-
2-B-B////AR16035:S02-615-1B-B///GEMS67
H99 ae ///GEMS67
KV 2010 :130
65.7
KV 2010 :118
36.8
SCR01:N1310-358-1-B-B
///GEMS67
KV 2010 :330
84.7
KV 2010 :331
82.1
UR10001:S1813-2571///GEMS67
KV 2010 :307
79.3
KV 2010 :327
76.8
KV 2010 :306
76.3
KV 2010 :309
76.0
KV 2010 :232
KV 2010 :308
KV 2010 :310
73.3
KV 2010 :313
70.0
KV 2010 :305
68.6
KV 2010 :329
65.1
KV 2010 :240
63.3
KV 2010 :312
Recovery of amylomaize VII
genotypes grown in Indiana
summer of 2010
IN:2010 56
47.1
IN:2010 86
55.9
76.0
IN:2010 55
65.1
75.7
IN:2010 51
64.1
IN:2010 77
51.3
IN:2010 79
47.1
IN:2010 74
36.8
63.3
IN:2010 59
55.0
KV 2010 :235
61.8
IN:2010 59
51.2
KV 2010 :314
60.6
IN:2010 60
36.6
KV 2010 :135
59.9
IN:2010 59
36.0
KV 2010 :311
56.7
KV 2010 :326
56.3
IN:2010 95
47.5
KV 2010 :325
46.7
KV 2010 :289
20.3
IN:2010 100
49.7
KV 2010 :236
19.0
IN:2010 99
15.0
KV 2010 :324
18.8
IN:2010 89
57.2
KV 2010 :288
14.6
KV 2010 :316
14.5
KV 2010 :332
13.3
IN:2010 73
61.2
KV 2010 :239
10.3
IN:2010 90
65.9
IN:2010 91
63.0
IN:2010 90
53.1
IN:2010 92
35.3
IN:2010 94
13.6
IN:2010 64
63.8
IN:2010 69
63.8
IN:2010 63
60.9
IN:2010 98
58.7
KV 2010 :237
6.5
KV 2010 :188
74.5
KV 2010 :131
68.0
KV 2010 :488
64.7
KV 2010 :119
62.2
UR13085:N0215-14-1B///GEMS67
IN:2010 61
46.6
IN:2010 71
41.2
IN:2010 68
38.3
IN:2010 70
23.5
AR03056:N09-182-1-B-B-B////
CH05015:N15-3-1-BB///GEMS67
AR03056:N09-191-1-B-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
AR03056:N09-24-1-B-B-B////
CH05015:N15-3-1-BB///GEMS67
AR03056:N09-24-1-B-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
AR16035:S02-615-1-BB///GEMS67
CH05015:N1204-57-1-B-B////
CH05015:N15-3-1-BB///GEMS67
CH05015:N1204-57-1-B-B////
CH05015:N15-3-1-BB///GEMS67
CH05015:N1204-57-1-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
CH05015:N15-3-1-BB///GEMS67
DKXL370:N11a20-234-2-B-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
FS8B T):N11a-322-1-BB////DKXL370:N11a20-31-1-BB-SIB///GEMS67
SCR01:N1310-358-1-BB///GEMS67
SDS – Slow disgestible ae wx starch improved with GEM germplasm
Our Slow Digestible Starch (SDS) corn utilizes a combination of ae with the waxy (wx) mutation. Potentially,
a therapeutic food additive, ae wx could reduce nocturnal hypoglycemia in type I diabetics using synthetic
insulin, patients with glycogen storage disease. Other benefits include prolonged energy for athletes and
reducing glucose spikes for borderline type II diabetes. Already, industrial starch modification
(heat/moisture or enzyme treated) has led to several SDS undergoing testing.
Naturally produced SDS rice starch was suggested as a low processed source of SDS. Alone wx starch is
entirely branched. Together with ae, elongated branch chains allow for crystalline double-helical formations
among branches. Like RS, enzyme hydrolysis is low, but with ae wx starch it is less extreme. Glucose
released slowly over a longer time may help regulate blood sugar levels (Figure 5).
Figure 5. Previous studies from maize
and rice suggest that among alter
starch genotypes, ae wx may serve as
the most effective natural source of
slowly digestible starch.
We speculate ae wx maize starch may also likely serve as a natural SDS. Gérard (2010) reported ae wx
maize starch digestion rates falling between AmyVII and normal starch. When converted to ae wx,
commercial US dent corn displays extremely poor grain quality. Collapsed seed and ear rot makes
production uneconomical. Improving ae wx grain quality by incorporated various tropical GEM germplasm
has already resulted in improvements with certain backgrounds (Fig. 5). Additionally, ae wx sbe1::gm67 and
ae sbe1::gm67 combinations exist in our stocks and may possess more pronounced SDS characteristics. The
use of our sbe1 specific PCR marker will be required to confirm the presence of sbe1::gems67 in these prior
to analysis
Germplasm
Materials have been generated and genotypes presumed to be ae wx grown in 2009, 2010 and 2011 are
being evaluated. Studies this past summer were done on individual ear samples from 2009 and 2010. For
2008 samples grain quality data was collected on representative samples free of kernel rot but otherwise
without regard for other visible grain quality attribute.
Observations of experimental 2009 and 2010 ae wx GEM germplasm:
Samples presumed to be homozygous for the recessive ae and wx alleles were examined for amylose,
grain physical properties and in some cases thermal properties by differential scanning calorimetry.
Wide variations in grain characteristics were seen which may be important when considering
applications in wet and dry milling.
Figure 6. Amylopectin in normal maize will not
form a complex with iodine and therefore will
show little absorbance at 600 nm. When
amylopectin branch chain-lengths reach or
exceed ~ 10 glucose units long, a helical
conformation can occur and thus, a complex
with iodine form giving a blue color and is
referred to as apparent amylose.
Of the material examined, amylose values ranged widely with few showing no amylose or high amylose
associated with the ae allele alone. Currently we are genotyping the material to determine if apparent
amylose is influenced by the sbe1:gm67 allele. The wide variation in amylose suggest that if these are
indeed ae wx a rapid test for homozygosity of ae wx is needed.
Figure 7. Analysis of selected individual ae wx F4 ears samples produced in harvested from the 2009
Kirksville nursery.
Materials advanced to 2010 and 2011 and currently being examined with a primary selection criteria
being survival of inbreeding, plump kernels and absence of kernel rots. These materials are shown in
Tables 6 and 7. In addition to use of a PCR marker, a simple iodine staining of ae wx can be established
as described by D. Glover at Purdue. The images below can be used to determine if the wx allele has not
been fixed by looking for blue/black stained (not homozygous ae) versus those stained red. It is
uncertain what influence sbe1::gm67 has although it is hypothesized that they will resemble ae wx
alone
Figure 8. A knife is used to scrape the crown and a drop of potassium iodine can be used to
discriminate ae kernels (E,F) from ae wx (A,B,C,D). Selection for unusually plump, non-collapsed
ae wx kernels resulting from desirable background effects from GEM releases will require simple
techniques to maintain the double mutant.
Table 6. Pedigrees of GEM lines presumed to be of the double mutant ae wx
harvested 2010 Kirksville, MO nursery with kernel weight values
Source
X
S
CV
N
10-501-1
15.6
1
10-503-1
12.6
1
10-504-1
16.3
0.2
1.1
10-505-1
14.8
5.9
39.7
10-507-1
17.3
10-508-1
18.3
1.8
9.6
8
10-509-1
15.4
2.4
15.3
4
10-510-1
17.2
2.7
15.9
10-513-1
19.6
5.4
27.5
6 UR13085:N0215-14-1-B GM0310/////UR11003:S0302-1011-1-B-
10-515-1
15.6
1.9
11.9
2 B////(CUBA164:S2012-444-1-B//GEMS67
10-516-1
11.1
0.6
5.8
10-518-1
22.0
10-521-1
19.1
6.1
32.0
2 B////(FS8A(S):S09-362-1-B/GEMS-67
10-522-1
28.1
9.2
32.6
2 SCR01:N1310-509-1-B
10-523-1
19.2
5.6
29.5
10-524-1
14.0
10-527-1
21.7
4.0
18.4
23
10-528-1
22.2
0.0
0.1
2
10-529-1
24.6
6.2
25.0
2
10-530-1
42.3
6,7.
15.9
14
10-531-1
16.2
3.0
18.6
9
10-532-1
15.6
3.2
20.6
6
10-533-1
12.0
1.3
10.6
3
10-534-1
20.8
0.1
0.7
2
10-535-1
17.3
2.7
15.6
7
10-536-1
17.6
0.3
2.0
2
10-537-1
13.9
10-538-1
27.4
5.2
19.0
7
10-541-1
16.5
1.9
11.6
11
10-543-1
19.9
2.5
12.4
3
10-545-1
19.3
1.9
9.6
18
10-546-1
12.0
10-547-1
18.2
6.9
37.8
3
10-548-1
16.3
10.6
64.9
5
10-517-1
10.4
1.4
13.2
4
10-549-1
14.8
3.3
21.9
2
10-550-1
16.4
0.6
3.7
3
10-551-1
0.8
10-552-1
19.8
10-553-1
0.6
10-554-1
13.8
Pedigree
UR13085:N0215-14-1-B GM0310/GEMS67
2 DKXL370:N11a20-36-2-BGM0308/////CUBA164:S2012-444-1-B-B5 sib////(CUBA164:S2012-444-1-B/GEMS67
1
DKXL370:N11a20-36-2-B GM0308/////CHIS740:S1411a-783-2-BB////(CUBA164:S2012-444-1-B/GEMS67
11 SCR01:N1310-509-1-B GM0309/////DKB844:S1601-73-1-B-B-B////(FS8A(S):S09-362-1-B/GEMS67
2 FS8B(T):N1802-525-2-B GM0306/////UR11003:S0302-1011-1-B-B////(CUBA164:S2012-444-1-B/GEMS67
1 DKXL370:N11a20-36-2-B GM0308/////CHIS740:S1411a-783-2-B-
GM0309/////UR11003:S0302-1011-1-B16 B////(FS8A(S):S09-362-1-B/GEMS-67
1
1
1
1
3.1
15.6
7
1
10.3
74.5
10
DKXL370:N11a20-36-2-B GM0308/GEMS67
DKXL370:N11a20-36-2-B GM0308/////FS8B(T):N11a-87-1-B-B////(CH05015:N15-3-1-B-B/GEMS-67
Cuba164:S2012-966-1-B GF0303/////FS8B(T):N11a-110-1-B-B
////(CH05015:N15-3-1-B-B/GEMS-67
AR16035:S02-615-1-B GF0301/////UR11003:S0302-937-1-B-B
////(CUBA164:S2012-444-1-B/GEMS-67
FS8A(S):S09-362-1-B GF0305/////AR03056:N09-182-1-B-B-B
////(CH05015:N15-3-1-B-B//GEMS-67
DK844:S1601-3-2-B GF0304/GEMS67
Cuba164:S2012-966-1-B GF0303/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B/GEMS-67
FS8A(S):S09-362-1-B GF0305/////CHRIS775:S1911b-120-1-B-B-B
////(CUBA164:S2012-444-1-B/GEMS-67
Cuba164:S2012-966-1-B GF0303/////DKXL370:N11a20-199-2-B-B-B
////CH05015:N15-3-1-B-B/GEMS-67
FS8B(T):N1802-525-2-B GM0306/////UR11003:S0302-1011-1-B-B////(CUBA164:S2012-444-1-B/GEMS-67
Cuba164:S2012-966-1-B GF0303/////UR11003:S0302-937-1-B-B
////(FS8A(S):S09-362-1-B/GEMS-67
DK844:S1601-3-2-BGF0304/////DKB844:S1601-73-1-B-BB////(FS8A(S):S09-362-1-B /GEMS-67
AR16035:S02-615-1-B GF0301/////CUBA164:S2012-966-1-B-B////(CUBA164:S2012-444-1-B/GEMS-67
10-555-1
34.5
10.6
30.7
4
10-556-1
19.5
3.6
18.7
15
10-557-1
33.6
10-559-1
16.9
2.1
10-560-1
16.0
10-561-1
1
Cuba164:S2012-966-1-B GF0303/////AR03056:N09-250-1-B-BB////(CH05015:N15-3-1-B-B/GEMS-67
12.3
5
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B/GEMS-67
6.0
37.6
6
17.4
5.6
32.1
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B14 B////(FS8A(S):S09-362-1-B/GEMS-67
10-563-1
25.6
14.8
57.7
14
10-564-1
18.9
2.5
13.3
4
10-567-1
12.0
10-568-1
18.9
2.8
15.0
4
10-569-1
17.6
0.8
4.7
3
10-570-1
18.5
4.7
25.4
8
10-571-1
34.2
14.9
43.7
2
Cuba164:S2012-966-1-B GF0303/////UR11003:S0302-1011-1-BB////(CUBA164:S2012-444-1-B/GEMS67
10-573-1
30.9
31.5
101.9
6
AR16035:S02-615-1-B GF0301/////CUBA164:S1511b-325-1-B-B////(FS8A(S):S09-362-1-B/GEMS-67
10-574-1
25.3
5.4
21.3
6
Cuba164:S2012-966-1-B GF0303/GEMS-67
10-576-1
23.1
2.9
12.6
9
10-577-1
24.8
4.6
18.4
2
FS8A(S):S09-362-1-B GF0305/////UR11003:S0302-937-1-BB////(FS8A(S):S09-362-1-B/GEMS-67
10-578-1
42.2
16.4
38.9
2
Cuba164:S2012-966-1-B GF0303/////CH05015:N1204-57-1-B-B////(CH05015:N15-3-1-B-B/GEMS-67
10-580-1
14.9
10-581-1
20.0
10.8
54.1
10-582-1
19.7
4.0
20.5
10-584-1
16.1
1
AR16035:S02-615-1-B GF0301/////DK212T:N11a12-191-1-B-B////(CH05015:N15-3-1-B-B/GEMS-67
Cuba164:S2012-966-1-B GF0303/////CUBA164:S1511b-325-1-BB////(FS8A(S):S09-362-1-B/GEMS-67
DK844:S1601-3-2-B GF0304/////UR11003:S0302-937-1-BB////(FS8A(S):S09-362-1-B/GEMS-67
1
AR16035:S02-615-1-B GF0301/////DKB844:S1601-3-2-B-B10 B////(FS8A(S):S09-362-1-B/GEMS-67
2
FS8A(S):S09-362-1-B GF0305/GEMS-67
1
DK844:S1601-3-2-B GF0304/////CUBA164:S2012-966-1-B-B////(FS8A(S):S09362-1-B/GEMS-67
Table 7. Pedigrees of GEM lines presumed to be of the double mutant
ae wx harvested 2010 Ames,IA nursery with kernel weight values
50 Kernel
wt.
S
CV
N
Cuba164:S2012-966-1-B GF0303/////FS8B(T):N11a-110-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
14.8 1.1
7.2
3
DKXL370:N11a20-36-2-B GM0308/////FS8B(T):N11a-87-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
14.0 2.8
20.0
6
FS8A(S):S09-362-1-B GF0305/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B///GEMS67
12.2 1.1
8.6
3
8.9
5.3
60.1
3
13.6 1.5
11.1
2
.
1
FS8A(S):S09-362-1-B GF0305/////AR03056:N09-182-1-B-B-B////(CH05015:N15-3-1-B-B///GEMS67
DK844:S1601-3-2-B GF0304/////CUBA164:S2012-966-1-B-B////(FS8A(S):S09-362-1-B///GEMS67
DKXL370:N11a20-36-2-BGM0308/////CUBA164:S2012-444-1-B-B-sib////(CUBA164:S2012-444-1-B///GEMS67
X
11.1
.
Cuba164:S2012-966-1-B GF0303/////AR03056:N09-250-1-B-B-B////(CH05015:N15-3-1-B-B///GEMS67
10.1 0.8
7.6
5
Cuba164:S2012-966-1-B GF0303/////FS8B(T):N11a-110-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
12.2 2.6
21.5
6
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(FS8A(S):S09-362-1-B///GEMS67
12.8 0.0
0.4
2
Cuba164:S2012-966-1-B GF0303/////AR03056:N09-250-1-B-B-B////(CH05015:N15-3-1-B-B///GEMS67
12.1 2.0
16.5
9
AR16035:S02-615-1-B GF0301/////DK212T:N11a12-191-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
13.1 2.4
18.6
4
8.2
3.0
36.3
8
FS8A(S):S09-362-1-B GF0305/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B///GEMS67
13.2 1.5
11.4
5
AR16035:S02-615-1-B GF0301/////UR11003:S0302-937-1-B-B////(CUBA164:S2012-444-1-B///GEMS67
10.4 0.2
2.1
2
8.3
1.4
17.3
2
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
13.4 0.1
1.1
2
DKXL370:N11a20-36-2-BGM0308/////CUBA164:S2012-444-1-B-B-sib////(CUBA164:S2012-444-1-B///GEMS67
10.1 0.6
5.5
5
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
AR16035:S02-615-1-B GF0301/////DKB844:S1601-3-2-B-B-B////(FS8A(S):S09-362-1-B///GEMS67
Cuba164:S2012-966-1-B GF0303/////FS8B(T):N11a-110-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
19.5 2.3
11.7
5
DKXL370:N11a20-36-2-B GM0308/////CHIS740:S1411a-783-2-B-B////(CUBA164:S2012-444-1-B///GEMS67
12.0 1.3
10.7
7
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
11.4 2.9
25.5
6
Cuba164:S2012-966-1-B GF0303/////DKXL370:N11a20-199-2-B-B-B////(CH05015:N15-3-1-B-B///GEMS67
13.2 2.2
16.6
3
Cuba164:S2012-966-1-B GF0303/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
11.4 2.6
23.0
19
8.1
2.8
34.2
3
Cuba164:S2012-966-1-B GF0303/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B///GEMS67
13.0 3.0
22.9
7
DKXL370:N11a20-36-2-BGM0308/////CUBA164:S2012-444-1-B-B-sib////(CUBA164:S2012-444-1-B///GEMS67
10.1 0.4
4.2
5
Cuba164:S2012-966-1-B GF0303/////DKXL370:N11a20-199-2-B-B-B////(CH05015:N15-3-1-B-B///GEMS67
12.1 1.8
14.9
5
DKXL370:N11a20-36-2-B GM0308/////FS8B(T):N11a-87-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
14.5 0.9
6.2
8
FS8A(S):S09-362-1-B GF0305/////AR03056:N09-182-1-B-B-B////(CH05015:N15-3-1-B-B///GEMS67
10.2 4.1
39.6
4
9.9
2.7
27.2
6
11.9 1.0
8.4
5
DK844:S1601-3-2-BGF0304/////DKB844:S1601-73-1-B-B-B////(FS8A(S):S09-362-1-B///GEMS67
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
FS8A(S):S09-362-1-B GF0305/////AR03056:N09-182-1-B-B-B////(CH05015:N15-3-1-B-B///GEMS67
Table 8. Selected ears presumed to be ae wx from 2010 in Ames IA and Kirksville, MO
analyzed for apparent amylose using the iodine-affinity method
Apparent
Source
Pedigree
IA10-5659-1
DKXL370:N11a20-36-2-BGM0308/////CUBA164:S2012-444-1-B-B-sib////(CUBA164:S2012-444-1-B/// GEMS67
IA10-56(51)-1
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5637-1
Cuba164:S2012-966-1-B GF0303/////FS8B(T):N11a-110-1-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5643-3
FS8A(S):S09-362-1-B GF0305/////AR03056:N09-182-1-B-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5644-1
DK844:S1601-3-2-B GF0304/////CUBA164:S2012-966-1-B-B////(FS8A(S):S09-362-1-B/// GEMS67
IA10-5646-1
Cuba164:S2012-966-1-B GF0303/////AR03056:N09-250-1-B-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5647-1
Cuba164:S2012-966-1-B GF0303/////FS8B(T):N11a-110-1-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5648-1
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(FS8A(S):S09-362-1-B/// GEMS67
IA10-5649-1
Cuba164:S2012-966-1-B GF0303/////AR03056:N09-250-1-B-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5650-1
AR16035:S02-615-1-B GF0301/////DK212T:N11a12-191-1-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5655-1
FS8A(S):S09-362-1-B GF0305/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B/// GEMS67
IA10-5656-1
AR16035:S02-615-1-B GF0301/////UR11003:S0302-937-1-B-B////(CUBA164:S2012-444-1-B/// GEMS67
IA10-5657-1
AR16035:S02-615-1-B GF0301/////DKB844:S1601-3-2-B-B-B////(FS8A(S):S09-362-1-B/// GEMS67
IA10-5658-1
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5666-1
Cuba164:S2012-966-1-B GF0303/////UR11003:S0302-1011-1-B-B////(CUBA164:S2012-444-1-B/// GEMS67
IA10-5667-1
Cuba164:S2012-966-1-B GF0303/////DKXL370:N11a20-199-2-B-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5668-1
Cuba164:S2012-966-1-B GF0303/////CUBA164:S1511b-325-1-B-B////(FS8A(S):S09-362-1-B/// GEMS67
IA10-5671-1
Cuba164:S2012-966-1-B GF0303/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5672-1
DK844:S1601-3-2-BGF0304/////DKB844:S1601-73-1-B-B-B////(FS8A(S):S09-362-1-B/// GEMS67
IA10-5673-1
Cuba164:S2012-966-1-B GF0303/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B/// GEMS67
IA10-5674-1
DKXL370:N11a20-36-2-BGM0308/////CUBA164:S2012-444-1-B-B-sib////(CUBA164:S2012-444-1-B/// GEMS67
IA10-5676-1
Cuba164:S2012-966-1-B GF0303/////DKXL370:N11a20-199-2-B-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5677-1
DKXL370:N11a20-36-2-B GM0308/////FS8B(T):N11a-87-1-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5679-1
AR16035:S02-615-1-B GF0301/////DKB844:S1601-3-2-B-B-B////(FS8A(S):S09-362-1-B/// GEMS67
Amylose
24.4
28.4
30.4
24.0
50.2
11.4
19.6
25.0
28.7
18.4
23.4
16.9
18.8
24.9
11.2
25.0
64.4
15.9
21.2
31.3
18.4
26.3
27.5
24.5
IA10-5680-1
FS8A(S):S09-362-1-B GF0305/////AR03056:N09-182-1-B-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IAT0-5681-1
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5682-1
FS8A(S):S09-362-1-B GF0305/////AR03056:N09-182-1-B-B-B////(CH05015:N15-3-1-B-B/// GEMS67
IA10-5695-1
DKXL370:N11a20-36-2-BGM0308/////CUBA164:S2012-444-1-B-B-sib////(CUBA164:S2012-444-1-B/// GEMS67
IA10-5742-1
FS8A(S):S09-362-1-B GF0305/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B/// GEMS67
KV10-R-505-1
DKXL370:N11a20-36-2-BGM0308/////CUBA164:S2012-444-1-B-B-sib////(CUBA164:S2012-444-1-B///GGEMS67
KV10-R-508-1
DKXL370:N11a20-36-2-B GM0308/////CHIS740:S1411a-783-2-B-B////(CUBA164:S2012-444-1-B///GEMS67
KV10-R-509-1
DKXL370:N11a20-36-2-B GM0308/////CHIS740:S1411a-783-2-B-B////(CUBA164:S2012-444-1-B///GEMS67
KV10-R-510-1
SCR01:N1310-509-1-B GM0309/////DKB844:S1601-73-1-B-B-B////(FS8A(S):S09-362-1-B///GEMS67
KV10-R-515-1
UR13085:N0215-14-1-B GM0310/////UR11003:S0302-1011-1-B-B////(CUBA164:S2012-444-1-B///GEMS67
KV10-R-516-2
FS8B(T):N1802-525-2-B GM0306/////UR11003:S0302-1011-1-B-B////(CUBA164:S2012-444-1-B///GEMS67
KV10-R-518-1
DKXL370:N11a20-36-2-B GM0308/////CHIS740:S1411a-783-2-B-B////(FS8A(S):S09-362-1-B///GEMS67
KV10-R-529-2
Cuba164:S2012-966-1-B GF0303/////FS8B(T):N11a-110-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
KV10-R-533-2
AR16035:S02-615-1-B GF0301/////UR11003:S0302-937-1-B-B////(CUBA164:S2012-444-1-B///GEMS67
KV10-R-535-3
FS8A(S):S09-362-1-B GF0305/////AR03056:N09-182-1-B-B-B////(CH05015:N15-3-1-B-B///GEMS67
KV10-R-537-3
DK844:S1601-3-2-B GF0304/GEMS-67
KV10-R-538-1
DK844:S1601-3-2-B GF0304/GEMS-67
KV10-R-543-1
FS8A(S):S09-362-1-B GF0305/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B///GEMS67
KV10-R-445-2
FS8A(S):S09-362-1-B GF0305/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B///GEMS67
KV10-R-552-2
DK844:S1601-3-2-BGF0304/////DKB844:S1601-73-1-B-B-B////(FS8A(S):S09-362-1-B///GEMS67
KV10-R-556-1
Cuba164:S2012-966-1-B GF0303/////AR03056:N09-250-1-B-B-B////(CH05015:N15-3-1-B-B///GEMS67
KV10-R-560-2
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(FS8A(S):S09-362-1-B///GEMS67
KV10-R-561-5
FS8A(S):S09-362-1-B GF0305/////BARBGP2:NO8a18-332-1-B-B////(FS8A(S):S09-362-1-B///GGEMS67
KV10-R-563-6
AR16035:S02-615-1-B GF0301/////DK212T:N11a12-191-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
KV10-R-564-1
Cuba164:S2012-966-1-B GF0303/////CUBA164:S1511b-325-1-B-B////(FS8A(S):S09-362-1-B///GEMS67
KV10-R-567-1
Cuba164:S2012-966-1-B GF0303/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B///GEMS67
KV10-R-568-1
DK844:S1601-3-2-B GF0304/////UR11003:S0302-937-1-B-B////(FS8A(S):S09-362-1-B///GEMS67
KV10-R-571-1
Cuba164:S2012-966-1-B GF0303/////UR11003:S0302-1011-1-B-B////(CUBA164:S2012-444-1-B///GEMS67
KV10-R-573-5
AR16035:S02-615-1-B GF0301/////CUBA164:S1511b-325-1-B-B////(FS8A(S):S09-362-1-B///GEMS67
KV10-R-574-3
Cuba164:S2012-966-1-B GF0303/GEMS-67
KV10-R-576-1
FS8A(S):S09-362-1-B GF0305/////UR11003:S0302-937-1-B-B////(FS8A(S):S09-362-1-B///GEMS67
KV10-R-577-1
FS8A(S):S09-362-1-B GF0305/////UR11003:S0302-937-1-B-B////(FS8A(S):S09-362-1-B///GEMS-67
KV10-R-581-9
AR16035:S02-615-1-B GF0301/////DKB844:S1601-3-2-B-B-B////(FS8A(S):S09-362-1-B///GEMS-67
KV10-R-582-1
FS8A(S):S09-362-1-B GF0305/GEMS-67
KV10-R-738-1
DK844:S1601-3-2-B GF0304/GEMS-67
KV10-R-740-1
DK844:S1601-3-2-B GF0304/GEMS-67
KV10-R-750-1
DKXL370:N11a20-36-2-B GM0308/GEMS-67
KV10-R-751-1
DKXL370:N11a20-36-2-B GM0308/GEMS-67
KV10-R-758-1
DKXL370:N11a20-36-2-B GM0308/GEMS-67
KV10-R-762-1
UR13085:N0215-14-1-B GM0310/GEMS-67
33.0
11.0
35.6
16.8
20.6
17.6
31.2
32.8
17.9
17.2
30.2
23.9
55.1
24.8
24.8
24.6
13.1
26.7
23.2
60.9
24.7
22.6
23.2
64.6
22.4
11.7
20.7
20.9
27.1
20.1
29.0
31.9
24.9
28.6
27.0
26.5
26.6
14.8
25.3
19.9
KV10-R-763-1
UR13085:N0215-14-1-B GM0310/GEMS-67
KV10-R-764-1
UR13085:N0215-14-1-B GM0310/GEMS-67
KV10-R-774-1
DK844:S1601-3-2-B GF0304/GEMS-67
KV10-R-775-1
DK844:S1601-3-2-B GF0304/GEMS-67
KV10-R-776-1
UR13085:N0215-14-1-B GM0310/GEMS-67
10KV-756-1
DKXL370:N11a20-36-2-B GM0308/GEMS-67
09-479-2/3
DKXL370:N11a20-31-1-B-B-SIB///GGEMS67
09-531
(UR13085:N0215-14-1-B///GEMS67
09-805
CHIS775:S1911b-120-1-B-B-B////AR16035:S02-615-1-B-B///GEMS67
(09)10-123-1
Cuba164:S2012-966-1-B GF0303/////CUBA164:S1511b-325-1-B-B////(FS8A(S):S09-362-1-B///GEMS67
(09)10-130-1
Cuba164:S2012-966-1-B GF0303/////CUBA164:S1511b-325-1-B-B////(FS8A(S):S09-362-1-B///GEMS67
(09)10-152-1
AR16035:S02-615-1-B GF0301/////DK212T:N11a12-191-1-B-B////(CH05015:N15-3-1-B-B///GEMS67
(09)10-160-1
DK844:S1601-3-2-BGF0304/////DKB844:S1601-73-1-B-B-B////(FS8A(S):S09-362-1-B///GEMS67
(09)10-168-1
DKXL370:N11a20-36-2-B GM0308/GEMS-67
(09)10-192-1
DKXL370:N11a20-36-2-B GM0308/////CHRIS775:S1911b-120-1-B-B-B////(CUBA164:S2012-444-1-B///GEMS67
33.5
23.7
19.6
54.8
35.4
34.8
42.0
21.6
Examples of ears of ae wx samples examined for amylose displaying a wide range of
grain quality attribute
s
54.3
29.5
17.3
27.6
57.9
45.2
20.6
Structural and functional observations of amylopectins in
experimental ae wx GEM lines
The following analysis of several experimental ae wx genotypes were done in Dr. Jay Lin’s lab
by Hanyu Yangcheng:
Starches of five ae-wx corn lines were received and analyzed: 09-148-3, 09-159-2, 09-167-1, 09-188-3,
and 09-201-2. Amylopectin branch-chain length distribution of the starches were analyzed using FACE ;
Starch thermal properties were analyzed using DSC; Starch digestibility after cooking was determined
following Englyst method (1992); And raw-starch hydrolysis was carried out using PPA and
amyloglucosidase.
Results
Table 1 Amylopectin Branch-chain length
distribution of the ae-wx starches.
DP – degree of polymerization (~branch
length - glucose molecule)
09-201-2 ae wx
09-167-1a
09-188-3 ae wx
09-159-2 ae wx
09-148-3 ae
DP(6-12)
11.9±0.0
N/A
12.5±0.1
11.7±0.1
12.0±2.8
DP(13-24)
42.1±0.2
N/A
41.0±1.2
41.6±0.6
32.5±0.5
DP(25-36)
15.1±0.1
N/A
16.1±0.7
15.7±0.4
13.6±0.4
DP>37
30.9±0.1
N/A
30.4±2.0
31.0±1.0
41.9±1.8
AVECL
29.4±0.3
N/A
29.3±0.2
29.4±0.0
33.4±0.1
Table 2. Digestibility of ae-wx starches after cooking (Englyst 1992)
09-201-2 ae wx
09-167-1 ae wx
09-188-3 ae wx
09-159-2 ae wx
09-148-3 ae
RDS%
87.5±0.7
84.0±1.0
88.3±1.1
87.5±2.2
69.5±0.1
SDS%
7.9±0.6
7.9±0.6
7.6±2.0
6.7±1.9
5.2±1.0
RS%
4.6±1.4
8.1±0.4
4.2±0.9
5.8±0.3
25.3±1.0
Starches sent to Iowa State for analysis were obtained from the summer nursery of 2009 in
Kirksville,,MO. Genotypes were selected visually by selecting wx kernels within F2 ears followed by ae
in F3 ears and resulting in double-mutant ae wx F4 ears. All samples show the expected Resistant
Starch value for ae wx except for sample 09-148-3 where values resemble that of a single mutant ae .
Misclassification will likely occur, however, simple diagnostic techniques such as kernel iodine staining
will be used to avoid this. The sample 09-167-1 unfortunately had no DP data however a slightly
elevated RS would raise the possibility of sbe1::gm67 being present,
24
Raw-starch hydrolysis of ae wx corn starches:
200mg starch (db) was suspended in 19mL of sodium phosphate buffer (0.1M, pH6.9). The
suspension was equilibrated at 37 ºC for 1 hr. 1mL of the freshly prepared porcine pancrease alphaamylase solution (200units/ml) was added to the suspension. The tube was vortexed and then
incubated in 37ºC with shaking (100rpm). At each time interval (0hr, 3hr, 6hr, 12hr, 24hr, 48hr, or
longer if needed), 0.3mL of the suspension was removed to a micro-centrifuge tube. The micro-tube
was centrifuged at 6,600g for 5 min. 0.1ml of the supernatant was transferred to the 0.89ml sodium
acetate buffer solution (0.1M, pH 4.5). 10µl of the amyloglucosidase (2-3 units of activity) was added
into the transferred solution. It was vortexed and then incubated at 50 ºC for at least 2 hr. 0.1mL of
the solution was taken out for GOPOD analysis.
Table 3 Percentages hydrolysis (%) of the ae wx corn starches using a raw-starch hydrolysis process
09-201-2 ae wx
09-167-1 ae wx
09-188-3 ae wx
09-159-2 ae wx
09-148-3 ae
B73
3h
6.7±0.3
6.7±0.1
9.1±0.4
7.1±0.2
9.0±0.1
14.8±0.0
6h
10.2±0.5
10.3±0.3
14.0±0.0
11.2±0.2
13.6±0.3
26.6±0.3
Hydrolysis values varied
and all had expected
lowered rates. The sample
09-167-1 showed the
slowest rate of digestion
and command further
genotyping and digestion
studies to determine the
influeence of novel GEM
alleles.
10h
14.8±0.6
14.8±0.3
19.7±0.1
15.9±0.3
18.4±0.5
41.1±0.1
24h
25.1±0.7
23.4±0.1
29.9±0.1
26.9±0.5
29.5±0.4
72.8±0.3
48h
31.8±0.7
29.8±0.2
34.5±0.2
31.9±0.6
34.7±0.5
82.1±0.5
Raw-starch hydrolysis of ae-wx corn starches
100
90
Hydrolysis (%)
80
70
60
50
40
30
20
10
Figure 1. Raw-starch
hydrolysis of the ae wx
corn starches
0
Time (hr) 0
6
201-2
Gelatinization
To
Tp
09-201-2
69.2±0.7
79.1±0.6
95.7±0.3
09-167-1
69.7±0.6
79.6±0.9
09-188-3
67.1±0.4
09-159-2
65.0±0.4
18
167-1
24
30
188-3
159-2
36
148-3
42
48
B73
Retrograded starch
To
Tp
Tc
18.7±0.5
46.5±1.9
65.1±0.0
87.9±0.1
14.2±0.4
75.9
95.2±1.8
18.6±0.1
55.6
69.8
84.2
14.4
77.2
79.9±0.2
93.0±0.0
17.2±0.0
46.3±1.9
65.0±0.0
86.5±0.6
13.9±0.7
81.3
78.4±0.0
91.2±0.1
18.9±0.2
47.3±0.4
65.0±0.0
87.7±0.3
13.2±0.2
70.2
105.3±0.2
9.3±0.0
50.2
60.6
71.4
1.1
11.6
09-148-3 66.5±0.9 83.2±1.4
a
Data need to be repeated.
Tc
12
H
a
H
Retrogradation
(%)
25
Figures 2 (a-e). The following are curves of the heat
flow of the starches analyzed using DSC. Starches of
two lines, 09-167-1 and 09-201-2, showed a little rightskewed heat flow peaks.
c. 09-167-1
DKXL370:N11a20-36-2-B GM0308/GEMS-67
a. 09-159-2
AR16035:S02-615-1-B GF0301/////CUBA164:S2012-966-1-BB////(CUBA164:S2012-444-1-B///GEMS67
d. 09-201-2
SCR01:N1310-509-1-B GM0309/////DKB844:S1601-73-1-B-BB////(FS8A(S):S09-362-1-B/// GEMS-67
b. 09-188-3
DKXL370:N11a20-36-2-B GM0308/////CHRIS775:S1911b-1201-B-B-B////(CUBA164:S2012-444-1-B///GEMS67
e. 09-148-3
Cuba164:S2012-966-1-B GF0303/////FS8B(T):N11a-110-1-BB////(CH05015:N15-3-1-B-B/// GEMS-67
26
Continued Molecular Marker Studies on the sbe1a::gem67 allele
By: Marianne Emery and Avinash Karn
In our previous GEM report (2010), we described the development of potential gene-specific marker for
the allele sbe1a::gem67 believed to serve as the main modifying gene in GEMS-0067. We suggested an
insertion at an invariant Adenine (A) site in one of the introns in sbe1a::gem67 allele. The insertion is believed
to interrupt the normal intron splicing during pre-mRNA transcriptional modification. Specifically, the
insertion may have occurred in a a critical position within the non-coding intron resulting in the continued
removal of the exon immediately upstream. The exon presumed to be absent in the altered sbe1a::gem67
mRNA transcript would appear to possess a region necessary for translation of the -amylase domain normally
found in starch branching enzymes. Over the past year our main goal was to further investigate this splicing
mutation hypothesis and simultaneously address difficulties of this marker for routine genotyping in our
selection and recovery sbe1a::gem67 following crosses. Mainly difficulties were in resolving heterozygote
inbred lines from those which are homozygous recessive. The implementation of various Bioinformatics tools
together with improved robust wet-lab analyses were used to accomplish this.
Identification of the homozygote parental types and heterozygotes through endonuclease
enzyme activity on the PCR product amplified from sbe1a::gem67 marker
During the summer of 2010, we were successfully able to design 23 potential gene specific primer sets
that collectively amplified the sbe1a::gem67 allele. Of these, only one marker, named primer I, successfully
amplifed a polymorphic PCR product between parental lines GEMS-67 and H99ae and the F1 used in our
original QTL mapping population. The efficacy of the primer I -amplified PCR product was determined using
several converted Amylomaize VII GEM lines and normal endosperm-type GEM releases lines which were
tested on 2.5% agarose and ran at 130 Volts for at least 1 hour (Fig. 1).
Fig.1. PCR products of the GEMS-0067 along with Amylomaize VII line (column 3, 5, 7, 9, 11) and GEM releases
(column 2, 4, 6, 8, 10, 12) were amplified with primer ‘I’ and digested with the restriction enzyme Rsal. The digested
DNA fragments were staggered from one another reflecting the alternating presence of the wild-type and normal Sbe1a
in the GEM releases used as recipient lines and the presence of the sbe1::gm67 allele from GEMS-0067 in the converted
Amylomaize VII lines as shown in the gel image and run in 2.5% agarose gel for an hour.
Primer I amplified a 400bp length PCR product which was then further digested with a restriction endonuclease
Rsal. Prior sequence analysis of the product indicated three restriction sites in the PCR product amplified, and
would generate three short fragments during separation during electrophoresis. When tested on our materials,
endonuclease enzyme performed as expected and also revealed that the converted Amylomaize VII lines
produced a fragment from sbe1a::gem67 with a likely insertion in one of the larger DNA fragments compared
27
to the normal lines as seen in Fig.1. The primer I therefore appeared at that point to produce a PCR product of
interest for the modifier allele sbe1a::gem67. Before confirming “the” marker, we decided to conduct some
more test analysis and the interpret data. The primer I was tested on lines believed to be heterozygous inferred
from quantitatively identified (Iodine colorimetric test) along with what were potentially converted
Amylomaize VII lines on 3.5 % agarose gel running for 4 hours at 120 Volts (Fig.2.).
Fig.2. Primer I was tested on parental lines GEMS0067 (column 2) and H99ae (column 3) along
with converted Amylomaize VII lines with row number 8-442 and 8-457 resp.(in column 6 and 8)
and heterozygote lines (in column 4, 5, 7, 9). The PCR product obtained was screened in 3.5 %
agarose gel and ran at 120V for 4 hours.
The screening of heterozygote lines with primer I did reveal the presence of the primer I modifier PCR product
and the presence of heterozygous ‘double banded’ lanes suggest that the modifier was displaying a co-dominant
pattern as desired (Fig.2.)
Despite the fact that primer I was successfully capable of amplifying both parental and heterozygote
type lines; the lengthy time required separate the heterozygote bands on 3.5% agarose gel was of concern. That
is, normal and sbe1a::gem67 PCR products obtained from heterozygote lines needed a full 4 hours to separate
and resolve in super fine agarose (SFR) gel and yet still, the two bands did not always resolve enough to be
easily categorized into heterozygote which would be undesirable especially if routinely used by future
undergraduate students with little experience. Our next strategy, therefore, was to overcome the above problem
and identify a restriction endonuclease enzyme with a restriction site only in the sequence of modifier allele.
This required the PCR products obtained from GEMS-0067 along with other converted Amylomaize VII lines
to be sequenced at Iowa State University. Sequences were analyzed via ‘BioEdit’ aligning software. The
sequences obtained from GEMS-0067 and converted Amylomaize VII lines along with sequence obtained from
B73 (as reference normal line) were aligned through the ‘ClustalW’ alignment algorithm. The alignment of
these three sequences revealed that the locus being amplified by primer I had an insertion of 8bp (shown in
Fig.3). Using Biology Workbench online bioinformatics tool, endonuclease enzyme/s with restriction site only
in the sequence with insertion was identified. We were capable of identifying MslI endonuclease enzyme that
28
has two restriction sites in the sequence of GEMS-0067 (shown in Fig.4) and no restriction site of the same
enzyme in normal ref. sequence of B73.(Fig. 4); the recognition site of MslI is ---CAYNNNNRTG---.
Fig.3. Two restriction sites (highlighted with red box) of MslI endonuclease enzyme in the
sequence of GEMS-0067 amplified by primer I; whereas, no restriction sited of MslI in the
reference sequence of B73.
Two weeks old leaf tissue obtained from GEMS0067, H99ae, converted Amylomaize VII lines and
heterozygote lines were harvested to isolate genomic DNA via SDS extraction method. The isolated DNA were
amplified with primer I at 95’C denaturing, 55’C annealing and 72’C extension for 30 cycles in Thermocycler
PCR machine. The PCR product was further treated with MslI endonuclease enzyme by adding 1uL of MslI
enzyme, 2.5uL of BSA and 2.5uL of 10X buffer for 1X reaction; and digested in water bath at 37’C for 10 min.
The final digested PCR product was screened on 1% (pre-stained with 10ul EtBr) SFR agarose gel at 120V for
an hour. The gel electrophoresis result shown in Fig.5 confirmed that maize lines with modifier gene has small
DNA fragments due to two endonuclease restriction sites; the normal lines lacking restriction site had larger
band whereas, the heterozygotes having two distinct bands. The marker data tested on the different lines shown
Fig.4 clearly indicates primer I being a gene specific marker for the allele sbe1a::gm67 and for that cause; we
have renamed our marker as Primer SBE1a::GM67 after the name of the modifier allele.
Fig.4. PCR products amplified from primer I were digested using endonuclease enzyme MslI and
screened on 1% SFR agarose gel at 120V for an hour.
Genotyping converted Amylomaize VII lines from year 2010 with the marker
Developed gene specific marker Sbe1::gems67 was utilized in screening 142 potential Amylomaize VII
lines from the year 2010 followed with MslI endonuclease digestion. During the screening process, genomic
DNA from 142 maize lines were isolated from two weeks leaf tissue via SDS DNA extraction method,
amplified by the primer SBE1a::GM67 at 95’C denaturing, 55’C annealing and 72’C extension for 30 cycles in
Thermocycler PCR machine. The PCR product was further treated with MslI endonuclease enzyme by adding
1uL of MslI enzyme, 2.5uL of BSA and 2.5uL of 10X buffer for 1X reaction; and digested in water bath at 37’C
29
for 10 min. The genotyping of 142 lines from year 2010 successfully screened 3 heterozygote lines, 3 lines with
no modifier allele and 136 lines with confirmed modifier allele. The collected marker data using primer
SBE1a::GM67 are shown below;
Table 1. Genotype screening results of Amylomaize VII inbreds from year 2010
Fig.5. PCR products obtained from 2010 Amylomaize VII lines were amplified with primer SBE1a::GM67
and were digested using endonuclease enzyme MslI. The digested PCR products were screened on 2.5% SFR agarose gel
at 120V for an hour. The gel electrophoresis analysis precisely identified the heterozygotes within less time expenditure
as well as the parental types.
30
Primer Sbe1a::Gm67 revealing the source of modifier allele in GEMS-0067
We screened the parental lines (GUAT209::S13 and H99ae) and GEMS-0067 along with potential
converted Amylomaize VII lines (10-64, 10-65, 10-69, 10-70, 10-71), and normal lines (GM-87, GM175) with
our primer Sbe1a::Gm67. Our marker data strongly indicate source of the modifier allele sbe1a::gem67 from
GUAT209::S13 (P1) as shown in Fig. 10.
Fig.10 Primer Sbe1a::Gm67 was utilized to identify the source of modifier allele
sbe1a::gem67. The PCR product obtained from primer SBE1a::67 after amplifying
Amylomaize VII maize lines (10-64, 10-65, 10-69, 10-70, 10-71) and normal lines (GM87, GM175) along with P1, P2 and F1 were staggered in 3% SFR agar gel columns from
one another in order to reflect the alternating presence of wild-type and normal Sbe1a in
GEM releases. The gel was run for 3hrs at 120 Voltage.
Genotyping lines possessing three mutant allele ae, sbe1a::gem67 and wx
Table 2 Marker score data of lines possessing three mutant allele ae, sbe1a::gem67 and
wx with the sbe1a::gem67 marker revealed three heterozygote lines (9-153-3, 10-775-4
and 10-173-2) and the rest with fixed modifier alleles.
31
Explained biochemistry of the Splicing Mutation and the missing codon
In our last year GEM report, we hypothesized splicing mutation phenomenon leading to splicing of a
domain coding region that explained truncated protein in Zymogram conducted in Dr. Alan Myer’s lab
(Fig.6). We now are very certain about the occurrence of splicing mutation phenomenon in our modifier
sbe1a::gm67 allele from received sequence data. The primer SBE1a::GM67 was used to amplify the
genomic DNA of GEMS-0067 and converted Amylomaize VII lines which were sent to be sequenced to
Iowa State University at Ames, IA. Obtained sequence data were aligned via ClustalW algorithm which
revealed an insertion of 8bp at two sites in modifier allele (Fig.3). The insertion at 5’ upstream region of the
sequence was 6bp and 3bp in length resp. at downstream region of the amplified region by the primer. The
insertion at upstream region was at invariant adenine (A) site of one of the introns in modifier allele that
interrupted in Pre-mRNA transcriptional modification, which subsequently caused the splicing of
neighboring exon.
Fig.6 Zymogram produced at Iowa
State University by Dr. Allen
Myers showing gene products of
the Sbe1 locus for sbe1::mu, wild
Type (Sbe1a), and GEMS-0067
sbe1::gm67
InterProScan conducted on the mRNA sequence of exon being spliced showed that, it being a domain
coding for Alpha-Amylase and is classified as family 13 of Glycosyl hydrolases. It has a structure of 8 stranded
alpha and beta barrel containing the active site which is interrupted by 70 amino acid calcium –binding domain
(Fig.7). The InterProScan summery concludes that the spliced exon is an active coding domain for Alpha
Amylase , which is responsible for branched chain in Amylopectin but when absent as such in our case, causes
long branches of α(1-4) glycosidic bond resulting in high amylose starch in GEMS0067.
Fig.7. InterProScan result of mRNA sequence of the splicing exon in the Sbe1a gene.
32
Identification of loci in Chromosome 4 and 6 with potential influence in kernel starch
composition
In a collaborative research conducted
with
Dr. Donald Auger and Dr. Yusheng Wu at
South Dakota State University, Brookings, SD,
on
identifying the modifier allele in GEMS-0067,
pointed towards two QTLs on Chromosome
5.03
and 6 respectively. The QTL data on
Chromosome 5 explained the 43% variance in
recombinant line whereas explaining only 6%
variance on Chromosome 6. Since then, Starch
Branching Enzyme Ia (SBE1a) allele in
GEMS067 has been identified as the modifier
allele
whereas, QTL from chromosome 6 has been
neglected due to its low significant LOD score
until
now. In our lab at Truman State, we conducted
few
bioinformatics analysis through Maize Genome
Fig.8. BLAST result of the sequence amplified by
Browsers such as MaizeGDB,
primer Sbe1::GM67 and its two significant hits in long
Maizesequence.org, CoGE and InterProScan on
arm of chromosome 5 and 6.
Chromosome 6. Upon BLAST analysis of the
sequence being amplified by primer SBE1a::GM67, we found two significant hit on long arms of chromosome
5 and 6 respectively (Fig. 8). We identified that the hit on Chromosome 6 being a duplicated gene of SBE1a
with Gene ID GRMZM2G169073 (SBE Ib). Similar approach towards the Amylose extender (ae) gene was
made and we found a duplicated copy of ae gene in long arm of the chromosome 4. mRNA expression data of
the individual paralogs and duplicated genes were collected from PLEXdb.org and an expression graph for
Sbe1a, Sbe1b, Sbe2a and Ae1 was plotted to study their expression throughout the developmental stages of
maize and found that most of the genes were significantly expressed during kernel development that is 12 DAP
and few expression during leaf development (Fig. 9).
Fig.9 mRNA expression graph depicting expression of genes involving in starch synthesis
throughout the developmental stages of maize.
33
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34