M. Campbell 06 Report
Mark Campbell
Associate Professor, Truman State
University
Division of Science, Kirksville, MO
63501
Undergraduate Research Assistants:
Morgan Bullock
Anna O’Brien
Kelly Limberg
Eric Witrock
Kendra Nilson
Omeiza Olumoye
Special Thanks to:
Dr. Kelly Nelson (University of
Missouri) and Staff at the Greenly
Memorial Research Station at
Novelty Missouri
O v e r r v i i e w
Starch is the major component of cereal grains, such as corn, making up approximately 75% of its dry matter. Starch is a polymer with the simple sugar glucose serving as the repeating unit. Normally, it is composed of two polymer types: amylose, a straight chained molecule making up 25% (dwb) of the starch and amylopectin, a branched form that comprises the remainder. High amylose corn is a non-GMO type specialty maize that utilizes traditional plant breeding techniques to increase the relative amount of amylose in the starch. It is generally grown under contract with wet-mills or exporters and commands a substantial premium, however, at least part of this is needed to off-set the fact that it yields only 75-80% as much as normal hybrids with lower test weights. There are a number of specific high amylose classes including amylomaize V (50% amylose) and amylomaize VII (70% amylose). In the U.S. high-amylose corn varieties have been grown and processed over the past 50 year mainly in the Eastern corn belt by two companies; National Starch and Chemical Company and Cargill. High-amylose varieties are highly protected and until recently, there has been no public germplasm source. Traditional uses of high amylose starches have been as an ingredient in gum candies, adhesives for corrugated cardboard and as ‘sizing’ to strengthen yarn during the weaving process to prevent breakage of the thread. There have been two recent advances that have increased interest in the use of high amylose starches that have resulted in increases in its production. The first has been in the development of thermoplastic starch-based biodegradable plastics
(TPS). With the use of specific plasticizing solvents, they can be used to produce thermoplastic materials such as shopping bags, bread bags, bait bags, over wrap, flushable sanitary product packing material, and mulch film. Second, many national and international food companies are looking at amylomaize as a source of resistant starch (RS), a type of starch that resist digestion. As a food additive, consumers could benefit from added RS in foods since it will lower the glycemic index. Additionally, RS behaves as dietary fiber and products of its fermentation in the lower digestive tract are believed to lower the risk of colon cancer.
Breeding studies at Truman State have led to the development of a corn line possessing amylomaize class VII starch designated as GEMS-0067 which is currently of the status “accepted with minor revisions” as a germplasm release in the Journal Crop Science ( PI 643420, NSSL 445438.01) .
GEMS-0067 possesses high-amylose modifier (HAM) gene(s?) that, together with the recessive amyloseextender (ae) allele, raises the starch-amylose percentage to at least 70%. To our knowledge GEM-0067 represents the only public source of Amylomaize VII to date. Development of GEMS-0067 began following the screening of many exotic varieties of corn maintained by the National Plant Germplasm
System (NPGS) and the Germplasm Enhancement of Maize (GEM) project. Only one line derived from the pedigree including a GEM line partially of Guatemalan origins [GUAT209:S13 x (H99ae x OH43ae)] was found to possess the desired modifiers and survived inbreeding . Subsequent studies have shown relatively high levels of resistant starch (40-45%) in GEMS-0067 compared to lines homozygous for ae alone (15-20%). The following report describes activities that have taken place over the past year towards further development of Amylomaize-VII germplasm using GEM germplasm.
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M. Campbell 06 Report
Figure 1. Amylose values for F4 lines from the pedigrees AR16035:S02-615-1-B-B
///GUAT209:S13//OH43ae/H99ae and DKXL370:N11a20-31-1-B-B-
SIB///GUAT209:S13//OH43ae/H99ae and nine public inbreds converted to ae.
-
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M. Campbell 06 Report
Using the same method for line development previously described, the analysis of F2 ears segregating for HAM genes are shown in Figure
2. The ears analyzed resulted from the crossing of GEMS-0067 onto six different GEM lines. In each case, at least one F2 ear possessed starch with an amylose content of 70% or greater. The ears having grain with >70% amylose have been selected and F3 seed from them were advanced this past summer (2006) or are currently growing in a winter nursery (2006/07). As modifiers are fixed in subsequent generations, they will be used in future testcrossing for yield evaluation and agronomic traits.
DKXL370:N11a20-31-1-
B-B-SIB
.
In order to develop additional amylomaize VII lines that are more distinctly of a stiff-stalk or non-stiff stalk heterotic background, the two lines described in Figure 1 ( AR16035:S02-615-1-
B-B ///GUAT209:S13//OH43ae/H99ae and
DKXL370:N11a20-31-1-B-B-
SIB///GUAT209:S13//OH43ae/H99ae) were used as donors of the ae allele and HAM genes onto released GEM lines. As expected, segregation for HAM genes occurred in the F2 and will be used as the main selection criterion (Figure 3A).
We have also started to routinely determine starch content using a traditional Polarimeter method. Data is given in ‘degrees of optical rotation’ only at this point since we do not yet have a standard curve to convert these to % starch, however, relative differences can be observed. Some of the samples described in
Figure 2 are currently being examined for starch content. Although we are working with limited data, it appears that materials with a background of DKXL370:N11a20-31-1-B-B-
SIB///GUAT209:S13//OH43ae/H99ae tend to also have slightly greater starch contents than those of
AR16035:S02-615-1-B-B
///GUAT209:S13//OH43ae/H99ae (Figures 3A and
3B
Figure 2. F2 ears segregating HAM genes from crosses between GEMS-0067 and six
GEM lines.
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M. Campbell 06 Report
Figure 3. A) F2 ears segregating HAM genes from crosses between DKXL370:N11a20-31-1-B-B-
SIB///GUAT209:S13//OH43ae/H99ae and six GEM lines and B) total starch determined for some of these.
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M. Campbell 06 Report
One of the main obstacles in developing amylomaize VII lines is fixing the HAM genes.
This is, in part, due to the fact that the inheritance and number of genes involved is unclear. Additionally, a time consuming laboratory assay is required that involves laboratory wet-milling followed by a colorimetric amylose-iodine binding method.
Our wet-milling process has routinely relied on using a toluene/saline “chemical wash” in order to physically separate and remove protein from the starch. Not only is this a time consuming step, but the toluene poses a health hazard and expensive chemical waste removal is required.
Working with Dr. Ya Jane Wang at the
University of Arkansas, a protein removal technique involving the use of two commercial proteases (Amano, Inc. Japan) were used to degrade protein safely and relatively effortlessly.
To test the method F2 ears from the crosses indicated in Table 1 were use in a comparison study, whereby starches purified by filtration only, removal by saline/toluene and protease degradation were compared (Table 1)
Table 1. A populations of F2 ears segregating for HAM genes was created by crossing GEMs-0067 onto two other
GEM lines removed with the protein. The data suggested that, possibly, that the amount of starch recovered from the protease method could be used to estimate the total amount of starch in the grain sample, which would be an important piece of information for germplasm development.
Figure 4. Three starch isolations procedures (no protein removal, protein removal by toluene/saline and protein removal by Amano Proteases are compared among 17 grain samples segregating HAM genes for A) amylose values for individual ears B) total grams of starch recovered C) average amylose percent and D) average percent contaminating protein.
The results were very promising. Amylose values where as high or higher for protease purified starch samples compared to those treated with toluene (Fig. 1 A and C). This data suggested that these samples may have even less protein contamination due to the high efficiency of the protease method. Pooled samples from each starch isolation procedure were then analyzed for protein content using a microkjehldahl procedure. As suspected, protease treated samples in fact had less contaminating protein (Fig. D). Another apparent advantage of this method is that a better yield of purified starch was observed with the protease treated samples (Fig. B). With the toluene method typically a large amount of starch will be suspended in the toluene and therefore, is
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M. Campbell 06 Report
As mentioned in previous GEM reports and work-plans, one challenge we have encountered is the lack of availability of public Amylomaize VII germplasm which limits our ability to find a suitable tester for our GEM Amylomaize VII germplasm. Therefore, our approach has been to inter-mate early GEM
Amylomaize lines (F3) that are homozygous for the ae allele, and possess, but have not yet had HAM genes fixed. In many cases, some of these F3 lines have already been tested in the laboratory and more advanced lines will have HAM genes fixed (see previous sections of this report.) Yield data of these early lines will give us an idea of which GEM-amylomaize parents will be good matches and allow us to prioritize the lines to advance and test in the laboratory. In 2006, forty-nine test hybrids and a commercial
Amylomaize VII variety were evaluated at two locations (Novelty, MO and Ames, IA). In general, the location in Iowa performed much better due probably to better soil, more timely rains and an earlier planting date.
Pooled yield and agronomic performance are provided in Table 3. As a reminder, it should be pointed out that parents designated as ‘stiff-stalk’ are printed in blue and ‘non-stiff-stalk’ in red. Also, keep in mind that the parents are essentially half-sibs of each other since the parents themselves share a parent:
GUAT209:S13//OH43ae/H99ae , aka, GEMS-0067. From Table 3 it can be seen that twenty (out of 49) of the test hybrids outyielded the proprietary amylomaize VII check hybrid. The top yield hybrid (2011-
01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1-2-1) – 1 X (CH05015:N15-3-1-B-
B///GUAT209:S13//OH43ae/H99ae 1-2-1) – 1 yielded an average of 120 bu/acre compared to the check (90 bu./acre). In fact, at the Iowa location, this same hybrid yielded 170 bu/ac. which substantially exceeded most all others (Table 4). One of the hybrids, simply referred to as ar x dk, is a hybrid made using the two lines described in Figure 1. Unlike other test hybrids, the parents used to make this hybrid have actually been selected over several generations for HAM genes. Another, designated as ar x Pa91ae, was included as well since Pa91ae has been found to typically have high-amylose (upper 60%). It yielded in the top 12 at Novelty, MO. Of the sixteen early lines evaluated (2011-01_SE32_S17_F2S4_9148-Blk22/00sib///GUAT209:S13//OH43ae/H99ae1-2-1) – 1 clearly performed best when used in hybrids and therefore is considered to have the best general combining ability as shown in Table 2. A yield trial is planned for the summer of 2007 that will include test hybrids made from parents that share GEMS-0067 as a grandparents
(see Figure 3) rather than as a parent as was done this year.
Table 2. General combining ability (determined from data in Table 3) shown for the sixteen different ‘early’ GEM
Amylomaize inbreds that are homozygous for ae but segregating HAM genes. The number of different hybrids the lines used in is indicated by ‘n’. Average yield and agronomic performance for each line when used as a hybrid is provided (Blue-SS;Red-NS).
STAND
SK
LDG
RT
LDG
PL
THT
EAR
HT
Plot
weight Yield
Moisture
Y/M
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(CUBA164:S1511b-325-1-B///GUAT209:S13//OH43ae/H99ae 1-2-1 ) - 1
(SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(AR16035:S02-450-1-B-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1
(FS8B(T):N1809-946-1-B-B///GUAT209:S13//OH43ae/H99ae1-1-1 ) - 1
(DKXL370:N11a20-322-B///GUAT209:S13//OH43ae/H99ae6-2-1-1) - 1
(2152-03_DK888_S11_F2S4_9205-Blk24/00-sib///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(DKB844:S1601-3-2-B///GUAT209:S13//OH43ae/H99ae 4-4-2-1) - 1
CUBA164:S1511b-325-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(FS8A(S):S09-362-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1
(CHIS740:S1411a-783-2-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
AR03056:N09-182-1-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(AR16026:S1704-153-1-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) – 1
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M . Campbell 06 Report
Table 3. Pooled Yield and agronomic data from 49 GEM-based high amylose hybrids made with inbred parents homozygous for ae and possessing (but not yet fixed) HAM genes and a commercial Amylomaize VII check from 2006 yield trials in Greenly, MO and Ames, IA
(Blue-SS;Red-NS).
parent 1
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(2152-03_DK888_S11_F2S4_9205-Blk24/00-sib///GUAT209:S13//OH43ae/H99ae 6-2-2-1) - 1
(2152-03_DK888_S11_F2S4_9205-Blk24/00-sib///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(2152-03_DK888_S11_F2S4_9205-Blk24/00-sib///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(2152-03_DK888_S11_F2S4_9205-Blk24/00-sib///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(AR16026:S1704-153-1-B///GUAT209:S13//OH43ae/H99ae 2-1-1 ) - 1
(AR16026:S1704-153-1-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(AR16026:S1704-153-1-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(AR16035:S02-450-1-B-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
X
X
X
X
X
X
X
X
X
X
X
X
(AR16035:S02-611-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(AR16035:S19-285-1-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CHIS740:S1411a-783-2-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(CHIS740:S1411a-783-2-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
CUBA164:S1511b-325-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
X
X
X
X
X
(CUBA164:S2012-444-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1 X
(CUBA164:S2012-444-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(CUBA164:S1511b-325-1-B///GUAT209:S13//OH43ae/H99ae 1-2-1 ) - 1
X
X
(CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
X
(CUBA164:S2012-966-1-B-B /// A61GUAT209:S13//OH43ae/H99ae 1-2-1 X
(CUBA164:S2012-966-1-B-B /// A61GUAT209:S13//OH43ae/H99ae 1-2-1 X
(CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
X
X
(CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(DKB844:S1601-3-2-B///GUAT209:S13//OH43ae/H99ae 4-4-2-1) - 1
X
X
(DKB844:S1601-3-2-B///GUAT209:S13//OH43ae/H99ae 4-4-2-1) - 1
(DKXL370:N11a20-322-B///GUAT209:S13//OH43ae/H99ae6-2-1-1) - 1
(FS8A(S):S09-362-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1
(FS8A(S):S09-362-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1
(UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
X
X
X
X
X
(UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
UR13085:N0215-14-1-B-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1
AR03056:N09-182-1-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
X
X
X
X
X
UR13085:N0215-14-1-B-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(DKXL370:N11a20-322-B///GUAT209:S13//OH43ae/H99ae6-2-1-1) - 1
CUBA164:S1511b-325-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
X
X
X
X
X
X
X
X
X
X parent 2
(DKXL370:N11a20-36-2-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1
(CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 1-2-1) - 1
(DKXL370:N11a20-322-B///GUAT209:S13//OH43ae/H99ae6-2-1-1) - 1
(SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(DKXL370:N11a20-322-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(DKXL370:N11a20-322-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(DKXL370:N11a20-199-2-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(FS8B(T):N11a-322-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
DKXL370:N11a20-234-2-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(FS8B(T):N11a-322-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(AR03056:N09-182-1-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-2-1) - 1
(2152-03_DK888_S11_F2S4_9205-Blk24/00-sib///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(DKXL370:N11a20-199-2-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(FS8B(T):N11a-322-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(DKXL370:N11a20-322-B///GUAT209:S13//OH43ae/H99ae6-2-1-1) - 1
(CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(DKXL370:N11a20-322-B///GUAT209:S13//OH43ae/H99ae 2-1-1 ) - 1
(UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(FS8B(T):N1809-946-1-B-B///GUAT209:S13//OH43ae/H99ae1-1-1 ) - 1
(SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CUBA164:S1511b-325-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(CHIS740:S1411a-783-2-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(CHIS740:S1411a-783-2-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(FS8A(S):S09-362-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1
(UR13085:N0215-14-1-B-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(2152-03_DK888_S11_F2S4_9205-Blk24/00-sib///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(AR16035:S02-450-1-B-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(CUBA164:S1511b-325-1-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(DKB844:S1601-3-2-B///GUAT209:S13//OH43ae/H99ae 2-1-1 ) - 1
(FS8A(S):S09-362-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1
(UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1 ar x pa91 ar x dk check average
LSD
STAND
SK
LDG
RT
LDG
PLT
HT
EAR
HT
Plot
weight Yield Moisture ba g no ent ry
(cm) (cm) (lbs/plot) (bu/ac) (%)
50.0 3.3
0.0
253.8
108.8
11.7
99.7
16.1
1 1
47.0 2.3
0.3
267.5
126.3
11.7
99.8
17.0
2 2
51.8 1.5
0.8
265.0
127.5
11.0
91.3
17.2
3 3
57.5 4.8
1.3
268.8
132.5
14.1
120.9
15.2
4 4
53.8 2.5
0.8
245.0
113.8
9.6
82.4
15.2
6 5
56.8 3.8
1.8
257.5
120.0
11.0
94.3
16.0
7 6
53.3 2.8
0.8
262.5
122.5
10.1
85.1
16.7
8 7
47.0 7.0
0.8
242.5
110.0
9.7
44.0 4.8
0.0
253.8
112.5
9.0
82.8
16.0
9 8
76.6
16.5
11 9
49.8 1.8
0.5
261.3
120.0
10.2
84.5
18.6
13 10
42.5 2.3
0.5
253.8
121.3
8.1
69.3
16.2
14 11
54.0 2.3
0.5
246.3
121.3
11.7
98.8
16.3
15 12
51.0 0.8
0.0
246.3
127.5
10.9
92.7
17.6
17 13
51.0 3.5
0.5
237.5
123.8
10.0
82.5
18.9
18 14
47.0 2.3
0.0
240.0
101.3
10.4
88.8
16.2
20 15
53.0 1.3
0.0
257.5
115.0
10.3
88.7
15.9
21 16
48.3 2.5
0.0
241.3
111.3
10.1
87.2
16.0
23 17
53.8 1.8
0.8
257.5
115.0
9.7
81.6
16.4
24 18
49.3 1.8
0.0
257.5
122.5
10.3
86.0
17.0
25 19
50.8 2.3
1.3
248.8
113.8
11.7
99.5
15.9
26 20
54.0 3.0
0.5
262.5
125.0
11.4
98.2
15.6
28 21
50.3 2.0
0.3
258.8
116.3
11.1
95.1
16.2
29 22
47.8 3.0
0.5
292.5
138.8
10.4
90.3
15.1
30 23
41.5 3.3
0.0
250.0
110.0
10.3
88.4
15.8
31 24
43.5 3.8
0.5
235.0
90.0
10.4
89.1
16.4
32 25
51.0 2.0
0.3
258.8
113.8
11.9
102.0
15.7
34 26
48.8 2.3
0.0
266.3
128.8
10.1
85.1
17.3
38 27
49.5 2.5
0.0
255.0
116.3
10.3
88.7
15.7
39 28
46.3 3.0
1.0
253.8
126.3
11.2
94.6
16.9
42 29
48.8 2.0
0.0
233.8
103.8
8.4
72.7
14.5
46 30
53.5 3.0
0.5
253.8
117.5
12.3
105.4
17.2
47 31
49.8 4.3
0.8
236.3
107.5
10.8
93.8
14.7
50 32
44.8 2.8
0.0
238.8
108.8
9.9
85.9
16.2
51 33
48.8 2.0
0.8
258.8
127.5
10.7
90.2
17.6
52 34
41.8 1.8
0.5
275.0
126.3
9.8
81.7
17.5
53 35
55.8 6.8
2.3
275.0
121.3
12.4
102.6
17.4
55 36
47.3
46.5
1.0
2.5
0.0
0.5
253.8
111.3
8.5
245.0
115.0
9.6
74.0
15.5
56 37
81.5
16.6
57 38
52.0 1.5
0.5
251.3
130.0
10.1
86.0
16.8
59 39
39.0 2.3
0.0
251.3
108.8
10.0
83.7
17.4
60 40
50.0 6.5
2.3
247.5
116.3
9.3
80.6
14.7
61 41
48.5 2.8
0.0
238.8
121.3
10.5
87.8
17.6
62 42
50.5 1.5
1.3
265.0
121.3
10.3
88.1
16.7
63 43
51.8 2.5
1.8
257.5
121.3
11.6
100.7
15.2
64 44
46.8 2.8
0.0
241.3
113.8
9.8
50.3 2.8
0.0
255.0
126.3
8.0
84.6
15.6
65 45
69.0
15.0
66 46
51.8 2.3
2.5
252.5
116.3
12.3
105.8
15.4
67 47
47.0 1.5
0.0
262.5
142.5
12.1
100.2
17.5
80 48
53.8 1.5
0.0
275.0
120.0
14.1
116.1
19.0
81 49
52.3 3.3
0.8
222.5
101.3
10.5
91.0
14.7
82 50
49.5 ns
2.7
ns
0.6
ns
253.7
118.2
10.6
37.4 3.2
90.1
27.4
16.4
2.3
Page 7 of 11

M . Campbell 06 Report
Table 4. Top twelve hybrids from each of the 2006 yield trial location (Greenley, MO and Ames, IA) made with inbred parents homozygous for ae and possessing, but not yet fixed, HAM genes and a commercial Amylomaize VII check.
Top 12 yield and check
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1- X (CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 1-2-1) - 1 ar x dk
STA
ND
SK
LDG
SK
LDG
RT
LDG
RT
LDG
PLT
HT
EAR
HT
Plot weight
(lbs/plot)
Yield
(bu/ac)
Moisture
(%) ave 47.7
1.3
1.0
222.3 103.9
13.2
111.3
17.4
min 34.0
0.0
0.0
192.5
7.7
65.4
14.5
max 59.0
8.0
5.0
265.0 150.0
19.9
170.7
21.5
STA
ND
PLT
HT
EAR
HT
Plot weight
(lbs/plot)
Yield
(bu/ac)
Moisture
(%)
(UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1 X (UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(2152-03_DK888_S11_F2S4_9205-Blk24/00-sib///GUAT209:S13//OH43ae/H99ae X (SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1 X (UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CUBA164:S1511b-325-1-B///GUAT209:S13//OH43ae/H99ae 1-2-1 ) - 1 X (CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-2-1) - 1 check
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1-2-1) - 1 X (UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1
(CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1 X (2152-03_DK888_S11_F2S4_9205-Blk24/00-sib///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1- X (DKXL370:N11a20-36-2-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1 X (AR16035:S02-450-1-B-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1
(CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1 X (CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
58 2.5
1.5
245.0 127.5
47 240.0 100.0
52.5
8 4.5
232.5
97.5
59 227.5 110.0
19.9 170.7
19.6
157.2
18.1
16.2
147.9
138.2
52 2.5
5.0
222.5 105.0
53.5
0 2.5
220.0
97.5
45.5
1.5
0.0
205.0 110.0
49 1.5
0.5
220.0
85.0
15.3
15.4
131.1
130.9
50.5
50.5
0
1
1.5
1.5
58
212.5
87.5
240.0 120.0
220.0 100.0
14.8
15.6
14.9
129.0
127.9
127.8
53.5
2.5
0.0
220.0
85.0 14.8
125.7
15.2 124.8
14.4
123.7
16.1
21.5
20.2
16.9
16.5
17.1
14.8
19.8
16.4
17.0
19.7
16.1
Top 12 yield and check
STA
ND
SK
LDG
SK
LDG
RT
LDG
RT
LDG
PLT
HT
EAR
HT ave 51.2
4.1
0.1
285.2 132.5
min 40.0
1.0
0.0
232.5 110.0
max 61.5 11.5 1.5
320.0 157.5
STA
ND
PLT
HT
EAR
HT
(FS8A(S):S09-362-1-B///GUAT209:S13//OH43ae/H99ae 2-2-2) - 1 X (UR13085:N0215-14-1-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1
(UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae1-2-1) - 1 X (SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1 X (CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CH05015:N12-183-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1 X (UR10001:S1813-257-1///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1 X (CH05015:N15-3-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(CHIS740:S1411a-783-2-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1 X (FS8B(T):N11a-322-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1
(2011-01_SE32_S17_F2S4_9148-Blk22/00-sib///GUAT209:S13//OH43ae/H99ae1- X (SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
CUBA164:S1511b-325-1-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1 X DKXL370:N11a20-234-2-B-B-B /// GUAT209:S13//OH43ae/H99ae 1-2-1 ar x pa91
(CUBA164:S2012-966-1-B///GUAT209:S13//OH43ae/H99ae7-2-2-1) - 1 X (SCR01:N1310-358-1-B-B///GUAT209:S13//OH43ae/H99ae 6-2-1-1) - 1
(AR16035:S02-450-1-B-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1 X (DKXL370:N11a20-322-B///GUAT209:S13//OH43ae/H99ae1-2-1) - 1 ar x dk check
53
47
52.5
53
54
44.5
4.5
51.5
2
0.0
4.5
0.0
252.5 122.5
4 0.0
0.0
282.5 127.5
2.5
0.0
297.5 142.5
55
4.5
0.5
305.0 137.5
3 0.0
44.5
3
290.0 135.0
290.0 140.0
292.5 137.5
280.0 135.0
0.0
290.0 135.0
40 277.5 125.0
61.5
4 0.5
275.0 140.0
60.5
3 0.0
310.0 140.0
54 6.5
0.0
232.5 115.0
Plot weight
(lbs/plot)
Yield
(bu/ac)
8.0
5.9
10.8
69.0
50.5
93.2
Plot weight
(lbs/plot)
Yield
(bu/ac)
Moisture
(%)
15.3
13.5
17.3
Moisture
(%)
10.8 93.2
9.4 81.1
9.4 81.1
9.4
9.3
9.3
9.2
80.6
80.2
17.3
16.8
15.5
14.4
15.3
79.8
15.3
79.4
16.8
9.2
9.2
9.1
8.8
8.7
6.2
79.4
79.4
15.7
14.3
78.5
15.7
75.5
75.0
53.0
14.5
16.5
14.6
Page 8 of 11

M. Campbell 06 Report
are shown in Figure 6 . These preliminary data suggest, at this point, that the gene action is strongly additive.
Figure 5 . Generation means analysis will include the following nine generations.
Generation Means Analysis
A generation means analysis (GMA) experiment is currently being conducted at SDSU. An experimental design as described by Bogyo et al
(1987, Heredity 60:61) will be used that takes advantage of the triploid nature of the endosperm which influences the starch phenotype. Results from this study should shed some light on the gene action of the high amylose modifier genes.
During the summer of 2006, nine generations, as described in Figure 5, were grown in a randomized complete block design in both
Kirksville, MO and Brookings, SD. The GMA is currently being done between three crosses
(H99ae x GEMS-0067, B99ae x GEMS-0067 and H108ae x GEMS-0067). We are currently processing these materials for laboratory analysis. Preliminary data was collected from material grown in a similar experiment in 2005 in South Dakota with only seven of the nine generations. The results for the amylose values
Figure 6. Results from a preliminary Generation Means Analysis using 7 of 9 possible generations
(shown in Figure 5) from ears produced in South Dakota during the summer of 2005 .
SD Linear (SD) y = 2.6179x + 56.571
R
2
= 0.9205
90.0
80.0
70.0
60.0
50.0
P1 (H99ae) BC1P1 F1 F2 F1r BC1P2 P2 (GEMS-
0067)
Generations
Page 9 of 11

M. Campbell 06 Report
QTL analysis of high amylose modifier (HAM) genes using SSR marker analysis.
Development of a mapping population : A mapping population derived from the cross of the two parents H99ae (55% amylose) and
GEMS-0067 (70% amylose) as been made and starch examined. Phenotypic data has already been collected from over 300 selfed F2 ears grown in 2005 in Brookings, SD and Kirksville,
MO. This phenotypic data will be merged with
SSR marker data and QTL analysis performed as described by Song et al. 2004. QTL Mapping of
Kernel Oil Concentration with High-oil Maize by SSR Markers. Maydica. 49: 41-48.
Figure 7. Distribution of starch amylose values among F2 ears from the cross between
H99ae x GEMS-0067. (n=315 in SD and n=283 in MO )
Identification of polymorphic SSR markers for the mapping populations: We have identified 46
SSR markers out of a total of 384 screened that are polymorphic for the parents (H99ae and GEMS-0067) and F1. There are 10, 4, 6, 4, 2, 4, 6, 3, 3 and 4 SSR markers on Chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 respectively. The location of these SSR marker loci are shown on the maize linkage groups in Figure 9.
We are currently using a Maize DNA extraction procedure using the CTAB method which works well. A subset (n=151) of F2 individuals grown in South Dakota (2005) ranging in amylose content from 53.7% to
82.7% and displaying a normal distribution will be used for SSR screening and QTL analysis. An example of a segregating SSR marker (p-454) for 23 of these F2 individuals is shown in Figure 10 where the polymorphism can be seen. P1(H99ae) has the upper band which is about 260 bp, P2 (GEMS-0067) has the lower band which is about 250 bp and the F1 has both (not shown). Seven SSR markers have been applied to the population which include p-1397 (bin: 1.03), p-1298 (bin: 1.09), and p-454 (bin: 1.11) on
Chromosome 1; p-100 (bin: 2.01) on Chromosome 2; p-1780 (bin: 3.01) on Chromosome 3; p-2313 (bin:
6.01) on Chromosome 6; p-1170 (bin: 9.02) on Chromosome 9. The molecular data have been collected, but not yet analyzed.
Particular attention will be given to the gene loci sh1, sh2, bt1, bt2, wx1 , su2 and su1 occurring in bin 9.01, 3.09, 4.05, 9.03, 5.04, and
4.05. Since these genes play a role in starch synthesis, they may be allelic to HAM genes.
Therefore, additional searches for SSR markers will be prioritized near these loci. Map manger
QTX and QTL cartographer will be used to find
QTLs of interest. Following data analysis, additional SSR markers will be identified on relevant chromosomes to narrow down the QTL associated with HAM genes.
F igure 8. Loci and gene products known to be involved in starch biosynthesis that will be considered when searching for QTLs serving as HAM genes
.
-
Page 10 of 11

M. Campbell 06 Report
Figure 9. Location of 46 polymorphic SSR markers currently identified for the mapping population derived from the cross between H99ae (55% amylose) and Guat209//H99ae/Oh43ae 1-2-1 (70% amylose) and nine ‘target’ loci (su2, sh2,su1, bt2, sh1, wx, bt1 and du) we intend to cover with additional markers. (Note: cM represents genetic distance based on IBM population).
Figure 10 Data set sample: Polymorphisms of the of p-454 SSR marker among F2 (1-23) individuals in the mapping population derived from the cross between parents H99ae (55% amylose) and
Guat209//H99ae/Oh43ae 1-2-1 (70% amylose).
Page 11 of 11