Germplasm Enhancement of Maize

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GEM - 2005 Public Cooperator's Report

NOTE: The information in this report is shared cooperatively. The data are not published, but are presented with the understanding that they will not be used in publications without specific consent of the public cooperator.


Development of Amylomaize VII hybrids, potential for resistant starch production and QTL mapping of high-amylose modifying genes from GEM germplasm

Mark Campbell, Courtney Bonney and Laban Kipkurui Tabartet

Truman State University, Division of Science, Kirksville, MO

Summary Statement: 

The purpose of the Truman-Iowa State GEM cooperative research project is to develop amylomaizeVII maize hybrids and explore the potential of using this materials as a source for the production of high-amylose corn starch.   These hybrids are typically processed by wet-mills in order to produce high-amylose starch which has a number of food and industrial applications.  High-amylose corn possesses starch having an amylose content of greater than 50% compared to normal starch (25% amylose).  A number of discrete classifications of high-amylose corn exists based on their specific amylose content including Amylomaize V (50-60% amylose) and Amylomaize VII (70-80% amylose). In the US, around 50,000 acres of high-amylose corn are produced, however, there are some indications that this may greatly increase in the future. The starch from high-amylose corn is used in the textile industry, in gum candies, production of biodegradable packaging material and as an adhesive in the manufacturing of corrugated cardboard.  There are a number of new applications of high-amylose starches especially because of their ‘nuetracuetical’ value.  Specifically it can be used as a food coating to minimize fat uptake, and therefore caloric value, of fried foods.   In addition, many national and international food companies are looking towards amylomaize as a source of resistant starch (RS) in order to address consumer concerns with obesity since it has a very low glycemic index and is believed to reduce colon cancer since RS behaves as dietary fiber.

We have already found GEM germplasm to be a source of high-amylose modifying genes to increase amylose content to ~70% in the presence of the amylose-extender (ae) allele.  In addition, yield evaluations from several locations in a single season (2004) suggest that GEM germplasm may serve as a genetic source with high yield potential.   The breeding strategy described in this report aims to continue in the development of amylomaize VII hybrids in several ways.  First, we need to address our main limitations which are that our GEM-amylomaize VII lines share 50% of their germplasm, represent a mixed heterotic classification and, we currently rely on private amylomaize VII testers for hybrid evaluation which compicates its use by breeders and processors just entering the high-amylose starch markets. For this reason, our first objective is to develop amylomaize VII lines at Truman State University into independent heterotic groups including stiff stalk and non-stiff stalk backgrounds by crossing existing amylomaize VII GEM lines back to released GEM lines of know heterotic class.   Amylomaize VII inbred lines will then be tested using the bulk of pollen from lines of the opposite heterotic group to eliminate the need for a private tester.   Secondly, in order to evaluate the potential utility of the GEM-amylomaize VII hybrids, we have examined the fine structures through chromatographic separation of amylose from these starches and explored commercial production of resistant starches of this material using a novel extrusion process in the laboratory of Dr. Jane at Iowa State University.  And thirdly, in the laboratory of Dr. Yen and graduate student Yusheng Wu, at South Dakota State University, a QTL mapping study involving the use of SSR markers has been initiated in order to identify the number and chromosomal locations of quantitative modifying genes identified from the GEM germplasm sourse (GUAT209S13 x (OH43 x H99 ae)) which could accelerate breeding efforts of this material through Marker Assisted Selection (MAS).  In addition, we will also continue to investigate improved laboratory methods for selection of amylose and total starch using Near Infrared calibrations development and continue in the development of GEM containing high-amylose double-mutant genotypes (amylose-extender [ae] /sugary-2 [su2] and amylose-exteder [ae] /dull [du]) which may serve some additional low gelatinization food applications in industry. 

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I. Detailed analysis of amylomaize VII starch identified from GEM germplasm in Dr. Jay-Lin Jay’s collaborating laboratory. 

The data from Dr. Jane’s laboratory is extremely promising in that it confirms our data collected at Truman State using a rapid amylose screening technique (third column).  Together the data that suggests that GEM materials possess amylomaize VII-type starch within three sister lines of the pedigree GUAT209:S13 x (OH43ae x H99ae) and an abundance of resistant starch.  More information can be seen in the GEM cooperators report submitted by Dr. Jane from Iowa State University.


Table 1. A summary of starch structural characteristics conducted at Truman State and Iowa State.



Amylose Content (%) based on colorimetric analysis at Truman State

Resistant Starch as a % of total starch Iowa

 State University

Amylose based on GPC chromatography (second peak: amylose + intermediate material) Iowa State University







GUAT209:S13//OH43ae/H99ae  1-2-1-2















H99 ae





OH43 ae





B89 ae










II. Collaborative project using SSR – QTL analysis to identify high amylose modifying genes previously identified from GEM Germplasm.

Over the past year Yusheng Wu, a graduate student from SDSU has been working with our lab in order to identify polymorphisms among a set of SSR markers among the parents H99ae (60% amylose) and Guat209//H99ae/Oh43ae 1-2-1 (70% amylose).   These parents will then be used to develop a mapping population to identify high amylose modifier genes. Figure 1A shows a list of 46 SSR markers found to be polymorphic among these parents.  Our goal has been to identify at least 85 markers, as recommended by Dr. Yen of SDSU, to be used to map the high amylose modifiers genes having an apparent quantitative effect conditioning starch amylose near 70%.   Successful identification of high amylose QTLs could lead to the application of marker assisted selection (MAS) for high amylose breeding programs.  While SSR analysis will be accomplished at South Dakota State University, we at Truman plan to collect phenotypic data by analyzing grain samples harvested from approximately 300 F2 ears grown in Missouri and another 300 F2 ears grown in South Dakota during the summer of 2005.  Phenotypic data will primarily include starch amylose values.  In addition starch content data and starch thermal properties will also be collected.

Figure 1. A list of SSR markers identified to date showing polymorphism among the parents H99ae [60% amylose]  and Guat209//H99ae/Oh43ae 1-2-1 [70% amylose]  (A). Our mapping study will be modeled on a related study by Song et al.  2004. QTL Mapping of Kernel Oil Concentration with High-oil Maize by SSR Markers. Maydica. 49: 41-48.  (B) Samples of F2 ears prior to starch analysis.

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III.  Yield Evaluation of GEM amylomaize VII lines using proprietary testers.

A number of F3 lines derived from crosses between the high-amylose modifier source Guat209//H99ae/Oh43ae, Zia Peublo//H99ae/Oh43ae, and Cochiti Peublo//H99ae/Oh43ae onto several released GEM lines previously selected for high yield were crossed onto an OH43-type amylomaize VII tester and a Stiff-stalk-type amylomaize VII tester.  In addition a proprietary amylomaize VII check hybrid was included for comparison.  In general, many of the GEM containing hybrids greatly out yielded the proprietary check.  Amylose data in Table II represents hybrid grain samples analyzed from only the 2004 growing season that was collected from selfed hybrid plants.  Although many amylose values are below 70%, it is likely that continued inbreeding and selection could be made to fix the genes since they probably carry many of the modifying genes in these genetically variable, early lines.  Of most immediate interest is the hybrid CHIS775:N1912-519-1-B-B///GUAT209:S13//OH43ae/H99ae //// ss tester   yielding 100.0 bu/A with a 2004 amylose analysis of 69.3%


Table II. Pooled yield and agronomic characteristics of GEM x Proprietary tester hybrids grown in 2004 and 2005 in Ames, IA and Novelty, MO.  (For explanation of pedigree notation see appendix A)


IV. Preparation of seed for a 2006 yield trial of hybrids made from GEM x GEM amylomaize VII parents with a 50% common genetic background and the hybrid of SS-GEM amylomaize VII lines  x Pa91ae

As mentioned earlier, we are trying to address the issue of making GEM amylomaize VII material more accessible to cooperators.  This is because the unavailability amylomaize VII germplasm can pose a great limitation since existing germplasm is privately held and highly protected.  Therefore, material having undergone one generation of the ‘heterotic backcross’ making them either 75% SS or non-SS, and selected for the recessive ae allele in the F3 generation were crossed this summer to produce GEM x GEM test hybrids.   Although parents will share 50% of there germplasm, it is hoped that this may help in guiding our decisions for future testcrosses between GEM x GEM materials.  In addition, SS-types GEM materials were crossed to Pa91ae, a converted public line with relatively high amylose (upper 60%’s).

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Figure 2.  Production of testcrosses among SS (blue) and non-SS (red) GEM materials (A) and SS-GEM and Pa91ae (B).


V.  Preparation of seed for 2007 yield trial of GEM x GEM amylomaize VII parents with only a 25% common genetic background

Many of the F3 lines for yield analysis described in section IV may or may not have been intensively selected for high amylose modifiers.  This selection work will continue especially when lines with desirable yields have been identified.  Several lines, however, have undergone intensive selection and display high amylose content similar to the original high amylose source - Guat209//H99ae/Oh43ae.  It was decided that these were good candidates to use as donor lines for high-amylose modifying genes in the  second ‘heterotic backcross’ in order to accomplish the independent developments of SS and non-SS amylomaize VII lines.  Data collected prior to planting confirms the amylomaize VII type donor lines as shown in Table III.


Table III. F5 lines grown from seed analyzed for amylose content as shown and used as the donor of high amylose modifier genes in the heterotic backcross procedures and for test crossing with Pa91ae.


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The F5 lines with ‘confirmed amylose of  >70%’ were grown in the 2005 summer breeding nursery in Kirksville, MO and crossed onto a number of released GEM lines to be used as the recurrent parent for the second heterotic backcross (HB2).  The material is currently being advanced as HB2 F1’s in a winter nursery and following harvest HB F2 plants homozygous for ae will be inter-mated for testcrossing and be selected for high amylose modifiers in subsequent generations.  These lines will be composed of 87.5% GEM germplasm, 87.5% of the pedigree will be of a specific heterotic background and will share only 25% of their total germplasm.


Figure 3.  Breeding scheme showing the independent development of stiffstalk and non-stiff stalk lines using GEM germplasm in which we currently are in the process of conducting the second heterotic backcross (HB2).

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VI. Dissemination of GEM data over the past year

Publications describing GEM materials: 

Amylose Determination of Native High-Amylose Corn Starches by Differential Scanning Calorimetry. Nathan W. Polaske, Amanda L. Wood, Mark R. Campbell, Maria C. Nagan, Linda M. Pollak. STARCH – STÄRKE. Volume 57, Issue 3-4, 2005. Pages 118-123.

Field day describing GEM materials. 

Yield and quality evaluation of amylomaize VII test hybrids using tropical corn germplasm from the USDA Germplasm Enhancement of Maize (GEM) Program.  2005.  Mark Campbell, Anna O’Brien, Laban Kipkurui Tabartet and Courtney Bonney.  Pages 44 – 46.  2005 Field Day Report, Greenley Memorial Research Center (University of Missouri)


VII. Appendix A:  Explanation of pedigree notation

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We are grateful to our Cooperators for their support!


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