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United States Department of Agriculture

Agricultural Research Service

Research Project: Continued Development of Specialty Starch Maize and Identification of Novel Alleles Involved in Starch Biosynthesis Using Gem Germplasm

Location: North Central Regional Plant Introduction Station, Ames, Iowa

Project Number: 3625-21000-059-03
Project Type: Specific Cooperative Agreement

Start Date: Aug 01, 2014
End Date: Jul 31, 2015

Objective:
Two maize specialty starches are of primary interest in the Truman State University breeding program. This includes Amylomaize VII with a focus on its prebiotic Resistant Starch (RS), and Slowly Digested Starch (SDS) having a unique internal structure that delays enzyme degradation in food and may help stabilize blood glucose. The objectives of this study are 1) to conduct a comprehensive characterization of a set ~10 GEM Amylomaize VII RS inbreds, and 2) to identify a set of partially inbred GEM lines with particular combinations of alleles believed to result in SDS. For the first objective an allele specific PCR genotyping will be used to confirm the presence of amylose-extender (ae1) and starch branching enzyme-1 (sbe1:gm67) resulting in elevated Apparent Amylose (Am) and RS. Grain data will be obtained from a set of self-pollinated plants grown in at least one common environment and phenotypic measures of grain traits including: wet-chemistry proximate analyses (total starch, protein and oil), test wt., seed wt., seed density, hardness tests, IR spectroscopy, and novel image analysis techniques will be collected. Phenotypic measures of starch structural and functional properties including Am, column separation of amylose/amylopectin/intermediate materials, granule image analysis), and functional properties determined by differential scanning spectroscosposy (DSC) and RS measurements. For the second objective maize genotypes will be investigated to determine what sources best meet SDS standards. It is uncertain what maize genotype would best serve as an SDS source although the literature suggests that ae wx starch may be most effective from in vitro hydrolysis studies. Starch of ae wx, have a collapsed kernel phenotype and are entirely amylopectin having unusually long branch-chains forming internal double-helices not present in normal amylopectin. Partially inbred ae wx inbreds have been developed at Truman State with improved grain type and verification of alleles will be part of this study. Additionally, these are derived from GEM Amylomaize VII lines crossed with GEM wx conversions by Ingredion Inc., and segregating F3 ae kernels were selected and planted. Despite initial high mortality, a set of ae wx lines surviving continued inbreeding via natural selection were identified. Confirmation of the ae wx genotype with allele-specific PCR markers will be part of this research. Candidate ae wx lines will then be subjected to phenotypic characterization for seed quality traits and assessment of starches as potential sources of SDS. Future mapping studies could then be planned to identify modifiers responsible for improved grain quality.

Approach:
Objective 1: The Kirksville breeding nursery will continue to maintain and select RS Amylomaize VII. Introgression of exotic maize germplasm from LAMP, GEM, Allelic Diversity Material and other plant introductions will be the source for trait improvement. Inbreeding following selection from high throughput grain and starch analyses will be made. These will include Am, total starch, RS, hydrolysis rate, DSC, and spectral studies for quality attributes. In addition, the development and modification of other analyses will be examined including grain density (100-kernal/volume), analysis of seed to determine special variability within the endosperm and starch granule using of digital image analysis. Continued development of in vitro fermentation bioassays will be investigated to better predict prebiotic efficacy. Improvements in our ability to collect molecular marker data will be prioritized to verify the presence of alleles using allele specific polymerase chain reaction (PCR) markers for future mapping studies. Also, evaluation of test crosses for yield, agronomic traits, and plant health will be taken in Ames, IA and Kirksville, MO. Parent line increases and crosses will be made in isolations and crossing blocks in Kirksville, MO. Objective 2: Breeding to generate and maintain potential SDS lines derived from GEM crosses will continue. In the Ames nursery 40 rows of partially inbred ae wx genotypes will be included. Continue collection of starch and grain phenotypic data will be accomplished from existing SDS inbreds to observe the extent of variation in grain and starch traits among materials believed to be homozygous ae wx. Molecular marker data will be used to verify genotypes using public marker data or self-designed allele-specific primer information. Improvements in generating marker data will be a priority. Technologies will be adopted to simplify, reduce or eliminate exposure to hazardous materials, yet provide undergraduates the skills to generate marker data in-house. Verification of other possible SDS genotypes will also be required where marker analysis can establish the presence and influence of the sbe1:gm67 allele. A comprehensive analysis of grain quality traits will be taken and selected lines grown in common environments to determine the extent of phenotypic variation. Analysis will include the determination of in vitro starch hydrolysis rates. Also, variations in Am values among ae wx genotypes will be studied to determine if variation in iodine affinities among ae wx materials can provide a rapid means of estimating starch branch chain lengths. Together with starch hydrolysis rates, Am values will be studied to determine if they can be used as a rapid method to estimate of digestibility rates. For Ae wx sbe1:gm67 starch, alterations in amylopectin structure can be determined following debranching of starches subjected to beta-amyolysis and verify possible hyperbranched amylopectin.

Last Modified: 9/23/2014
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