2011 GEM Report: Truman State University, Annual GEM Cooperator meeting, ASTA, Dec. 7, Chicago, IL

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1 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 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::gems 67 ) 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::gems 67 ) for future release. 2. Identify other genetic factors that influence starch properties and grain quality of ae or the combination of ae and sbe 1 ::gems 67 a. Improve marker assisted selection for recovery of our major high-amylose modifier (sbe 1 ::gems 67 ) 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::gems 67 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::gems 67 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.

2 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.

3 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::gems 67. 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:S B-B///GEMS _SE32_S17_F2S4_9148/////CHIS775:S1911b B-B-B////AR16035:S B-B///GEMS-67 2 CHIS775:S1911b B-B-B////AR16035:S B-B///GEMS _SE32_S17_F2S4_9148-Blk22/00/////CUBA164:S1511b B-B////AR16035:S B-B///GEMS-67 4 CUBA164:S1511b B-B////AR16035:S B-B///GEMS-67 5 CUBA164:S B-B////AR16035:S B-B///GEMS-67 6 DKB844:S B-B-B-B-B/////CUBA164:S1511b B-B////AR16035:S B-B///GEMS _SE32_S17_F2S4_9148-Blk22/00/////DKB844:S B-B-B////AR16035:S B-B///GEMS-67 8 BR105:S B-B/////DKB844:S B-B-B////AR16035:S B-B///GEMS-67 9 DKB844:S B-B-B////AR16035:S B-B///GEMS _DK212T_S11_F2S4_9157-Blk29/00-sib-B-B-B/////DKB844:S B-B-B////AR16035:S B-B///GEMS _SE32_S17_F2S4_9148-Blk22/00////AR16035:S B-B///GEMS CHIS740:S1411a B-B////AR16035:S B-B///GEMS CHIS775:S1911b B-B-B-B/////CHIS740:S1411a B-B////AR16035:S B-B///GEMS DKB844:S B-B-B-B-B/////CHIS740:S1411a B-B////AR16035:S B-B///GEMS GUAT209:S13 08a B-B////CHIS740:S1411a B-B////AR16035:S B-B///GEMS _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS CHIS775:S1911b B-B-B-B//// _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS _SE32_S17_F2S4_9148-Blk22/00//// _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS CUBA164:S B-B-Sib//// _SE32_S17_F2S4_9148-Blk22/00-sib/// GEMS CHIS740:S11411a b-b-b//// _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS CUBA164:S B///GEMS CHRIS775:S1911b B-B-B////CUBA164:S B///GEMS CUBA164:S1511b B-B-B-B-B-Sib/////CHRIS775:S1911b B-B-B//// CUBA164:S B///GEMS _SE32_S17_F2S4_9148-Blk22/00////CUBA164:S B///GEMS FS8A(S):S B///GEMS _SE32_S17_F2S4_9148-Blk22/00////FS8A(S):S B///GEMS CHIS740:S11411a b-b-b/////CUBA164:S1511b B-B////FS8A(S):S B///GEMS BVIR155:S B-B////UR10001:S ///GEMS CHIS740:S11411a b-b-b////UR10001:S ///GEMS DKB844:S B-B-B-B-B////UR10001:S ///GEMS UR10001:S ///GEMS _SE32_S17_F2S4_9148-Blk22/00////UR10001:S ///GEMS UR11003:S b-b-b////UR10001:S ///GEMS BR51675:N ////UR10001:S ///GEMS BR52060:S b-b-b////UR10001:S ///GEMS GUAT209:S13 08a B-B////UR10001:S ///GEMS CUBA164:S2008c B-B/////AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS-67 38

4 NS Amylomaize Inbreds No. DKXL370:N11a B-B-SIB///GEMS-67 1 CH05015:N B-B////DKXL370:N11a B-B-SIB///GEMS /97_DK888N11F2S3_ b-b/////CH05015:N B-B////DKXL370:N11a B-B-SIB///GEMS-67 3 BARBGP2:N08a B-B-B/////CH05015:N B-B////DKXL370:N11a B-B-SIB///GEMS-67 4 DK212T:N11a B-B-B/////CH05015:N B-B////DKXL370:N11a B-B-SIB///GEMS-67 5 FS8B(T):N11a b-b-sib-b-b/////CH05015:N B-B////DKXL370:N11a B-B-SIB///GEMS-67 6 CH05015:N B-B-B////DKXL370:N11a B-B-SIB///GEMS-67 7 DKXL370:N11a B-B-B-Sib/////CH05015:N B-B////DKXL370:N11a B-B-SIB///GEMS-67 8 AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS-67 9 AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS CHO5015:N b-b-b/////AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS CHO5015:N b-b-b/////AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS CHO5015:N b-b-b/////AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS DKXL370:N11a B-B-B-Sib/////AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS BR51675:N /////AR03056:N B-B-B////DKXL370:N11a B-B-SIB///GEMS AR03056:N B-B-B-Sib/////DKXL370:N11a B-B-B////DKXL370:N11a B-B-SIB///GEMS CL-G1607(CML420):N /////DKXL370:N11a B-B-B////DKXL370:N11a B-B-SIB///GEMS DKXL370:N11a B-B-B////DKXL370:N11a B-B-SIB///GEMS MDI022:N21-B ///// DKXL370:N11a B-B-B////DKXL370:N11a B-B-SIB///GEMS DK212T:N11a B-B-B////DKXL370:N11a B-B-SIB///GEMS CH0515:N b-b-b/////UR13085:N B-B-B-B////DKXL370:N11a B-B-SIB///GEMS DK888:N11-B B-001/////DREP150:N2011d B-B////DKXL370:N11a B-B-SIB///GEMS DREP150:N2011d B-B////DKXL370:N11a B-B-SIB///GEMS FS8B T):N11a B-B////DKXL370:N11a B-B-SIB///GEMS UR13085:N B///GEMS CHO5015:N b-b-b/////UR13085:N B///GEMS /97_DK888N11F2S3_ b-b////UR13085:N B///GEMS BARBGP2:N08a b-b-b/////UR13085:N B///GEMS BR51675:N ////UR13085:N B///GEMS CL-G1607(CML420):N ////UR13085:N B///GEMS CH0515:N b-b-b////UR10001:S ///GEMS CH05015:N B-B///GEMS CH05015:N B-B////CH05015:N B-B///GEMS AR03056:N B-B-B////CH05015:N B-B///GEMS AR03056:N B-B-B////CH05015:N B-B///GEMS BR51403(PE001):N16-B G-003/////AR03056:N B-B-B////CH05015:N B-B///GEMS CH05015:N B-B-B/////AR03056:N B-B-B////CH05015:N B-B///GEMS CH05015:N B-B-B/////AR03056:N B-B-B////CH05015:N B-B///GEMS DK212T:N11a B-B-B/////AR03056:N B-B-B////CH05015:N B-B///GEMS DK212T:N11a B-B-B/////AR03056:N B-B-B////CH05015:N B-B///GEMS DKXL370:N11a B-B-B-Sib/////AR03056:N B-B-B////CH05015:N B-B///GEMS AR03056:N B-B-B-Sib/////AR03056:N B-B-B////CH05015:N B-B///GEMS BR51403(PE001):N16-B C-001////CH05015:N B-B///GEMS BR51721(RN07):N20-B ////CH05015:N B-B///GEMS CH05015:N B-B-B////FS8A(S):S B///GEMS DKXL370:N11a B-B-B-Sib////FS8A(S):S B///GEMS DKXL380:N11-B B-002/////CHRIS775:S1911b B-B-B////CUBA164:S B///GEMS SCR01:N B-B///GEMS-67 50

5 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:N B-B-B////DKXL370:N11a B-B-SI as a NS tester or CHIS775:S1911b B-B-B////AR16035:S B- 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 ( Bu/Ac) and 2011 (170.2 Bu/Ac) was observed for the hybrid CHIS775:S1911b B-B-B////AR16035:S B- X AR03056:N B-B-B-Sib/////AR03056:N B-B-B////CH05015:N B-. 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. WISE_international_soil_profile_data

6 Table 2. Pooled yield and agronomic performance of GEMN x GEMS amylomaize hybrids grown in Ames, IA 2011 and NS GEM amylomaize VII Tester Wt Yield MOIST Y/M TWT Stand Skldg Rtldg AR03056:N B-B-B////DKXL370:N11a B-B-SI Lb. (bu/ac) % lb/bu % % % CHRIS775:S1911b B-B-B////CUBA164:S _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS UR10001:S ///GEMS CHIS740:S1411a B-B////AR16035:S B CUBA164:S1511b B-B////AR16035:S B CUBA164:S1511b B-B////AR16035:S B CUBA164:S B-B////AR16035:S B DKB844:S B-B-B////AR16035:S B-B///GEM AR16035:S B-B/GEMS CUBA164:S CHIS775:S1911b B-B-B////AR16035:S B CUBA164:S B-B-Sib//// _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS CHIS775:S1911b B-B-B-B//// _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS GUAT209:S1308a B-B////UR10001:S ///GEMS DKB844:S B-B-B-B-B/////CUBA164:S1511b B-B////AR16035:S B _DK212T_S11_F2S4_9157-Blk29/00-sib-B-B-B/////DKB844:S B-B-B////AR16035:S B GUAT209:S1308a B-B/////CHIS740:S1411a B-B////AR16035:S B

7 SS GEM amylomaize VII Tester Wt Yield MOIST Y/M TWT Stand Skldg Rtldg CHIS775:S1911b B-B-B////AR16035:S B- Lb. (bu/ac) % lb/bu % % % AR03056:N B-B-B////CH05015:N B AR03056:N B-B-B////CH05015:N B CH05015:N B-B////CH05015:N B UR13085:N AR03056:N B-B-B////DKXL370:N11a B-B-SI AR03056:N B-B-B////DKXL370:N11a B-B-SI DKXL370:N11a B-B-B////DKXL370:N11a B-B-SI FS8BT:N11a B-B////DKXL370:N11a B-B-SI CH05015:N B-B////DKXL370:N11a B-B-SI DREP150:N2011d B-B////DKXL370:N11a B-B-SI DKXL370:N11a B-B-SI UR13085:N DKXL370:N11a B-B-SI DK212T:N11a B-B-B/////CH05015:N B-B////DKXL370:N11a B-B-SIB///GEM DK212T:N11a B-B-B/////AR03056:N B-B-B////CH05015:N B-B///GEM AR03056:N B-B-B-Sib/////AR03056:N B-B-B////CH05015:N B SS GEM amylomaize VII Tester Wt Yield MOIST Y/M TWT Stand Skldg Rtldg CUBA164:S1511b B-B////AR16035:S B- Lb. (bu/ac) % lb/bu % % % AR03056:N B-B-B////CH05015:N B CH05015:N B-B////CH05015:N B FS8BT:N11a B-B////DKXL370:N11a B-B-SI DKXL370:N11a B-B-SI AR03056:N B-B-B////CH05015:N B

8 Amylomaize/normal check hybrid AMY VII Check # AMY VII Check # Asgrow normal ave ave Entry (p-value) Year (p-value) Figure 4. Ears of hybrid CHIS775:S1911b B-B-B////AR16035:S B- X AR03056:N B-B-B-Sib/////AR03056:N B-B- B////CH05015:N B- displaying good ear quality in addition to being the top yielding amylomaize hybrid in both 2010 and 2011 in Ames, IA.

9 Table 3. Grain physical properties from two hybrid ears taken per plot in Ames, IA 2010 yield trial. NS GEM amylomaize VII Tester AR03056:N B-B-B////DKXL370:N11a B-B-SI Density (g/ml) Ear wt. (g) HKW (g) displacement vol (ml) Grit #200 separation % CHRIS775:S1911b B-B-B//// CUBA164:S _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS UR10001:S ///GEMS CHIS740:S1411a B-B////AR16035:S B-B///GEM CUBA164:S B-B////AR16035:S B CUBA164:S B-B////AR16035:S B-B///GEM CUBA164:S B-B////AR16035:S B DKB844:S B-B-B////AR16035:S B-B///GEM AR16035:S B-B/GEMS FS8AS:S B///GEMS CUBA164:S CHIS775:S1911b B-B-B////AR16035:S B CUBA164:S B-B-Sib//// _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS CHIS775:S1911b B-B-B-B//// _SE32_S17_F2S4_9148-Blk22/00-sib///GEMS GUAT209:S1308a B-B////UR10001:S ///GEMS DKB844:S B-B-B-B-B/////CUBA164:S1511b B-B////AR16035:S B _DK212T_S11_F2S4_9157-Blk29/00-sib-B-B-B/////DKB844:S B-B-B////AR16035:S B GUAT209:S1308a B-B/////CHIS740:S1411a B-B////AR16035:S B-B///GEMS BVIR155:S B-B////UR10001:S ///GEMS ave SS GEM amylomaize VII Tester Grit #200 CHIS775:S1911b B-B-B////AR16035:S B- Density Ear wt. HKW displacement separation AR03056:N B-B-B////CH05015:N B AR03056:N B-B-B////CH05015:N B CH05015:N B-B////CH05015:N B UR13085:N AR03056:N B-B-B////DKXL370:N11a B-B-SI DK212T:N11a B-B////DKXL370:N11a B-B-SI DKXL370:N11a B-B-B////DKXL370:N11a B-B-SIB///GEMS FS8BT:N11a B-B////DKXL370:N11a B-B-SI CH05015:N B-B////DKXL370:N11a B-B-SI DREP150:N2011d B-B////DKXL370:N11a B-B-SI DKXL370:N11a B-B-SI H99ae///GEMS UR13085:N DKXL370:N11a B-B-SI DK212T:N11a B-B-B/////CH05015:N B-B////DKXL370:N11a B-B-SI DK212T:N11a B-B-B/////AR03056:N B-B-B////CH05015:N B-B///GEM AR03056:N B-B-B-Sib/////AR03056:N B-B-B////CH05015:N B AR03056:N B-B-B////CH05015:N B ave

10 SS GEM amylomaize VII Tester CUBA164:S1511b B-B////AR16035:S B-B/// GEMS67 Density Ear wt. HKW 100 K displacem ent Grit #200 separation CH05015:N B-B////CH05015:N B-B///GEM AR03056:N B-B-B////DKXL370:N11a B-B-SI FS8BT:N11a B-B////DKXL370:N11a B-B-SI DREP150:N2011d B-B////DKXL370:N11a B-B-SI DKXL370:N11a B-B-SI UR13085:N DKXL370:N11a B-B-SI AR03056:N B-B-B////CH05015:N B UR13085:N DKXL370:N11a B-B-B////DKXL370:N11a B-B-SI ave K displacem Grit #200 ent separation Amylomaize/normal check hybrid Density Ear wt. HKW AMY VII Check # AMY VII Check # Normal Check ave overall ave Entry (p-value) Year (p-value) 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:N B-B-B////DKXL370:N11a B-B-SI x FS8AS:S B///GEMS-67 to 1.5 g/ml CUBA164:S1511b B-B////AR16035:S B- x DK212T:N11a B-B-B/////CH05015:N B-B////DKXL370:N11a B-B- SI. 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.

11 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). 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. Table 4. Nepali recipient populations for GEM amylomaize VII in 2011 Kirksville nursery 28. Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Ames Ames Zea mays subsp. mays (TUSA RANI) -- PI Zea mays subsp. mays No PI Zea mays subsp. mays (NGAWAL) -- PI Zea mays subsp. mays (SANKHU) -- PI Zea mays subsp. mays (MAJERIPATAN) -- PI Zea mays subsp. mays (POKHARA) -- PI Zea mays subsp. mays (KATHMMANDV VALLEY) -- PI Zea mays subsp. mays -- PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal 3903B -- PI Zea mays subsp. mays Nepal 3904B -- PI Zea mays subsp. mays Nepal 3905A -- PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI Zea mays subsp. mays Nepal PI

12 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::gems 67. 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 : _SE32_S17_F2S4 KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : _9148-Blk22/00-si ///GEMS67 KV 2010 : _DK212T_S11_ F2S4_9157-Blk29/00-sib-B-B- BGEMS-0006///// DKB844:S B-B-B//// AR16035:S B-B/// GEMS67 KV 2010 : AR03056:N B-B- B////(CH05015:N B- KV 2010 : AR03056:N B-B-B KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : ////DKXL370:N11a B-B- SI KV 2010 : B////DKXL370:N11a B- KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : AR03056:N B-B-B- KV 2010 : SibGEMN- 0089/////AR03056:N B-B-B////CH05015:N B- KV 2010 : AR03056:N B-B-B- KV 2010 : SibGEMN- 0089/////DKXL370:N11a20- KV 2010 : B-B- B////DKXL370:N11a B- B-SI KV 2010 : AR03056:N B-B-B- SibGEMN- 0089////DKXL370:N11a B-B-SI KV 2010 : AR03056:N B-B- KV 2010 : B////DKXL370:N11a B- B-SI KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : AR03056:N B-B-

13 KV 2010 : B////(CH05015:N B- KV 2010 : AR03056:N B-B- KV 2010 : B////DKXL370:N11a B- B-SI KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : AR03056:N B-B- B////DKXL370:N11a B- B-SI KV 2010 : BARBGP2:N08a B-B- KV 2010 : BGEMN- 0110/////CH05015:N B-B////DKXL370:N11a B-B-SI KV 2010 : BVIR155:S B- KV 2010 : BGEMS- 0175////UR10001:S KV 2010 : ///GEMS67 KV 2010 : CH05015:N B- B////(CH05015:N B- KV 2010 : CH05015:N B- KV 2010 : B////DKXL370:N11a B- B-SI KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : CH05015:N B-B- KV 2010 : BGEMN- 0112/////AR03056:N B- B-B////CH05015:N B- KV 2010 : CH05015:N B-B- BGEMN- 0112////DKXL370:N11a B-B-SI KV 2010 : CH05015:N B- KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : CHIS740:S1411a B- KV 2010 : B////AR16035:S B- KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : CHIS775:S1911b B-B- KV 2010 : B-BGEMS-0113//// _SE32_S17_F2S4_9148- Blk22/00-sib///GEMS67 KV 2010 : CHIS775:S1911b B-B- KV 2010 : B-BGEMS- 0113/////CHIS740:S1411a-783- KV 2010 : B-B////AR16035:S B- KV 2010 : CHIS775:S1911b B-B- KV 2010 : B////AR16035:S B- KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 :

14 KV 2010 : KV 2010 : CHRIS775:S1911b B-B- KV 2010 : B////(CUBA164:S KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : CUBA164:S1511b B-B- KV 2010 : B-B-B-SibGEMS- 0063/////CHRIS775:S1911b B-B- B////CUBA164:S KV 2010 : CUBA164:S1511b B- KV 2010 : B////AR16035:S B- KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : CUBA164:S B-B- KV 2010 : SibGEMS-0092//// _SE32_S17_F2S4_9148- Blk22/00-sib///GEMS67 KV 2010 : CUBA164:S KV 2010 : KV 2010 : KV 2010 : KV 2010 : CUBA164:S B- KV 2010 : B////AR16035:S B- KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : DK212T:N11a B-B- BGEMN- 0114/////AR03056:N B-B-B////CH05015:N B- KV 2010 : DKB844:S B-B-B- KV 2010 : B-BGEMS- KV 2010 : /////CUBA164:S1511b B-B////AR16035:S B- KV 2010 : DKB844:S B-B-B- KV 2010 : B-BGEMS- 0115////UR10001:S ///GEMS67 KV 2010 : DKB844:S B-B- KV 2010 : B////AR16035:S B- KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : DKXL370:N11a B-B- KV 2010 : B-SibGEMN- 0094/////AR03056:N KV 2010 : B-B-B////DKXL370:N11a KV 2010 : B-B-SI KV 2010 : DKXL370:N11a B-B- KV 2010 : B////DKXL370:N11a B- B-SI KV 2010 : KV 2010 : KV 2010 : KV 2010 : DKXL370:N11a B-B- KV 2010 : SI KV 2010 : KV 2010 : KV 2010 : DREP150:N2011d B- KV 2010 : B////DKXL370:N11a B- B-SI KV 2010 : KV 2010 : KV 2010 : KV 2010 : FS8A(S):S KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : FS8B(T):N11a B- KV 2010 : B////DKXL370:N11a B- B-SI KV 2010 : GUAT209:S13 08a B- BGEMS- 0184////CHIS740:S1411a-783-

15 2-B-B////AR16035:S B- KV 2010 : H99 ae ///GEMS67 KV 2010 : SCR01:N B-B ///GEMS67 KV 2010 : UR10001:S KV 2010 : ///GEMS67 KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : KV 2010 : UR13085:N KV 2010 : KV 2010 : KV 2010 : Recovery of amylomaize VII genotypes grown in Indiana summer of 2010 IN: AR03056:N B-B-B//// CH05015:N B- IN: AR03056:N B-B- B////DKXL370:N11a B- B-SI IN: AR03056:N B-B-B//// IN: CH05015:N B- IN: AR03056:N B-B- IN: B////DKXL370:N11a B- B-SI IN: AR16035:S B- IN: IN: CH05015:N B-B//// CH05015:N B- IN: CH05015:N B-B//// IN: CH05015:N B- IN: CH05015:N B- B////DKXL370:N11a B- B-SI IN: CH05015:N B- IN: IN: DKXL370:N11a B-B- B////DKXL370:N11a B- B-SI IN: IN: FS8B T):N11a B- IN: B////DKXL370:N11a B- B-SI IN: IN: IN: IN: SCR01:N B- IN: IN: IN: IN: IN: IN: IN:

16 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 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.

17 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.

18 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.

19 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 Pedigree UR13085:N B GM0310/GEMS DKXL370:N11a BGM0308/////CUBA164:S B-Bsib////(CUBA164:S B/GEMS DKXL370:N11a B GM0308/////CHIS740:S1411a B B////(CUBA164:S B/GEMS SCR01:N B GM0309/////DKB844:S B-B-B////(FS8A(S):S B/GEMS UR13085:N B GM0310/////UR11003:S B- B////(CUBA164:S B//GEMS FS8B(T):N B GM0306/////UR11003:S B-B////(CUBA164:S B/GEMS DKXL370:N11a B GM0308/////CHIS740:S1411a B- B////(FS8A(S):S B/GEMS SCR01:N B GM0309/////UR11003:S B- B////(FS8A(S):S B/GEMS DKXL370:N11a B GM0308/GEMS DKXL370:N11a B GM0308/////FS8B(T):N11a-87-1-B-B////(CH05015:N B-B/GEMS , Cuba164:S B GF0303/////FS8B(T):N11a B-B ////(CH05015:N B-B/GEMS-67 AR16035:S B GF0301/////UR11003:S B-B ////(CUBA164:S B/GEMS-67 FS8A(S):S B GF0305/////AR03056:N B-B-B ////(CH05015:N B-B//GEMS-67 DK844:S B GF0304/GEMS Cuba164:S B GF0303/////BARBGP2:NO8a B-B////(CH05015:N B-B/GEMS FS8A(S):S B GF0305/////CHRIS775:S1911b B-B-B ////(CUBA164:S B/GEMS Cuba164:S B GF0303/////DKXL370:N11a B-B-B ////CH05015:N B-B/GEMS FS8B(T):N B GM0306/////UR11003:S B-B////(CUBA164:S B/GEMS Cuba164:S B GF0303/////UR11003:S B-B ////(FS8A(S):S B/GEMS DK844:S BGF0304/////DKB844:S B-B B////(FS8A(S):S B /GEMS AR16035:S B GF0301/////CUBA164:S B-B////(CUBA164:S B/GEMS-67

20 Cuba164:S B GF0303/////AR03056:N B-B- B////(CH05015:N B-B/GEMS FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(CH05015:N B-B/GEMS FS8A(S):S B GF0305/////BARBGP2:NO8a B B////(FS8A(S):S B/GEMS AR16035:S B GF0301/////DK212T:N11a B-B////(CH05015:N B-B/GEMS Cuba164:S B GF0303/////CUBA164:S1511b B B////(FS8A(S):S B/GEMS DK844:S B GF0304/////UR11003:S B B////(FS8A(S):S B/GEMS Cuba164:S B GF0303/////UR11003:S B- B////(CUBA164:S B/GEMS AR16035:S B GF0301/////CUBA164:S1511b B-B////(FS8A(S):S B/GEMS Cuba164:S B GF0303/GEMS FS8A(S):S B GF0305/////UR11003:S B B////(FS8A(S):S B/GEMS Cuba164:S B GF0303/////CH05015:N B-B////(CH05015:N B-B/GEMS AR16035:S B GF0301/////DKB844:S B-B B////(FS8A(S):S B/GEMS FS8A(S):S B GF0305/GEMS DK844:S B GF0304/////CUBA164:S B-B////(FS8A(S):S 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. X S CV N Cuba164:S B GF0303/////FS8B(T):N11a B-B////(CH05015:N B DKXL370:N11a B GM0308/////FS8B(T):N11a-87-1-B-B////(CH05015:N B FS8A(S):S B GF0305/////CHRIS775:S1911b B-B-B////(CUBA164:S FS8A(S):S B GF0305/////AR03056:N B-B-B////(CH05015:N B DK844:S B GF0304/////CUBA164:S B-B////(FS8A(S):S DKXL370:N11a BGM0308/////CUBA164:S B-B-sib////(CUBA164:S Cuba164:S B GF0303/////AR03056:N B-B-B////(CH05015:N B Cuba164:S B GF0303/////FS8B(T):N11a B-B////(CH05015:N B FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(FS8A(S):S Cuba164:S B GF0303/////AR03056:N B-B-B////(CH05015:N B AR16035:S B GF0301/////DK212T:N11a B-B////(CH05015:N B FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(CH05015:N B FS8A(S):S B GF0305/////CHRIS775:S1911b B-B-B////(CUBA164:S AR16035:S B GF0301/////UR11003:S B-B////(CUBA164:S AR16035:S B GF0301/////DKB844:S B-B-B////(FS8A(S):S FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(CH05015:N B DKXL370:N11a BGM0308/////CUBA164:S B-B-sib////(CUBA164:S

21 Cuba164:S B GF0303/////FS8B(T):N11a B-B////(CH05015:N B DKXL370:N11a B GM0308/////CHIS740:S1411a B-B////(CUBA164:S FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(CH05015:N B Cuba164:S B GF0303/////DKXL370:N11a B-B-B////(CH05015:N B Cuba164:S B GF0303/////BARBGP2:NO8a B-B////(CH05015:N B DK844:S BGF0304/////DKB844:S B-B-B////(FS8A(S):S Cuba164:S B GF0303/////CHRIS775:S1911b B-B-B////(CUBA164:S DKXL370:N11a BGM0308/////CUBA164:S B-B-sib////(CUBA164:S Cuba164:S B GF0303/////DKXL370:N11a B-B-B////(CH05015:N B DKXL370:N11a B GM0308/////FS8B(T):N11a-87-1-B-B////(CH05015:N B FS8A(S):S B GF0305/////AR03056:N B-B-B////(CH05015:N B FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(CH05015:N B FS8A(S):S B GF0305/////AR03056:N B-B-B////(CH05015:N B 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 Amylose IA DKXL370:N11a BGM0308/////CUBA164:S B-B-sib////(CUBA164:S B/// GEMS IA10-56(51)-1 FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(CH05015:N B-B/// GEMS IA Cuba164:S B GF0303/////FS8B(T):N11a B-B////(CH05015:N B-B/// GEMS IA FS8A(S):S B GF0305/////AR03056:N B-B-B////(CH05015:N B-B/// GEMS IA DK844:S B GF0304/////CUBA164:S B-B////(FS8A(S):S B/// GEMS IA Cuba164:S B GF0303/////AR03056:N B-B-B////(CH05015:N B-B/// GEMS IA Cuba164:S B GF0303/////FS8B(T):N11a B-B////(CH05015:N B-B/// GEMS IA FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(FS8A(S):S B/// GEMS IA Cuba164:S B GF0303/////AR03056:N B-B-B////(CH05015:N B-B/// GEMS IA AR16035:S B GF0301/////DK212T:N11a B-B////(CH05015:N B-B/// GEMS IA FS8A(S):S B GF0305/////CHRIS775:S1911b B-B-B////(CUBA164:S B/// GEMS IA AR16035:S B GF0301/////UR11003:S B-B////(CUBA164:S B/// GEMS IA AR16035:S B GF0301/////DKB844:S B-B-B////(FS8A(S):S B/// GEMS IA FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(CH05015:N B-B/// GEMS IA Cuba164:S B GF0303/////UR11003:S B-B////(CUBA164:S B/// GEMS IA Cuba164:S B GF0303/////DKXL370:N11a B-B-B////(CH05015:N B-B/// GEMS IA Cuba164:S B GF0303/////CUBA164:S1511b B-B////(FS8A(S):S B/// GEMS IA Cuba164:S B GF0303/////BARBGP2:NO8a B-B////(CH05015:N B-B/// GEMS IA DK844:S BGF0304/////DKB844:S B-B-B////(FS8A(S):S B/// GEMS IA Cuba164:S B GF0303/////CHRIS775:S1911b B-B-B////(CUBA164:S B/// GEMS IA DKXL370:N11a BGM0308/////CUBA164:S B-B-sib////(CUBA164:S B/// GEMS IA Cuba164:S B GF0303/////DKXL370:N11a B-B-B////(CH05015:N B-B/// GEMS IA DKXL370:N11a B GM0308/////FS8B(T):N11a-87-1-B-B////(CH05015:N B-B/// GEMS IA AR16035:S B GF0301/////DKB844:S B-B-B////(FS8A(S):S B/// GEMS

22 IA FS8A(S):S B GF0305/////AR03056:N B-B-B////(CH05015:N B-B/// GEMS IAT FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(CH05015:N B-B/// GEMS IA FS8A(S):S B GF0305/////AR03056:N B-B-B////(CH05015:N B-B/// GEMS IA DKXL370:N11a BGM0308/////CUBA164:S B-B-sib////(CUBA164:S B/// GEMS IA FS8A(S):S B GF0305/////CHRIS775:S1911b B-B-B////(CUBA164:S B/// GEMS KV10-R DKXL370:N11a BGM0308/////CUBA164:S B-B-sib////(CUBA164:S B///GGEMS KV10-R DKXL370:N11a B GM0308/////CHIS740:S1411a B-B////(CUBA164:S KV10-R DKXL370:N11a B GM0308/////CHIS740:S1411a B-B////(CUBA164:S KV10-R SCR01:N B GM0309/////DKB844:S B-B-B////(FS8A(S):S KV10-R UR13085:N B GM0310/////UR11003:S B-B////(CUBA164:S KV10-R FS8B(T):N B GM0306/////UR11003:S B-B////(CUBA164:S KV10-R DKXL370:N11a B GM0308/////CHIS740:S1411a B-B////(FS8A(S):S KV10-R Cuba164:S B GF0303/////FS8B(T):N11a B-B////(CH05015:N B KV10-R AR16035:S B GF0301/////UR11003:S B-B////(CUBA164:S KV10-R FS8A(S):S B GF0305/////AR03056:N B-B-B////(CH05015:N B KV10-R DK844:S B GF0304/GEMS KV10-R DK844:S B GF0304/GEMS KV10-R FS8A(S):S B GF0305/////CHRIS775:S1911b B-B-B////(CUBA164:S KV10-R FS8A(S):S B GF0305/////CHRIS775:S1911b B-B-B////(CUBA164:S KV10-R DK844:S BGF0304/////DKB844:S B-B-B////(FS8A(S):S KV10-R Cuba164:S B GF0303/////AR03056:N B-B-B////(CH05015:N B KV10-R FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(FS8A(S):S KV10-R FS8A(S):S B GF0305/////BARBGP2:NO8a B-B////(FS8A(S):S B///GGEMS KV10-R AR16035:S B GF0301/////DK212T:N11a B-B////(CH05015:N B KV10-R Cuba164:S B GF0303/////CUBA164:S1511b B-B////(FS8A(S):S KV10-R Cuba164:S B GF0303/////CHRIS775:S1911b B-B-B////(CUBA164:S KV10-R DK844:S B GF0304/////UR11003:S B-B////(FS8A(S):S KV10-R Cuba164:S B GF0303/////UR11003:S B-B////(CUBA164:S KV10-R AR16035:S B GF0301/////CUBA164:S1511b B-B////(FS8A(S):S KV10-R Cuba164:S B GF0303/GEMS KV10-R FS8A(S):S B GF0305/////UR11003:S B-B////(FS8A(S):S KV10-R FS8A(S):S B GF0305/////UR11003:S B-B////(FS8A(S):S B///GEMS KV10-R AR16035:S B GF0301/////DKB844:S B-B-B////(FS8A(S):S B///GEMS KV10-R FS8A(S):S B GF0305/GEMS KV10-R DK844:S B GF0304/GEMS KV10-R DK844:S B GF0304/GEMS KV10-R DKXL370:N11a B GM0308/GEMS KV10-R DKXL370:N11a B GM0308/GEMS KV10-R DKXL370:N11a B GM0308/GEMS KV10-R UR13085:N B GM0310/GEMS

23 KV10-R UR13085:N B GM0310/GEMS KV10-R UR13085:N B GM0310/GEMS KV10-R DK844:S B GF0304/GEMS KV10-R DK844:S B GF0304/GEMS KV10-R UR13085:N B GM0310/GEMS KV DKXL370:N11a B GM0308/GEMS /3 DKXL370:N11a B-B-SIB///GGEMS (UR13085:N CHIS775:S1911b B-B-B////AR16035:S B (09) Cuba164:S B GF0303/////CUBA164:S1511b B-B////(FS8A(S):S (09) Cuba164:S B GF0303/////CUBA164:S1511b B-B////(FS8A(S):S (09) AR16035:S B GF0301/////DK212T:N11a B-B////(CH05015:N B (09) DK844:S BGF0304/////DKB844:S B-B-B////(FS8A(S):S (09) DKXL370:N11a B GM0308/GEMS (09) DKXL370:N11a B GM0308/////CHRIS775:S1911b B-B-B////(CUBA164:S Examples of ears of ae wx samples examined for amylose displaying a wide range of grain quality attribute s

24 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: , , , , and 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) DP(6-12) DP(13-24) DP(25-36) DP>37 AVECL ae wx 11.9± ± ± ± ± a N/A N/A N/A N/A N/A ae wx 12.5± ± ± ± ± ae wx 11.7± ± ± ± ± ae 12.0± ± ± ± ±0.1 Table 2. Digestibility of ae-wx starches after cooking (Englyst 1992) RDS% SDS% RS% ae wx 87.5± ± ± ae wx 84.0± ± ± ae wx 88.3± ± ± ae wx 87.5± ± ± ae 69.5± ± ±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 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 unfortunately had no DP data however a slightly elevated RS would raise the possibility of sbe1::gm67 being present, 24

25 Hydrolysis (%) 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 3h 6h 10h 24h 48h ae wx 6.7± ± ± ± ± ae wx 6.7± ± ± ± ± ae wx 9.1± ± ± ± ± ae wx 7.1± ± ± ± ± ae 9.0± ± ± ± ±0.5 B ± ± ± ± ±0.5 Hydrolysis values varied and all had expected lowered rates. The sample showed the slowest rate of digestion and command further genotyping and digestion studies to determine the influeence of novel GEM alleles. Figure 1. Raw-starch hydrolysis of the ae wx corn starches Raw-starch hydrolysis of ae-wx corn starches Time (hr) B73 Gelatinization Retrograded starch T o T p T c H T o T p T c H Retrogradation (%) ± ± ± ± ± ± ± ± ± ± ± ± a ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± a Data need to be repeated. 25

26 Figures 2 (a-e). The following are curves of the heat flow of the starches analyzed using DSC. Starches of two lines, and , showed a little rightskewed heat flow peaks. c DKXL370:N11a B GM0308/GEMS-67 a AR16035:S B GF0301/////CUBA164:S B- B////(CUBA164:S d SCR01:N B GM0309/////DKB844:S B-B- B////(FS8A(S):S B/// GEMS-67 b DKXL370:N11a B GM0308/////CHRIS775:S1911b B-B-B////(CUBA164:S e Cuba164:S B GF0303/////FS8B(T):N11a B- B////(CH05015:N B-B/// GEMS-67 26

27 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 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

28 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 and 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

29 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

30 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

31 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 (GM- 87, 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 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 ( ,

32 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

33 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 Browsers such as MaizeGDB, Maizesequence.org, CoGE and InterProScan on Chromosome 6. Upon BLAST analysis of the Fig.8. BLAST result of the sequence amplified by primer Sbe1::GM67 and its two significant hits in long arm of chromosome 5 and 6. 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 GRMZM2G (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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