Serial analysis of zein by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis

Zein, the major storage protein of maize (Zea mays L.) endosperm, was extracted from a number of inbreds with alcohol plus a reducing agent. Isoelectric focusing (IEF) separated total zeins into 41 components, while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated total zeins into about 15 components. Each procedure gave characteristic patterns of zein bands for a number of maize inbreds. IEF and SDS-PAGE were used serially so that each band separated by IEF could be assayed as an individual SDS-PAGE sample. Some IEF bands revealed only a single band after SDS-PAGE, while others revealed two or more bands. A nomenclature system is presented which integrates the two separation systems with information about chromosome locations of zein genes, maize mutations which affect zein synthesis, and inbred sources for different zeins. SDS-PAGE of zein gives apparent molecular masses which vary widely according to the standards used and the properties of the gels, therefore an artificial nomenclature for identifying zein bands after SDS-PAGE is presented. The new nomenclature provides a flexible system which is useful and can be conveniently used in different laboratories.     

喀斯特高海拔山区玉米骨干自交系遗传多样性RAPD标记分析

以代表我国玉米6个主要杂种优势群旅大红骨、Lancaster、Reid、苏湾、墨白和塘四平头的标准测验种丹340、自330、7922、苏37、449、黄早四和适应我国喀斯特高海拔山区的玉米骨干自交系为材料,利用RAPD标记对其进行遗传多样性和杂种优势群划分.从90个随机引物中筛选出21个多态性好的引物扩增材料,共产生146条谱带,其中124条谱带有多态性,占86.3%,说明喀斯特高海拔山区的玉米骨干自交系具有较丰富的遗传多样性.通过UPGMA聚类分析,以遗传相似系数为0.632,可将我国喀斯特高海拔山区玉米种质资源的16个骨干自交系划分为5个类群.     

Change in ribosomal DNA intergenic spacer-length composition in maize recurrent selection populations. 1. Analysis of BS13, BSSS,and BSCB1

Five different ribosomal DNA (rDNA) intergenic spacer-length variants (slvs) were detected among the maize inbreds which were the progenitors of Iowa Stiff Stalk Synthetic (BSSS). One rDNASstI restriction site polymorphism in the 3 region of the 26S gene was detected. Nine different rDNA intergenic spacer (IGS) hybridization fragment patterns (assigned letter designations A-I) were observed among the BSSS progenitors. Following 7 cycles of half-sib recurrent selection in BSSS using the Ia13 double cross as a tester, hybridization fragment pattern E became predominant in the population. In contrast, 11 cycles of reciprocal recurrent selection in BSSS with the Iowa Corn Borer Synthetic No. 1 (BSCB1) population resulted in hybridization pattern D becoming predominant. Hybridization pattern E is present in the elite inbreds B14, B37, B73, and B84, which were derived from different cycles of the BSSS half-sib recurrent selection program with Ia13. Hybridization pattern D is present in the elite inbreds B89 and B94, which were derived from different cycles of the BSSS reciprocal recurrent selection program with BSCB1. Therefore, two different forms of recurrent selection on BSSS resulted in different hybridization patterns becoming predominant in the selected populations and present in elite inbreds derived from the populations. These results also suggest that rDNA IGS hybridization fragment patterns D and E, which both have the longest slv detected, may have a selective or adaptive advantage in BSSS materials grown in the Corn Belt.     

玉米重要自交系的肿囊腐霉茎腐病抗性鉴定与评价

由肿囊腐霉菌(Pythium inflatum Matthews)引起的玉米茎腐病是影响玉米产量的一种重要病害。为进一步拓展可利用的抗源,于2010-2011年在田间采用人工接种方法对287份重要的玉米自交系种质进行了玉米茎腐病的抗性鉴定评价。结果表明,287份鉴定材料中有171份自交系对茎腐病的抗性达到中抗以上水平,占鉴定材料的59.58%,其中高抗自交系共43份,占鉴定材料总数的14.98%;感病类型自交系共116份,占鉴定材料的40.42%,其中高感自交系共95份,占鉴定材料总数的33.10%。Lancaster、Reid及P群种质中具有丰富的茎腐病抗源,而塘四平头种质群中茎腐病抗源相对缺乏,多为感病类型。该研究结果可为今后我国玉米茎腐病抗性种质的引进和改良提供重要参考。     

RFLP analyses of early-maturing European maize germ plasm

Summary Thirty inbred lines representing a wide range of early-maturing European elite germ plasm of maize (Zea mays L.) were assayed for RFLPs using 203 clone-enzyme combinations (106 DNA clones with restriction enzymes EcoR1 and HindIII). The genetic materials comprised 14 flint, 12 dent, and 4 lines of miscellaneous origin. Objectives were to (1) characterize the genetic diversity for RFLPs in these materials, (2) compare the level of genetic diversity found within and between the flint and the dent heterotic groups, and (3) examine the usefulness of RFLPs for assigning inbreds to heterotic groups. All but two DNA clones yielded polymorphism with at least one restriction enzyme. A total of 82 and 121 clone-enzyme combinations gave single-banded and multiple-banded RFLP patterns, respectively, with an average of 3.9 and 7.7 RFLP patterns per clone-enzyme combination across all 30 inbreds, respectively. Genetic similarity (GS) between lines, estimated from RFLP data as Dice's similarity coefficient, showed considerable variation (0.32 to 0.58) among unrelated inbreds. The mean GS for line combinations of type flint x dent (0.41) was significantly smaller than for unrelated flint lines (0.46) and dent lines (0.46), but there was considerable variation in GS estimates of individual line combinations within each group. Cluster and principal coordinate analyses based on GS values resulted in separate groupings of flint and dent lines in accordance with phylogenetic information. Positioning of lines of miscellaneous origin was generally consistent with expectations based on known breeding behavior and pedigrees. Results from this study corroborated that RFLP data can be used for assigning inbreds to heterotic groups and revealing pedigree relationships among inbreds.     

玉米自交系幼胚培养能力与遗传关系研究(简报)

玉米幼胚培养获得的胚性愈伤组织具有较强的长期继代能力和再生植株能力,常被用于构建转基因受体系统。然而基因型是制约玉米组织培养植株再生的重要因素之一,不同玉米基因型的幼胚培养能力关系到其遗传转化研究的结果[1]。     

Identification of Corn Varieties Using Lactate Polyacrylamide Gel Electrophoresis of Seed Albumins and Globulins

Systematic electrophoretic analysis of albumins and globulins of the inbred and hybrid corn (Zea mays) seeds was carried out on an improved lactate-polyacrylamide gel electrophoresis, a method with high resolving power, good reproducibility and stability. The electrophoregram was classified into four groups designated as α、β、γandω respectively. Each inbred or hybrid had its own unique band pattern distinguishable from the others, regarding as its "fingerprint”. The band pattern of the whole kernel was basically similar to that of its embryo, except that of the endosperm showing less bands with weaker staining intensity; and most of the patterns overlapped with those of the embryo. The band number of the Fl hybrid was exactly equivalent to the number of the common bands and the specific bands of the two parents, indicating that the difference of band patterns was a genetic trait controlled by the nuclear genes. The F1 electrophoregram could be predicted by those of the two parents. The band pattern of the Fl hybrids was identical with that produced from mechanically mixed extract of the two parent inbreds. This procedure could be used in corn cultivar identification and as a test for genetic purity.     

Studies on human thyroxine-binding globulin. 8. Isoelectric focusing evidence for microheterogeneity of thyroxine-binding globulin

Highly purified thyroxine-binding globulin from pooled human serum homogeneous by conventional criteria, subjected to isoelectric focusing in polyacrylamide gels in a pH gradient from 3–6, produced a pattern of at least nine stainable protein bands. All of these bands appeared to bind thyroxine. Completely desialylated thyroxine-binding globulin subjected to isoelectric focusing produced the same number and pattern of bands located at a different area in the pH gradient. Thyroxine-binding globulin purified from the serum of a single donor was subjected to isoelectric focusing. This thyroxine-binding globulin had the same pattern of protein bands with the exception that one of the major bands seen in the thyroxine-binding globulin from pooled serum was absent. Several possible explanations for these phenomena are discussed.     

Multiple zeins from maize endosperms characterized by reversed-phase high performance liquid chromatography

The major storage proteins of maize (Zea mays L.) endosperm are located in protein bodies, and may be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) into two major classes and four minor classes of polypeptides. The two major classes (commonly known as zeins) have been separated previously into a large number of components by isoelectric focusing (IEF). Reversed-phase high performance liquid chromatography (HPLC) further separated the major classes into additional components, and gave distinctive peaks for each minor zein class. Some IEF bands produced two or more HPLC fractions, while some HPLC fractions produced two or more IEF bands. Apparently identical IEF bands from different inbreds may appear in different fractions after HPLC. Thus the total number of zeins revealed by separations based on apparent size (SDS-PAGE), net charge (IEF), and hydrophobicity (HPLC) is very large. Different laboratories have developed diverse nomenclatures which cause much confusion. A key is presented to provide a flexible and expandable nomenclature for this complex group of proteins.     

Linkages among zein genes determined by isoelectric focusing

Summary Genetic control of the major zein polypeptides in maize (Zea mays L.) was studied by isoelectric focusing (IEF) in agarose. Linkage relationships were determined by making a number of crosses, then determining the expression of zein polypeptides in backcross seeds. Chromosome linkages were determined by using the markers sugary-1 (for chromosome 4), yellow-8, and a waxy 7–9 translocation (for chromosome 7). Nine zeins were in one linkage group on chromosome 4, six in another linkage group on chromosome 4, and four zeins were in one linkage group on chromosome 7. Some IEF single bands consisted of at least two polypeptides, which were detected by subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis, by aberrant ratios in backcrosses, or by differing recombination percentages. One zein occurred only in homozygous sugary-1 seeds. Three sets of closely-linked zeins were noted that occurred together almost exclusively in certain inbreds.Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the Illinois Agricultural Experiment Station, Department of Agronomy, University of Illinois, Urbana, USAMention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned     

Plant Nucleases: V. Survey of Corn Ribonuclease II Isoenzymes

Corn (Zea mays L.) ribonuclease II of the root microsomal fraction was isolated from 11 inbreds and seven hybrids. Polyacrylamide gel electrophoresis revealed a total of five bands of activity among corn lines tested. Most inbreds had only one isoenzyme, but three had two isoenzymes. The hybrids tested contained all of the isoenzymes found in the parental inbreds.     

Chloroplast and nuclear DNA variation among homozygous plants in a population of the autogamous annualMicroseris douglasii (Asteraceae,Lactuceae)

The autogamous diploid annualMicroseris douglasii of California occurs in many isolated populations. The populations consist of one to many highly inbred biotypes. Morphological variation among populations usually is greater than within populations. In spite of the virtual absence of gene flow even within populations, genetically determined character differences are randomly distributed and associated throughout the range of the species. Recent evidence even suggests introgression of chloroplasts from the relatedM. bigelovii. Offspring families from 25 plants of a very variable population were raised and examined for segregation of morphological and molecular (RAPD) markers. All 25 original plants were completely homozygous for all markers, but each differed from all others at least in some markers. The population consisted of two genetically isolated groups of plants: a distinct inbred line (3 plants) and 22 plants with random associations of a common set of markers and characters, possibly recombinant inbreds from a past hybridization event. One of these 22 plants contained a chloroplast genome found inM. bigelovii, the other 24 plants a chloroplast genome found only inM. douglasii.     

RAPD and ISSR markers in the evaluation of genetic divergence among accessions of elephant grass

Considering the expected genetic variability of elephant grass (Pennisetum purpureum), due to its cultivation in different continents, we characterized and estimated the genetic divergences between 46 accessions of elephant grass with different edaphoclimatic adaptations, using RAPD and ISSR markers. We evaluated, comparatively, the consistency of the information achieved with these markers. Twenty-six RAPD and 25 ISSR primers were employed. The RAPD markers produced 185 bands, 72% of which were polymorphic, with a mean of 5.11 polymorphic bands per primer. The 25 ISSR starters produced 216 bands; 76% were polymorphic, with a mean of 6.56 polymorphic bands per primer. The correlation between the genetic distances achieved by the RAPD and ISSR markers was 0.76, which is highly significant by the Mantel test. Based on UPGMA grouping, considering the point of sudden change, five and six groups were formed for the data from the RAPD and ISSR markers, respectively. These markers provided partially concordant groups, indicating that these techniques can provide consistent information and consequently could be used in studies of genetic diversity among accessions.     

Distribution of pigments in the shell of the gastropod <Emphasis Type="Italic">Littorina obtusata</Emphasis> (Linnaeus, 1758)

The patterns of pigment distribution in the shell of the gastropod Littorina obtusata were studied. It was determined that the background coloration of the periwinkle shell resulted from the coloration interaction of the external and internal parts of the ostracum. The coloration of the latter can be monochromatic or twolayer. The external and/or internal zones of the ostracum can be colored purple, white, orange, yellow and greenish. Only 11 of 25 possible color combinations were recorded. Four additional versions of shell background coloration resulted from partial depigmentation of external or internal parts of ostracum. The mottling pattern corresponded to white or depigmented lenticular sites in the external part of the ostracum. The bands on the shell may be subdivided into two groups according to their means of formation. The bands that appeared due to redistribution of pigments forming background coloration belonged to the first group, the bands arising as a layer of additional (white, yellow, orange) pigment belonged to the second group. Various band versions could occur simultaneously in the same individual.     

N-linked oligosaccharide analysis of glycoprotein bands from isoelectric focusing gels

Glycoproteins often display a complex isoelectric focusing profile because of the presence of negatively charged carbohydrates, such as sialic acid, phosphorylated mannose, and sulfated GalNAc. Until now, understanding the role of these charged carbohydrates in determining the isoelectric focusing profile has been limited to observing pattern shifts following complete removal of the sugars in question. We have developed a simple and sensitive method for analyzing N-linked oligosaccharides from the individual isoelectric focusing bands of a glycoprotein using recombinant human thyroid-stimulating hormone as a model system. N-linked oligosaccharides were released and profiled from individual bands following electroblotting of isoelectric focusing gels. As might be predicted, high-pH anion-exchange chromatography-pulsed amperometric detection and matrix-assisted laser desorption/ionization-time of flight analyses indicated that the bands that migrated closer to the positive electrode contained more sialylated N-linked oligosaccharides. The sialic acid content of these bands correlated with that predicted from the corresponding oligosaccharide analyses.     

Diversity of seed storage protein patterns in wild peanut (Arachis,Fabaceae) species

55 accessions of wild peanuts (Arachis spp.) introduced from South America were analyzed for seed storage protein composition using SDS-PAGE electrophoresis. The objectives of the study were to evaluate variability within sect.Arachis and to classify taxa based on protein composition. 25 different band positions were resolved. Individual accessions had 11 to 18 bands which included the conarachin region (MW > 50 kD), two to five bands in the acidic arachin region (MW 38–49.9 kD), three to seven in the intermediate MW region (23 to 37.9 kD), two to five bands in the basic arachin region (18–22.9 kD), and one to three bands in the low MW protein region (14–17.9 kD). These data were utilized in a principal coordinate analysis based on the matrix of genetic distances between all pairs of the 55 accessions. Several groups of accessions conformed to expected species classification includingA. batizocoi, A. stenosperma, andA. monticola; whileA. duranensis, A. cardenasii, A. helodes, andA. correntina did not form good groups. The study showed that great diversity exists for protein profiles and seed storage proteins have potential for aiding species classification and for serving as markers for interspecific hybridization studies.     

Genetic diversity among progenitors and elite lines from the Iowa Stiff Stalk Synthetic (BSSS) maize population: comparison of allozyme and RFLP data

Summary Data for restriction fragment length polymorphisms (RFLPs) of 144 clone-enzyme combinations and for 22 allozyme loci from 21 U.S. Corn Belt maize (Zea mays L.) inbreds were analyzed. The genetic materials included 14 progenitors of the Iowa Stiff Stalk Synthetic (BSSS) maize population, both parents of one missing BSSS progenitor, four elite inbreds derived from BSSS, and inbred Mo17. Objectives were to characterize the genetic variation among these 21 inbreds for both allozymes and RFLPs, to compare the results from both types of molecular markers, and to estimate the proportion of unique alleles in the BSSS progenitors. Genetic diversity among the 21 inbreds was substantially greater for RFLPs than for allozymes, but the percentages of unique RFLP variants (27%) and unique allozyme alleles (25%) in the BSSS progenitors were similar. Genetic distances between inbreds, estimated as Rogers' distance (RD), were, on average, twice as large for RFLP (0.51) as for allozyme data (0.24). RDs obtained from allozyme and RFLP data for individual line combinations were only poorly correlated (r = 0.23); possible reasons for discrepancies are discussed. Principal component analysis of RFLP data, in contrast to allozyme data, resulted in separate groupings of the ten BSSS progenitors derived from the Reid Yellow Dent population, the four BSSS elite lines, and Mo17. The remaining six BSSS progenitors were genetically rather diverse and contributed a large number of rare alleles to BSSS. The results of this study corroborate the fact that RFLPs are superior to allozymes for characterizing the genetic diversity of maize breeding materials, because of (1) the almost unlimited number of markers available and (2) the greater amount of polymorphisms found. In particular, RFLPs allow related lines and inbreds with common genetic background to be identified, but a large number of probe-enzyme combinations is needed to estimate genetic distances with the precision required.Joint contribution from Cereal and Soybean Research Unit, USDA, Agricultural Research Service, and Journal Paper No. J-14236 of the Iowa Agricultural and Home Economics Experiment Station, Projects 2818 and 2778     

Isolation of S-alleles from a wild population of Brassica campestris L. at Balcesme,Turkey and their characterization by S-glycoproteins

Summary S-alleles of self-incompatibility were isolated from a wild population of Brassica campestris growing at Balcesme, Turkey. Out of 88 plants observed, 73 were self-incompatible and 4 were self-compatible. In certain families, selfed progenies from a self-incompatible plant segregated into fewer than three incompatibility classes, which is consistent with a one-locus sporophytic genetic control of self-incompatibility. Out of 25 combinations of S-alleles tested, dominance interactions were observed in 6 of them on the pollen side and on 5 of them on the stigma side. The 35 S-homozygotes thus isolated consisted of 18 independent S-alleles. The number of S-alleles in this population was estimated to be more than 30. The S-locus glycoproteins (SLGs) corresponding to the respective S-alleles were identified by iso-electric focusing (IEF)-gel immunoblotting with a polyclonal antiserum against SLG⁸. SLGs in a stigma were generally composed of several bands, one major and a few minor ones, whose molecular weight was similar to each other, and the major and minor bands were heritable in correlation with each other. SDS-PAGE analysis of SLGs differentiated a few juxtaposed bands between 50 and 60 kDa, and the variations in these bands were considered to be due to differences in the number of polysaccharide residues. General features of the variation of S-genes and their SLGs between the populations in Balcesme, Turkey and Oguni, Japan, were comparatively similar to one another, despite the different surroundings and history of these populations.     

Characteristics of glutamate dehydrogenase, a new diagnostic marker for the genus Fusobacterium

Enzymes representative of carbohydrate and nitrogen metabolism were screened for their presence and activity amongst species of the genus Fusobacterium. Glutamate dehydrogenase (GDH) was reliably detected in all 25 strains studied. The pH profile of this enzyme and the DNA base composition of selected strains were also determined. DNA base composition of selected strains ranged between 28-32.9 mol% G + C. GDH was active between pH 7.5-11.5 but two pH profiles of activity, with optima at 9.5 and 10.5, were discernible among species. Apart from Fusobacterium nucleatum, which had a heterogeneous enzyme pattern, the GDH electrophoretic mobility was constant within a species but in a few cases the enzyme bands overlapped. A combination of the pH profile, the GDH electrophoretic pattern and the DNA base composition provided clear separation of the test organisms into discrete groups; however, a larger number of strains must be examined before the full potential of these tests can be evaluated.