Chromosomal Location by Use of Trisomics and New Alleles of an Endopeptidase in ZEA MAYS

An association was found earlier between the Ep₁ gene locus coding for an endopeptidase and the endosperm color gene Y₁ on chromosome 6 of Zea mays. By employing primary trisomics we have unequivocally placed the Ep₁ gene on chromosome 6, closely linked to the Y₁ locus. Additionally we describe new alleles at the Ep₁ locus, and present evidence for the existence of a second endopeptidase system in maize.     

Inheritance of qualitative and quantitative trypsin inhibitor variants in Pisum

A trypsin inhibitor locus (Tri) has been mapped close to Vc-2 on Pisum (pea) linkage group 5 using recombinant inbred lines derived from crosses of genotypes showing qualitative variation in seed trypsin inhibitors. F₂ seed populations derived from crosses between lines showing qualitative variation in trypsin inhibitors as well as quantitative variation in inhibitor activity showed an association between the segregation of the structural variation and relative activity levels. Clones complementary to Pisum trypsin inhibitor mRNA were used in hybridization analyses which showed that the segregation of protein polymorphisms reflected directly the segregation of polymorphisms associated with the structural genes.     

Identification of a candidate gene for the wheat endopeptidase <Emphasis Type="Italic">Ep-D1</Emphasis> locus and two other STS markers linked to the eyespot resistance gene <Emphasis Type="Italic">Pch1</Emphasis>

Wheat is prone to strawbreaker foot rot (eyespot), a fungal disease caused by Oculimacula yallundae and O. acuformis. The most effective source of genetic resistance is Pch1, a gene derived from Aegilops ventricosa. The endopeptidase isozyme marker allele Ep-D1b, linked to Pch1, has been shown to be more effective for tracking resistance than DNA-based markers developed to date. Therefore, we sought to identify a candidate gene for Ep-D1 as a basis for a DNA-based marker. Comparative mapping suggested that the endopeptidase loci Ep-D1 (wheat), enp1 (maize), and Enp (rice) were orthologous. Since the product of the maize endopeptidase locus enp1 has been shown to exhibit biochemical properties similar to oligopeptidase B purified from E. coli, we reasoned that Ep-D1 may also encode an oligopeptidase B. Consistent with this hypothesis, a sequence-tagged-site (STS) marker, Xorw1, derived from an oligopeptidase B-encoding wheat expressed-sequence-tag (EST) showed complete linkage with Ep-D1 and Pch1 in a population of 254 recombinant inbred lines (RILs) derived from a cross between wheat cultivars Coda and Brundage. Two other STS markers, Xorw5 and Xorw6, and three microsatellite markers (Xwmc14, Xbarc97, and Xcfd175) were also completely linked to Pch1. On the other hand, Xwmc14, Xbarc97, and Xcfd175 showed recombination in the W7984 × Opata85 RIL population suggesting that recombination near Pch1 is reduced in the Coda/Brundage population. In a panel of 44 wheat varieties with known eyespot reactions, Xorw1, Xorw5, and Xorw6 were 100% accurate in predicting the presence or absence of Pch1 whereas Xwmc14, Xbarc97, and Xcfd175 were less effective. Thus, linkage mapping and a germplasm survey suggest that the STS markers identified here should be useful for indirect selection of Pch1.     

Characterization of asparaginyl endopeptidase activity in endosperm of developing and germinating castor oil seeds

A spectrophotometric assay was devised to characterize the asparaginyl (Asn) endopeptidase activity from the endosperm of castor oil seeds. (Ricinus communis L. var. Baker 296). The assay measures the release of p-nitroaniline from the hydrolysis of benzoyl-l-Asn-p-nitroanilide. Assay sensitivity was improved through diazotization of the reaction product with N(]-napthy])-ethylenediamine dihydrochloride: diazotized p-nitroaniline was determined spectrophotometrically at 548 nm (?₅₄₈= 1.64 × 10^?1M^?1 cm^?2). By using this assay. Asn endopeptidase activity was detected in endosperm extracts of developing, mature and germinating castor seeds. Comparison of the Asn endopeptidase activities of developing and germinating castor endosperms revealed that they: 1) have identical pH-activity profiles with optimal activity occuring at pH 5.4: 2) are heat-labile proteins displaying comparable thermal stability profiles, and 3) are activated and inhibited by dithiothreitol and thiol modifying reagents, respectively. Thus, the Asn endopeptidases of developing and germinating castor seeds are very similar, if not identical, cysteine proteases. The most significant increase in the activity of endosperm Asn endopeptidase occurs during the full coryledon to maturation stage of seed development, this period coincides with the most active phase of reserve protein accumulation by ripening castor oil seeds. Asn endopeptidase activity of fully mature (dry) castor seeds was about 2-fold lower than that of muturation stage ripening castor oil seed. Asn endopeptidase activity showed a slight reduction over the inicial 2-day period following seed imbibition, and then rapidly decreased over the next several days of germination. The results are compatible with the proposal that Asn endopeptidase functions both to process storage preproteins following their import into protein bodies of developing seeds, as well as to participate in the mobilization of storage proteins during the early phase of seed germination.     

Inheritance,intracellular localization,and genetic variation of phosphoglucomutase isozymes in maize (Zea mays L.)

Phosphoglucomutase (PGM; EC 2.7.5.1) isozyme variants were studied in a large number of inbred lines, crosses, and races of maize (Zea mays L.). Patterns of Mendelian inheritance demonstrated for PGM isozyme variants indicated that they are encoded by nuclear genes. Two unlinked loci, Pgm1 and Pgm2, located on the long arm of chromosome 1 and the short arm of chromosome 5, respectively, specify the observed electrophoretic variation on starch gels. No intra- or interlocus hybrid bands were found, suggesting that each isozyme band consists of a single polypeptide. PGM isozymes were present in all plant parts studied and the activity specified by both loci appears to reside in the cytoplasm. In studies of 520 racial collections of maize from Latin America, a single allele at each locus predominated in most collections. Likewise, the same alleles predominated in a set of 406 inbred lines of maize from the United States and Canada.This work was supported in part by NIH Research Grant GM 11546.Paper No. 8496 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina.     

Transgenic Expression of Trypsin Inhibitor CMe from Barley in Indica and Japonica Rice,Confers Resistance to the Rice Weevil Sitophilus Oryzae

Indica and japonica rice (Oryza sativa L.) plants were transformed by particle bombardment with the Itr1 gene encoding the barley trypsin inhibitor BTI-CMe, under the control of its own promoter that confers endosperm specificity, and the maize ubiquitin promoter. From 38 independent transgenic lines of indica (breeding line IR58) and 15 of the japonica (cv Senia) selected, 22 and 11, respectively, expressed the barley inhibitor at detectable levels. The transgene was correctly translated as indicated by western blot analysis with a level of expression in R₃ seeds up to 0.31% (IR58) and 0.43% (Senia) of the total extracted protein. The functional integrity of BTI-CMe was confirmed by trypsin activity assays in liquid media and by activity staining gels, performed with seed extracts. The significant reduction of the survival rate of the rice weevil (Sitophilus oryzae, Coleoptera: Curculionidae) reared on homozygous transgenic indica and japonica rice seeds expressing the BTI-CMe, compared to non-transformed controls, and the decrease in the trypsin-like activity of insect crude midgut extracts, confirmed the utility of this proteinase inhibitor gene for the control of important storage pests.     

Molecular cloning,characterization and expression analysis of two catalase isozyme genes in barley

Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and-3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).     

Identification of genes alternatively spliced in developing maize endosperm

• The process of alternative splicing is critical for the regulation of growth and development of plants. Thus far, little is known about the role of alternative splicing in the regulation of maize (Zea mays L.) endosperm development.
• RNA sequencing (RNA‐seq) data of endosperms from two maize inbred lines, Mo17 and Ji419, at 15 and 25 days after pollination (DAP), respectively, were used to identify genes that were alternatively spliced during endosperm development. Intron retention (IR) in GRMZM2G005887 was further validated using PCR and re‐sequencing technologies.
• In total, 49,000 alternatively spliced events and ca. 20,000 alternatively spliced genes were identified in the two maize inbred lines. Of these, 30 genes involved in amino acid biosynthesis and starch biosynthesis were identified, with IR occurring only in a specific sample, and were significantly co‐expressed with ten well‐known genes related to maize endosperm development. Moreover, IR in GRMZM2G005887, which encodes a cysteine synthase, was confirmed to occur only in the endosperm of Mo17 at 15 DAP, resulting in the retention of a 121‐bp fragment in its 5′ untranslated region. Two cis‐acting regulatory elements, CAAT‐box and TATA‐box were observed in the retained fragment in Mo17 at 15 DAP; this could regulate the expression of this gene and influence endosperm development.
• The results suggest that the 30 genes with IR identified herein might be associated with maize endosperm development, and are likely to play important roles in the developing maize endosperm.

     

Studies of dotted,a regulatory element in maize

Summary Dotted (Dt) is the regulatory element of a two-unit controlling system in maize. Dt causes the inherited change from the recessive a ₁ (colorless) to its dominant allele, A ₁ (anthocyanin production), during the development of the stalk, leaves, and endosperm. The mutation events are observed as sectors of color in an anthocyaninless background.One of the most puzzling, but perhaps significant, aspects of controlling elements in maize is that they originate in conjunction with chromosome or chromatid breaks. This fact invokes a requirement that either an existing regulatory mechanism is disturbed by the breakage or that a foreign element is incorporated before fusion of the broken chromatids.Experimental crosses were made between Dt tester stocks and a pollen parent, a large proportion of whose chromosomes 9 were undergoing the chromatid type of bridge-breakage-fusion cycle. New Dt's were induced in endosperm sectors of 250 of 154,422 kernels tested (1/600); among these, two germinal Dt's (Dt ^crown ₄ and Dt ₅) were recovered, presumably due to chromatid breaks during meiosis or the first microspore division. Dt ₅ produces a mutation pattern very similar to the original Dt ₁ and is located 0.33 crossover units away from the yg ₂ locus. This is close to the known location of Dt ₁ (7 crossover units distal to the yg ₂ locus) and is suggestive of a specific site for Dt inductive breaks. Dt ^crown ₄, on the other hand, is inherited independently of the yg ₂ locus and does not support this contention. Dt ^crown ₄ represents a new state causing a high concentration of fine dots in the crown of the kernel, with little or no dotting at the base.The phase variation of Dt ^crown ₄ is discussed together with the tissue-dependent expression of Dt ^in-ac ₁ (Dotted, inactive-active). Dt ^in-ac ₁ is a new state of Dt ₁ and shows inactive (no a ₁ to A ₁ mutations) and active (a ₁ to A ₁ mutations) phases in the endosperm, whereas it is only in the active phase in the diploid scutellum. The observed phase variation was shown to be a property of the regulatory elements, Dt, responding to differences in the cellular environment.In appreciation of his help and guidance, I dedicate this article to Professor Marcus M. Rhoades on his birthday.Journal Paper No. J-7454 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1880.     

Allelic forms of mouse transcobalamin 2

Transcobalamin 2 is the only vitamin B12-binding protein found in mouse serum. Two allelic forms of mouse transcobalamin 2 are described. The two forms differ in their mobilities on polyacrylamide gel electrophoresis. The slowly migrating form has been found in serum from 25 inbred mouse strains. The more rapidly migrating form was detected in 3 inbred mouse strains (NZB, ST/bJ, and CPB-WV). Both parental variants were expressed in F₁ progeny of appropriate interstrain crosses, showing codominant expression of the transcobalamin 2 alleles. In backcrosses between F₁ and parental individuals, the two electrophoretic variants were inherited as single Mendelian traits. The strain distribution pattern of the two variants in recombinant inbred lines likewise suggested a single-gene mode of inheritance and indicated a lack of close linkage with a number of genetic loci on chromosomes 1, 2, 4, 5, 6, 7, 9, 12, 14, 15, and 17. We propose the symbol Tcn-2 for the polymorphic gene locus coding for transcobalamin 2 in the mouse and Tcn-2 ^s and Tcn-2 ^f for the two alleles.     

Purification and partial characteristic of a major gliadin-degrading cysteine endopeptidase from germinating triticale seeds

The endopeptidase of the highest electrophoretic mobility was the main endopeptidase hydrolyzing gliadin in the endosperm of germinated triticale (X Triticosecale Wittm.) grains after three days of imbibition. Activity of this endopeptidase, named EP8 starts to be detectable after two days of imbibition. The appearance of its activity in the endosperm on a second day of imbibition may suggest that EP8 is synthesized in aleurone during germination and/or secreted into the starchy endosperm as an inactive polypeptide during grains development and then activated. EP8 was isolated from the endosperm of germinating triticale seeds and purified 257-fold using ammonium sulphate, ion exchange chromatography on DEAE Sepharose CL-6B and gel filtration on Sephadex G-100. The enzyme was totally inhibited by E-64—class-specific cysteine proteinases inhibitor and activated by thiol compounds. Molecular weight estimated by SDS-PAGE was 39.5 kDa. The optimum pH for the hydrolysis of gliadin was 4.2 and for hemoglobin 5.2. High activity of EP8 against wheat gliadin in vitro suggests that this cysteine endopeptidase plays a major role in the mobilization of storage proteins in the endosperm of germinating triticale grains.     

Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^–1 and 3.1 × 10⁷ M^–1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Phosphoglucomutase electrophoretic variants in the mouse

The phosphoglucomutase (PGM) electrophoretic phenotype of the mouse (Mus musculus) consists of several distinct components which can be grouped into two major zones designated PGM-1 and PGM-2. Evidence presented here indicates that each zone is controlled by a single genetic locus denoted Pgm-1 and Pgm-2, respectively. Two variant forms segregated at the Pgm-1 locus. They were codominantly expressed and inherited as alleles at an autosomal locus. The alleles were termed Pgm-1 ^a (fast) and Pgm-1 ^b (slow). These alleles were separately fixed in a number of inbred strains of mice. Preliminary evidence based on wild mouse phenotypes indicates that variant forms also exist for PGM-2 which are inherited as alleles at an autosomal locus. Genetic linkage relationships have not been determined for these loci. PGM-1 variants and PGM-2 were expressed in mouse fibroblasts in vitro.Supported by U.S. Public Health Service grants GM-09966 and GM-07249 from General Medical Sciences and 5 F2 HD-35,531 from Child Health and Human Development; and Atomic Energy Commission contract AT(30-1)-3671.Postdoctoral Fellow of the U.S. Public Health Service.     

Genetics of an esterase in Aedes aegypti

Zymograms of single individuals of Aedes aegypti were obtained by means of starch gel electrophoresis, using alpha-naphthyl acetate as substrate. Inbred lines gave consistently homogeneous patterns; earlier results from random-breeding laboratory strains had shown considerable variability. Six distinct bands were observed. The furthest moving band, designated Esterase 6, showed differential migration in two inbred lines. Reciprocal crosses between these lines gave F₁ progeny showing both bands. Backcrosses of F₁ to either parental line gave a 1:1 segregation. These results are consistent with the hypothesis that the two forms of Esterase 6 are controlled by a single pair of codominant alleles at a single gene locus (Est 6 ^a and Est 6 ^b). Linkage tests with marker genes have demonstrated that Est 6 is on linkage group 2, with the following alignment: spot-abdomen (9.0±1.0) yellow-larva (17.4±1.3) Est 6. Crosses with another inbred line demonstrated a third band with intermediate mobility, designated Est 6 ^c. An additional electrophoretic variant which seems to have a simple Mendelian basis was found in esterase band 1.This work was supported by NIH Research Grant No. A1-02753.     

Genetic control and intracellular localization of glutamate oxaloacetic transaminase in maize

Glutamate oxaloacetic transaminase (L-aspertate: 2-oxoglutarate aminotransferase, E.C. 2.6.1.1; GOT) was found to occur in five distinct electrophoretic forms in different tissue extracts from a number of highly inbred strains of Zea mays L. No major qualitative differences were detected in the various tissues examined, and the isozyme patterns did not undergo changes during temporal development of any given inbred strain. Cell fractionation studies showed one isozyme to be associated with the mitochondria (mGOT), another to be exclusively associated with the soluble fraction (sGOT), and a third to be associated with the glyoxysomes (gGOT). The glyoxysomal form occurs as two electrophoretically distinct variants which exist in different inbred strains of maize. The gGOT variants are under the control of two codiminant alleles (Got1A and Got1B) at the Got1 locus (isozyme5, gGOT). The genetic data and gene dosage effects suggest that GOT in maize is functionally a dimer.     

Nucleotide Diversity of Maize ZmBT1 Gene and Association with Starch Physicochemical Properties

Cereal Brittle1 protein has been demonstrated to be involved in the ADP-Glc transport into endosperm plastids, and plays vital roles in the biosynthesis of starch. In this study, the genomic sequences of the ZmBT1 gene in 80 elite maize inbred lines were obtained, and the nucleotide polymorphisms and haplotype diversity were detected. A total of 30 variants, including 22 SNPs and 8 indels, were detected from the full sequences of this gene. Among these polymorphic sites, 9 SNPs and 2 indels were found to be located in the coding region. The polymorphisms of CDS sequences classified the maize ZmBT1 gene into 6 haplotypes, which encode 6 different ZmBT1 proteins. Neutrality tests revealed a decrease in population size and/or balancing selection on the maize ZmBT1 locus. To detect the association between sequence variations of this gene and the starch physicochemical properties, 7 pasting and 4 gelatinization traits of starch were measured for the tested inbred lines using rapid visco analyzer (RVA) and differential scanning calorimeter (DSC), respectively. The result of association analysis revealed that an indel in the coding region was significantly associated with the phenotypic variation of starch gelatinization enthalpy.     

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SW_XL and G3000SW_XL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SW_XL and G4000SW_XL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl₂ and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type. Received: 29 October 1998 / Accepted: 10 December 1998     

Three alcohol dehydrogenase genes in wild and cultivated barley: Characterization of the products of variant alleles

Barley (Hordeum vulgare) and its wild progenitor (H. spontaneum) have three loci for alcohol dehydrogenase (EC 1.1.1.1; ADH). The Adh1 locus is constitutively expressed in seed tissues, whereas expression of the loci Adh2 and Adh3 requires anaerobic induction. The Adh3 gene is well expressed in aleurone and embryo tissues kept under N₂ for 2–3 days. Using N₂-treated embryos, a diverse collection of H. spontaneum was screened in starch gels for electrophoretic variants at the Adh3 locus. Four variants were found: two were conventional mobility variants (Adh3 S, Adh3 V); one was a null variant (Adh3 n); and the fourth (Adh3 I) variant lacked active homodimers and showed reduced heterodimer activity. The ³⁵S-labeled monomers induced under N₂ in the lines homozygous for Adh1, Adh2, or Adh3 variants were immunoprecipitated with antiserum raised against maize ADH. Fluorography after separation by SDS-PAGE and by urea-isoelectric focusing indicated that the Adh3 n allele was CRM- and that the Adh3 I gene product was smaller than normal. The Adh1 and Adh3 variants showed independent segregation.     

Purification and characterization of proteolytic enzymes from normal and opaque-2Zea mays L. developing endosperms

Purification and characterization of proteases from developing normal maize endosperm and high lysine opaque-2 maize endosperm have been carried out with a view to understand their role in storage protein modification. At day 15, normal maize endosperm had two types of proteolytic enzymes, namely, protease I and protease II, while at day 25 protease n disappeared and in place protease III appeared. However, in opaque-2 maize endosperm at both the stages only one type of enzyme (protease I) was present. These proteases had many properties in common-optimum pH and temperature were respectively, 5.7and 40°C; their activity was inhibited to the extent of 75 –93 % by p-chloromercuribenzoate; trypsin inhibitor inhibited the activity more at early stages of endosperm development; all proteases cleaved synthetic substrates p-tosyl-L-arginine methylesler and N-benzoyl-L-tyrosine ethyl ester and poly-L-glutamic acid. TheKm values of day 15 and 25 normal maize endosperm proteases ranged from 2.73–3.30, while for opaque-2 maize endosperm protease I it was 3.33 mg azocasein per ml assay medium. These enzymes, however, differed with respect to proteolytic activity towards poly-L-lysine. Only normal maize endosperm protease III at day 25 followed by protease II at day 15 showed high activity towards this homopolypeptide suggesting thereby their role in determining the quality of normal maize endosperm protein. Part of Ph.D. thesis submitted by the first author     

Estimation of coefficient of coancestry using molecular markers in maize

Summary The coefficient of coancestry (f_AB) between individuals A and B is the classical measure of genetic relationship. f_AB is determined from pedigree records and is the probability that random alleles at the same locus in A and B are copies of the same ancestral allele or identical by descent (ibd). Recently, the proportion of molecular marker variants shared between A and B (S_AB) has been used to measure genetic relationship. But S_AB is an upwardly-biased estimator of f_AB, especially between distantly-related lines. f_AB, S_AB, and adjusted (to remove bias) estimates of molecular marker similarity (f _AB ^M ) were compared. RFLP banding patterns at 46 probe-restriction enzyme combinations were obtained for 23 maize inbred lines derived from the Iowa Stiff Stalk Synthetic (BSSS) maize (Zea mays L.) population, and for 4 non-BSSS lines. f _AB ^M was estimated as , where _A (or _B) was the average proportion of RFLP variants shared between inbred A (or inbred B) and the non-BSSS lines. The average f_AB among 253 pairwise combinations of BSSS lines was 0.212, whereas the average S_AB was 0.397. The average f _AB ^M was 0.162, indicating that the upward bias in S_AB was effectively removed. S_AB and f_AB were significantly different ( = 0.05) in 76.3% of the comparisons, whereas 24.9% of the f _AB ^M values differed significantly from f_AB. The latter result suggests that selection and/or drift were present during inbred line development and that f_AB may not be an accurate measure of the true proportion of ibd alleles between two lines. Cluster analyses based on S _AB ^M and f _AB ^M grouped lines according to pedigree, although several exceptions were noted. The presence of shared molecular marker variants between unrelated lines must be considered when setting S_AB-based minimum distances for varietal protection. Under simplified conditions, more than 250 molecular marker loci are necessary to obtain sufficiently precise estimates of coefficient of coancestry using molecular markers.A contribution from Limagrain Genetics, a Group Limagrain company