Identification of two opaque2 modifier loci in Quality Protein Maize

Genetic modifiers of opaque2 convert the soft, starchy endosperm of opaque2 maize mutants to a hard, vitreous phenotype, while maintaining the enhanced lysine content of the grain. Genetic analysis of F2 segregating seeds from crosses of opaque2 by modified opaque2 genotypes indicated that the modifiers are complex traits that act codominantly. We developed two different segregating F2 populations and mapped the modifier loci by restriction fragment length polymorphism (RFLP) analysis. A relationship was found between formation of vitreous endosperm and the locus encoding the gamma-zein storage protein, which maps near the centromere of chromosome 7. Endosperm modification was consistently associated with the presence of two rather than one gamma-zein gene at this locus. A second modifier locus was mapped near the telomere of chromosome 7L.     

Characterization of <Emphasis Type="Italic">opaque2</Emphasis> modifier QTLs and candidate genes in recombinant inbred lines derived from the K0326Y quality protein maize inbred

Quality protein maize (QPM) is a high lysine-containing corn that is based on genetic modification of the opaque2 (o2) mutant. In QPM, modifier genes convert the starchy endosperm of o2 to the vitreous phenotype of wild type maize. There are multiple, unlinked o2 modifier loci (Opm) in QPM and their nature and mode of action are unknown. We previously identified seven Opm QTLs and characterized 16 genes that are differentially up-regulated at a significant level in K0326Y QPM, compared to the starchy endosperm mutant W64Ao2. In order to further characterize these Opm QTLs and the genes up-regulated in K0326Y QPM, we created a population of 314 recombinant inbred lines (RILs) from a cross between K0326Y QPM and W64Ao2. The RILs were characterized for three traits associated with endosperm texture: vitreousness, density and hardness. Genetic linkage analysis of the RIL population confirmed three of the previously identified QTLs associated with o2 endosperm modification in K0326Y QPM. Many of the genes up-regulated in K0326Y QPM showed substantially higher levels of expression in vitreous compared with opaque RILs. These included genes associated with the upstream regulation of the ethylene response pathway, and a gene encoding a regulatory subunit of pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase, an adaptive enzyme of the glycolytic pathway.     

Identification of two opaque2 modifier loci in Quality Protein Maize

Genetic modifiers of opaque2 convert the soft, starchy endosperm of opaque2 maize mutants to a hard, vitreous phenotype, while maintaining the enhanced lysine content of the grain. Genetic analysis of F2 segregating seeds from crosses of opaque2 by modified opaque2 genotypes indicated that the modifiers are complex traits that act codominantly. We developed two different segregating F2 populations and mapped the modifier loci by restriction fragment length polymorphism (RFLP) analysis. A relationship was found between formation of vitreous endosperm and the locus encoding the gamma-zein storage protein, which maps near the centromere of chromosome 7. Endosperm modification was consistently associated with the presence of two rather than one gamma-zein gene at this locus. A second modifier locus was mapped near the telomere of chromosome 7L.     

Identification of opaque-2 genotypes in segregating populations of Quality Protein Maize by analysis of restriction fragment length polymorphisms

Quality Protein Maize (QPM) is a name given to genetically modified opaque-2 maize with hard endosperm. The opaque-2 mutation conditions a reduction in the amount of zein seed storage protein; zeins are deficient in the essential amino acids lysine and tryptophan, and mutant seed have a higher nutritional value. To utilize the potential of opaque-2 maize, elite inbreds can be converted to o2/o2 forms and subsequently to hard endosperm opaque-2. Since opaque-2 is recessive and endosperm specific, conventional backcross procedures to convert elite inbreds to opaque-2 forms are inefficient. To alleviate this problem, a marker-assisted selection procedure was developed for the Texas A&M University Quality Protein Maize breeding program. Hybridization of an O2 cDNA probe to blots of DNA from plants carrying O2 and o2 alleles showed that restriction fragment length polymorphisms (RFLPs) exist between the W64A o2 allele and O2 alleles of Mo17 and TX5855 inbred lines. To identify the opaque2 genotypes in segregating populations, an RFLP marker assay combining the O2 cDNA probe and HindIII-digestion of genomic DNA was developed. The effectiveness of the O2 RFLP marker assay was tested under field conditions using F₂ and backcross populations of several hard endosperm opaque-2 lines. A comparison of the genotypes identified by RFLP analysis with the seed phenotypes of the next generation indicated that this procedure is accurate and can be used for identifying O2/O2, O2/o2, and o2/o2 genotypes of individual juvenile plants in breeding populations.     

Two-generation marker-aided backcrossing for rapid conversion of normal maize lines to quality protein maize (QPM)

The low nutritive value of maize endosperm protein is genetically corrected in quality protein maize (QPM), which contains the opaque2 gene along with numerous modifiers for kernel hardness. We report here a two generation marker-based backcross breeding program for incorporation of the opaque2 gene along with phenotypic selection for kernel modification in the background of an early maturing normal maize inbred line, V25. Using the flanking marker distances from opaque2 gene in the cross V25×CML176, optimum population size for the BC₂ generation was computed in such a way that at least one double recombinant could be obtained. Whole genome background selection in the BC₂ generation identified three plants with 93 to 96% recurrent parent genome content. The three BC₂F₂ families derived from marker identified BC₂ individuals were subjected to foreground selection and phenotypic selection for kernel modification. The tryptophan concentration in endosperm protein was significantly enhanced in all the three classes of kernel modification viz., less than 25%, 25–50% and more than 50% opaqueness. BC₂F₃ lines developed from the hard endosperm kernels were evaluated for desirable agronomic and biochemical traits in replicated trials and the best line was chosen to represent the QPM version of V25, with tryptophan concentration of 0.85% in protein. The integrated breeding strategy reported here can be applied to reduce genetic drag as well as the time involved in a conventional line conversion program, and would prove valuable in rapid development of specialty corn germplasm.     

Genetic analysis of opaque2 modifier loci in quality protein maize

Quality protein maize (QPM) was created by selecting genetic modifiers that convert the starchy endosperm of an opaque2 (o2) mutant to a hard, vitreous phenotype. Genetic analysis has shown that there are multiple, unlinked o2 modifiers (Opm), but their identity and mode of action are unknown. Using two independently developed QPM lines, we mapped several major Opm QTLs to chromosomes 1, 7 and 9. A microarray hybridization performed with RNA obtained from true breeding o2 progeny with vitreous and opaque kernel phenotypes identified a small group of differentially expressed genes, some of which map at or near the Opm QTLs. Several of the genes are associated with ethylene and ABA signaling and suggest a potential linkage of o2 endosperm modification with programmed cell death.     

The inheritance of rye seed peroxidases

Summary Genetic analyses were conducted on peroxidase of the embryo and endosperm of seeds of one open pollinated and six inbred lines of cultivated rye (Secale cereale L.), and one line of Secale vavilovii Grossh. The analyses of the individual parts of the S. cereale seed yield a total of 14 peroxidase isozymes. Isozymes m, a, b, c, d, e, f and g (in order from faster to slower migration) were found in the embryo plus scutellum, while isozymes 1, 2, 3, 4, 5 and 6 (also from faster to slower migration) were peculiar of the endosperm. S. vavilovii has isozymes m, c₁, d, e, f and g in its embryo plus scutellum, and isozyme 2 in the endosperm. Segregation data indicated that at least 13 different loci would be controlling the peroxidase of S. cereale. Isozymes a and b are controlled by alleles of the same locus, all the other loci have one active and dominant allele coding for one isozyme, and other null and recessive allele. The estimation of linkage relationships shows that five endosperm loci are linked, and tentative maps are shown. A possible dosage effect and the existence of controlling gene(s) for endosperm isozyme 4 is reported. All these data and the high frequency of null alleles found are discussed in relation to recent reports.     

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 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.

     

Purification and properties of an endospermic protein of maize associated with the Opaque-2 and Opaque-6 genes

Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.Abbreviations b-32 endosperm protein of M_r 32000 - cDNA complementary DNA - EDTA ethylenediaminetetraacetic acid - O2, O6 opaque 2, opaque-6 genes - PMSF phenylmethylsulfonylfluoride - RSP reduced soluble proteins - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Genetic control of the opaque-2 gene and background polygenes over some kernel traits in maize (Zea mays L.)

Some kernel traits of agronomical importance in maize are affected by the opaque-2 (o2) gene and background polygenes, which express in different genetic systems such as embryo, endosperm, cytoplasm and maternal plant. A genetic model for seed quantitative traits with the o2 gene effects and polygenic effects as well as their GE interactions was used for protein content, lysine content, oil content and kernel density in maize. The results suggested that the o2 gene was involved in the traits investigated but the effects of the o2 gene were distinctive on various traits. The effects of the o2 gene were large on lysine content and protein content while minor on oil content. There was a substantially wide quantitative variation from polygenes expressing in different genetic systems for the traits evaluated. Significant GE interactions of the o2 gene and background polygenes declared that not only the main effects but also specific expressions depending on environments were responsible for variation of the traits studied. There seemed to have strong maternal heterosis and slight embryo heterosis for kernel density.     

Molecular genetic mapping of a high-lysine mutant gene (<Emphasis Type="Italic">opaque-16</Emphasis>) and the double recessive effect with <Emphasis Type="Italic">opaque-2</Emphasis> in maize

The lysin content in maize endosperm protein is considered to be one of the most important traits for determining the nutritional quality of food and feed. Improving the protein quality of the maize kernel depends principally on finding a mutant with a higher lysine content. Two high-lysine mutant lines with opaque endosperm, QCL3024 and QCL3021, were isolated from a self-cross population derived from Robertsons Mutator stocks. The gene controlling this mutation is temporarily termed opaque-16 (o16). In order to illuminate the genetic locus and effect of the o16 gene, two F_2:3 populations, one developed from a cross between QCL3024 and QCL3010 (a wild type line) and another from a cross between Qi205 (opaque-2 line) and QCL3021, were created, and F₃ seeds from the F₂ plants in the two populations were evaluated for lysine content. The distributions of lysine content and tests for their normality indicate that the lysine content in the two populations is regulated by the major gene of o16 and genes of o2 and o16, respectively. Based on two data sets of the linkage maps of the F₂ plant marker genotypes and the lysine content of F₃ seeds originating from the two F_2:3 populations, the o16 gene was located within 5 cM, at either 3 or 2.2 cM from umc1141 in the interval between umc1121 and umc1141 on the long arm of chromosome 8, depending on the recombination rate in the two populations as determined by composite interval mapping. According to the data of the F_2:3 population constructed from the o2 and o16 lines, the double recessive mutant effect was analyzed. The average lysine content of the F₃ o2o2o16o16 families identified by the umc1066 and umc1141 markers was approximately 30% higher than that of the F₃ o2o2 and o16o16 families, respectively. The lysine content of seven F₃ families among nine F₃ double recessive mutant families showed different increments, with an average increase of some 6% compared with that of the maternal o2 line. The potential application of the o16 mutant for maize high-lysine breeding may be to combine it with the o2 mutant bearing modifier genes, thus obtaining a mutant with much higher lysine content. For the purpose of pyramiding the o16 with o2 genes, the availability of closely linked markers of the o16 and o2 loci will facilitate marker-assisted selection and greatly reduce breeding time and effort.     

A novel tubular array associated with protein bodies in the rough endoplasmic reticulum ofopaque-2 maize

Summary The seed storage proteins of maize (Zea mays L.) are synthesized during endosperm development on membrane-bound polyribosomes. Protein body formation in normal genotypes occurs via a sequential deposition of the various types of zeins, and leads to the formation of spherical structures with a diameter of about l m. In the endosperm mutantopaque-2 the level of one zein class is reduced; these kernels exhibit an opaque phenotype instead of the vitreous phenotype displayed in normal genotypes, presumably due to the decrease in total zein protein at the time of desiccation. Previous microscopic examination ofopaque-2 protein bodies at 22 DAP (days after pollination) showed that the protein bodies were morphologically similar to those of normal genotypes. However, the endosperm ofopaque-2 maize at 14 DAP contains tubular arrays within the rough endoplasmic reticulum. These tubular arrays are tightly associated with the developing protein bodies. Long strands of tubules, sometimes 10 m in length, are observed in the endosperm, and partially formed protein bodies often seem to be forming directly from these tubular arrays. No immunostaining is associated with this tubular material when any of the anti-zein antibodies are used.Abbreviations BSA bovine serum albumin - DAP days after pollination - IgG immunoglobulin G Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology