Oleosin genes in maize kernels having diverse oil contents are constitutively expressed independent of oil contents

In seeds, the subcellular storage oil bodies have a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We used two maize (Zea mays L.) strains having diverse kernel (seed) oil contents to study the effects of varying the oil and oleosin contents on the structure of the oil bodies. Illinois High Oils (IHO, 15% w/w oils) and Illinois Low Oils (ILO, 0.5%) maize kernels were the products of breeding for diverse oil contents for about 100 generations. In both maize strains, although the genes for oil synthesis had apparently been modified drastically, the genes encoding oleosins appeared to be unaltered, as revealed by Southern blot analyses of the three oleosin genes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with immunoblotting of the oleosins. In addition, both strains contained the same three oleosin isoforms of a defined proportion, and both accumulated oils and oleosins coordinately. Oleosins in both strains were restricted to the oil bodies, as shown by analyses of the various subcellular fractions separated by sucrosedensity-gradient centrifugation. Electron microscopy of the embryos and the isolated organelles revealed that the oil bodies in IHO were larger and had a spherical shape, whereas those in ILO were smaller and had irregular shapes. We conclude that in seeds, oleosin genes are expressed independent of the oil contents, and the size and shape of the oil bodies are dictated by the ratio of oils to oleosins synthesized during seed maturation. The extensive breeding for diverse oil contents has not altered the apparent mechanism of oil-body synthesis and the occurrence of hetero-dimer or -multimer of oleosin isoforms on the oil bodies.Abbreviations IHO Illinois High Oils - ILO Illinois Low Oils This work was supported by a USDA NRICGP grant. We thank Dr. J.W. Dudley of the University of Illinois for the IHO and ILO maize kernels, and Dr. W. Thomson for discussion on the stereological method.     

Quantitative trait locus analysis of fatty acid concentrations in maize.

A study was conducted to determine the number and chromosomal location of quantitative trait loci (QTL) influencing the concentration of five fatty acids in 200 F2S1 lines derived from an Illinois High Oil (IHO) by Illinois Low Oil (Early Maturity) (ILO(EM)) cross. Restriction fragment length polymorphism (RFLP) analysis was performed on the 200 S1 lines and concentrations of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids were determined in self-pollinated kernels harvested from plants grown in replicated field trials during 1992 and 1993. A series of 74 cDNA and genomic clones were used and these revealed 80 polymorphic loci spaced, on average, 24 cM apart throughout the maize genome. Analysis of variance detected significant (p < 0.05) associations between several RFLP loci and the concentration of each fatty acid. A total of 15 RFLP loci clustered in 12 chromosomal regions were associated with the concentration of 16:0, 17 loci clustered in 10 regions were associated with the concentration of 18:0, 12 loci clustered in eight regions were associated with the concentration of 18:1 and 18:2, and 17 loci clustered in eight regions were associated with the concentration of 18:3. Multiple linear regression models consisting of four RFLP loci explained 24 and 62% of the total phenotypic and genotypic variation (R2) among the 200 F2S1 lines for 16:0, five loci explained 51 and 71% of the variation for 18:0, three loci explained 67 and 79% of the variation for 18:1, two loci explained 67 and 81% of the variation for 18:2, and seven loci explained 52 and 78% of the variation for 18:3 in these 200 F2S1 lines. The ratio of 18:1 to 18:2 was tightly interrelated as the same QTL were associated with the concentrations of 18:1 and 18:2. A quantitative trait locus that explained 63% of the phenotypic variation in the ratio of 18:1 to 18:2 is tightly linked to umc65 on chromosome 6 in the region of the linoleic acid1 locus.     

Genetic linkage mapping in peach using morphological,RFLP and RAPD markers

We have constructed a genetic linkage map of peach [Prunus persica (L.) Batsch] consisting of RFLP, RAPD and morphological markers, based on 71 F₂ individuals derived from the self-fertilization of four F₁ individuals of a cross between New Jersey Pillar and KV 77119. This progeny, designated as the West Virginia (WV) family, segregates for genes controlling canopy shape, fruit flesh color, and flower petal color, size and number. The segregation of 65 markers, comprising 46 RFLP loci, 12 RAPD loci and seven morphological loci, was analyzed. Low-copy genomic and cDNA probes were used in the RFLP analysis. The current genetic map for the WV family contains 47 markers assigned to eight linkage groups covering 332 centi Morgans (cM) of the peach nuclear genome. The average distance between two adjacent markers is 8 cM. Linkage was detected between Pillar (Pi) and double flowers (Dl) RFLP markers linked to Pi and flesh color () loci were also found. Eighteen markers remain unassigned. The individuals analyzed for linkage were not a random sample of all F₂ trees, as an excess of pillar trees were chosen for analysis. Because of this, Pi and eight other markers that deviated significantly from the expected Mendelian ratios (e.g., 121 or 31) were not eliminated from the linkage analysis. Genomic clones that detect RFLPs in the WV family also detect significant levels of polymorphism among the 34 peach cultivars examined. Unique fingerprint patterns were created for all the cultivars using only six clones detecting nine RFLP fragments. This suggests that RFLP markers from the WV family have a high probability of being polymorphic in crosses generated with other peach cultivars, making them ideal for anchor loci. This possibility was examined by testing RFLP markers developed with the WV family in three other unrelated peach families. In each of these three peach families respectively 43%, 54% and 36% of RFLP loci detected in the WV family were also polymorphic. This finding supports the possibility that these RFLP markers may serve as anchor loci in many other peach crosses.     

RFLP analysis of soybean seed protein and oil content

Summary The objectives of this study were to present an expanded soybean RFLP map and to identify quantitative trait loci (QTL) in soybean [Glycine max (L.) Merr.] for seed protein and oil content. The study population was formed from a cross between a G. max experimental line (A81-356022) and a G. soja Sieb. and Zucc. plant introduction (PI 468916). A total of 252 markers was mapped in the population, forming 31 linkage groups. Protein and oil content were measured on seed harvested from a replicated trial of 60 F₂-derived lines in the F₃ generation (F₂₃ lines). Each F₂₃ line was genotyped with 243 RFLP, five isozyme, one storage protein, and three morphological markers. Significant (P<0.01) associations were found between the segregation of markers and seed protein and oil content. Segregation of individual markers explained up to 43% of the total variation for specific traits. All G. max alleles at significant loci for oil content were associated with greater oil content than G. soja alleles. All G. soja alleles at significant loci for protein content were associated with greater protein content than G. max alleles.     

Molecular marker analysis of Helianthus annuus L. 2. Construction of an RFLP linkage map for cultivated sunflower

A detailed linkage map of Helianthus annuus was constructed based on segregation at 234 RFLP loci, detected by 213 probes, in an F₂ population of 289 individuals (derived from a cross between the inbred lines HA89 and ZENB8). The genetic markers covered 1380 centiMorgans (cM) of the sunflower genome and were aranged in 17 linkage groups, corresponding to the haploid number of chromosomes in this species. One locus was found to be unlinked. Although the average interval size was 5.9 cM, there were a number of regions larger than 20 cM that were devoid of markers. Genotypic classes at 23 loci deviated significantly from the expected ratios (121 or 31), all showing a reduction in the ZENB8 homozygous class. The majority of these loci were found to map to four regions on linkage groups G, L and P.     

Establishment of molecular markers and linkage groups in two F2 populations of Upland cotton

Two F₂ populations of cotton (Gossypium hirsutum L.) from the crosses of HS46 x MARCABUCAG8US-1-88 (MAR) and HS46 x Pee Dee 5363 (PD5363) were characterized for restriction fragment length polymorphisms (RFLPs) using DNA probes. Seventy-three probe/enzyme combinations were used in the HS46 x MAR population analysis, which resulted in 42 informative polymorphic fragments. These 42 moleclar markers represented 26 polymorphic loci, which consisted of 15 codominant and 11 dominant (+/-) genotypes. Chi-square analyses of these loci fit expected genotypic ratios of 121 and 31, respectively An analysis of these loci with the MAPMAKER program resulted in the establishment of four linkage groups A, B, C, and D with 4,2,2, and 2 loci, respectively, as well as 16 unlinked loci. Six probe-enzyme combinations were assayed on the HS46 x PD5363 population, which resulted in 11 informative polymorphic fragments. These 11 fragments represented 6 polymorphic loci, 1 dominant (+/-) and 5 codominant genotypes. The MAPMAKER analysis of these loci yielded 2 linked loci. Thus, a total of 53 polymorphic fragments and 32 polymorphic loci, representing five linkage groups, were identified among the two families.Contribution of the USDA-ARS in cooperation with the Miss Agric For Exp Stn.     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

Genetic analysis of morphological variation in Brassica oleracea using molecular markers

A cross between the open-pollinated Brassica oleracea cabbage cultivar Wisconsin Golden Acre and the hybrid broccoli cultivar Packman was used with molecular markers to investigate the genetic control of morphological variation. Twenty-two traits derived from leaf, stem, and flowering measurements were analyzed in 90 F₂ individuals that were also classified for genotype by restriction fragment length polymorphism (RFLP) markers. Seventy-two RFLP loci, which covered the mapped genome at an average of 10 map-unit intervals on all nine linkage groups, were tested individually for associations to phenotypic measurements by single factor ANOVA, and markers with significant associations (P<0.05) were used to develop multilocus models. These data were utilized to describe the location, parental contribution of alleles, magnitude of effect, and the gene action of trait loci. Single marker loci that were significantly associated (P<0.05) with trait measurements accounted for 6.7–42.7% of the phenotypic variation. Multilocus models described as much as 60.1% of the phenotypic variation for a given trait. In some cases, different related traits had common marker-locus associations with similar gene action and genotypic class ranking. The numbers, action, and linkages, of genes controlling traits estimated with marker loci in this population corresponded to estimates based on classical genetic methods from other studies using similar, or similarly-wide, crosses. There was no evidence that genome duplication accounted for a significant portion of multiple genes controlling trait loci over the entire genome, but possible duplications of trait loci were identified for two regions with linked, duplicated marker loci.     

Mapping of a gene determining linolenic acid concentration in rapeseed with DNA-based markers

Rapeseed ranks third in world oil production. An important breeding objective to improve oil quality in this crop is to lower linolenic acid concentration in the seeds. Previous reports indicate that the concentration of this acid in Brassica napus is determined by two or three nuclear genes. Using DNA-based markers, we have successfully mapped a gene determining linolenic acid concentration in an F₂ population derived from crossing the cultivar Duplo and alow linolenic acid line, 3637-1. Linolenic acid concentration in this population ranged from 2.1% to 10.5% with-amean of 6.2%. A RAPD marker, K01-1100, displayed significantly different frequencies between two subpopulations consisting of either high or low linolenic acid concentration individuals sampled from the two extremes of the F₂ distribution. Marker K01-1100 segregated in a codominant fashion when used as an RFLP probe on DNA from individuals of this F₂ population. The linolenic acid concentration means for the three resulting RFLP genotypes in the F₂ population were 4.8% (homozygous 3637-1 allele), 6.4% (heterozygous), and 7.5% (homozygous Duplo allele), respectively. It is estimated that this marker accounts for 26.5% of the genetic variation of linolenic acid concentration in this population.     

Segregation of allozymes in megagametophytes of viable seed from a natural population of jack pine,Pinus banksiana Lamb.

Summary Segregation ratios of allozymes in haploid female gametophytes obtained from viable seed were studied in a natural population of jack pine, Pinus banksiana. Stability of these ratios was assessed for three levels of the sexually reproductive crown as well as for four years of natural fertilization. Analyses of observed segregation ratios of four of five polymorphic isozyme loci showed good correspondence to the overall 11 ratios expected for simple Mendelian inheritance. Allozymes of glucose-6-phosphate dehydrogenase did not segregate in the expected 11 ratio. In addition, there were significant deviations from the expected segregation ratio for all the loci at some sampling positions on individual trees. Heterogeneity of segregation among trees, strata and years could be the result of pollen pool heterogeneity, segregation distortion and/or recessive lethal and semi-lethal gene combinations resulting in early embryo abortion. These types of segregation deviations in viable seed can affect the estimation of allele frequencies from bulked samples of a small number of individuals, the inference of heterozygosity/homozygosity of parental trees, and estimates of selfing rates.     

Towards an integrated linkage map of common bean 2. Development of an RFLP-based linkage map

Summary A restriction fragment length polymorphism (RFLP)-based linkage map for common bean (Phaseolus vulgaris L.) covering 827 centiMorgans (cM) was developed based on a F₂ mapping population derived from a cross between BAT93 and Jalo EEP558. The parental genotypes were chosen because they exhibited differences in evolutionary origin, allozymes, phaseolin type, and for several agronomic traits. The segregation of 152 markers was analyzed, including 115 RFLP loci, 7 isozyme loci, 8 random amplified polymorphic DNA (RAPD) marker loci, and 19 loci corresponding to 15 clones of known genes, 1 virus resistance gene, 1 flower color gene, and 1 seed color pattern gene. Using MAPMAKER and LINKAGE-1, we were able to assign 143 markers to 15 linkage groups, whereas 9 markers remained unassigned. The average interval between markers was 6.5 cM; only one interval was larger than 30 cM. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios (121 or 31) and mapped into four clusters. Probes of known genes belonged to three categories: seed proteins, pathogen response genes, and Rhizobium response genes. Within each category, sequences homologous to the various probes were unlinked. The I gene for bean common mosaic virus resistance is the first disease resistance gene to be located on the common bean genetic linkage map.     

Genetic mapping of the barley nitrate reductase-deficient nar1 and nar2 loci

Summary The nar2 locus that codes for a protein involved in molybdenum cofactor function in nitrate reductase and other molybdoenzymes was mapped to barley chromosome 7. F₂ genotypic data from F₃ head rows indicated nar2 is located 8.4±2.1 and 23.0± 4.6 cm from the narrow leaf dwarf (nld) and mottled seedling (mt2) loci, respectively. This locates the nar2 locus at 54.7±3.1 cm from the short-haired rachilla (s) locus near the centromere of chromosome 7. Close linkage of nar2 with DDT resistance (ddt) and high lysine (lys3) loci was detected but could not be quantified due to deviations from the individual expected 121 segregations for the ddt and lys3 genes. Southern blots of wheat-barley addition lines probed with a nitrate reductase cDNA located the NADH : nitrate reductase structural gene, nar1, to chromosome 6.Scientific Paper No. 7762. College of Agriculture and Home Economics Research Center, Washington State University, Project No. 0745. This investigation was supported in part by United States Department of Agriculture Grant No. 86-CRCR-1-2004     

Construction of a molecular linkage map in coffee

A linkage map for coffee (Coffea canephora P.) totalling 1402 cM has been developed on the basis of a population of doubled haploids. Both RFLP markers and PCR-based markers (RAPD) were used to construct 15 linkage groups. Coffee genomic and cDNA clones provided the source of the probes. In total, 47 RFLP and 100 RAPD loci have been placed on the linkage map. A rather low DNA polymorphism rate (18% for RFLP markers and 29% for RAPD primers) was detected. Only 81% of RAPD markers and 85% of RFLP markers fit an expected 11 ratio (P<0.01). The availability of a molecular linkage map has many implications for the future development of the genetics and breeding of this commercially important crop species.     

Enzymatic variation at seven loci in nine natural populations of Drosophila melanogaster

Allozyme polymorphisms at seven loci have been studied in nine natural populations of Drosophila melanogaster from the Saône and Rhône valleys sampled in 1973 and 1974. A great deal of polymorphism was observed; an individual was on the average heterozygous at 20.2% of its loci. The populations were genetically very homogeneous throughout the region sampled. The number of ovariolae per female varied from one group of populations to another depending on their geographical separation. Yet the number of ovariolae remained constant from one year to the next. The results show that migration alone cannot explain the homogeneity of the allozyme frequencies. It seems reasonable to conclude that selection plays a major role in maintaining the homogeneity of populations living in proximal biotopes.E.R.A. No. 406: Analyse et mécanismes de maintien du polymorphisme.     

RFLP mapping of the sugary enhancer1 gene in maize

RFLP marker data from an F₂₃ population derived from a cross between a sugary1 (su1) and a sugary enhancer1 (su1, sel) inbred were used to construct a genetic linkage map of maize. This map includes 93 segregating marker loci distributed throughout the maize genome, providing a saturated linkage map that is suitable for linkage analysis with quantitative trait loci (QTL). This population, which has been immortalized in the form of sibbed F₂₃ families, was derived from each of the 214 F₂ plants and along with probe data are available to the scientific community. QTL analysis for kernel sucrose (the primary form of sugar) concentration at 20 days after pollination (DAP) uncovered the segregation of seven major QTL influencing sucrose concentration; a locus linked to umc36a described the greatest proportion of the variation (24.7%). Since maltose concentration has previously been reported to be associated with the se1 phenotype, an analysis of probe associations with maltose concentration at 40 DAP was also conducted. The highly significant association of umc36a with maltose and sucrose concentrations provided evidence that this probe is linked to se1. Phenotypic evaluation for the se1 genotype in each F₂₃ family enabled us to map the gene 12.1 cM distal to umc36a. In contrast to previous work where se1 was reported to be located on chromosome four, our data strongly suggest that the sugary enhancer1 locus maps on the the distal portion of the long arm of chromosome 2 in the maize genome.     

Comparative mapping in F2∶3 and F6∶7 generations of quantitative trait loci for grain yield and yield components in maize

This study was conducted to compare maize quantitative trait loci (QTL) detection for grain yield and yield components in F₂₃ and F₆₇ recombinant inbred (RI) lines from the same population. One hundred and eighty-six F₆₇ RIs from a Mo17×H99 population were grown in a replicated field experiment and analyzed at 101 loci detected by restriction fragment length polymorphisms (RFLPs). Single-factor analysis of variance was conducted for each locus-trait combination to identify QTL. For grain yield, 6 QTL were detected accounting for 22% of the phenotypic variation. A total of 63 QTL were identified for the seven grain yield components with alleles from both parents contributing to increased trait values. Several genetic regions were associated with more than one trait, indicating possible linked and/or pleiotropic effects. In a comparison with 150 F₂₃ lines from the same population, the same genetic regions and parental effects were detected across generations despite being evaluated under diverse environmental conditions. Some of the QTL detected in the F₂₃ seem to be dissected into multiple, linked QTL in the F₆₇ generation, indicating better genetic resolution for QTL detection with RIs. Also, genetic effects at QTL are smaller in the F₆₇ generation for all traits.Abbreviations RFLPs Restriction fragment length polymorphisms - QTL quantitative trait loci - RIs recombinant inbreds Journal Paper no. J-16261 of the Iowa Agric and Home Economics Exp Stn Project no. 3134     

Isolation and characterization of oil-desulphurizing bacteria

The objective of this study was to isolate local bacterial strains capable of removing sulphur from oil fractions without degrading the hydrocarbon. Oil biodesulphurization is an important step in combating pollution problems emanating from burning fossil fuels. Organisms which survive on oil are plentiful in local Kuwaiti soils; however, those that selectively only attack the carbon–sulphur bond are more difficult to find. Three strains were isolated based on their ability to use dibenzothiophene (DBT) as a sole source of sulphur for growth at 30 °C. Similar to other biodesulphurization organisms, the strains convert DBT to [2-hydroxybiphenyl (2-HBP) as detected by gas chromatography (GC). The specific desulphurization activity was in the range 5–13 mol 2-HBP/g-cell × h. Identification of the strains, based on 16 rRNA gene sequence similarity, showed the strains to be Rhodococcus erythropolis and Rhodococcus globerulus. The biodesulphurization activity was enhanced by promoting oxidore-ductase enzyme co-expression through the addition of a carbon source. The desulphurization was limited by the availability of DBT to the organism. Interfacial mass transfer through the aqueous-organic layer was confirmed to be a limiting factor.     

Allozyme polymorphisms within and among open-pollinated and adapted exotic populations of maize

Summary Twelve U.S. Corn Belt open-pollinated and five adapted exotic populations of maize (Zea mays L.) were assayed for allozyme (allele) variation at 13 enzyme marker loci. Extensive allozyme variability was observed in all populations studied. No locus was monomorphic over all populations. Each of the lociIdh2, Got1, Mdh2, Pgd1, andPgd2 expressed two allozymes over all populations,Adh1, Acp1, Prx1, andEst1 each had three allozymes present,Est4, Glu1, andEnp1 had five allozymes, andAcp4 had six allozymes present. Significant deviations of genotypic frequencies were detected from Hardy-Weinberg equilibrium frequencies and 94% of average Fixation Index values indicated heterozygote deficiencies, which suggested that nonrandom mating and/or natural selection favoring homozygotes were possible factors affecting the maintenance or loss of genetic variability marked by these enzyme loci. Genetic distance and cluster analyses indicated that the observed genetic variability at the 13 enzyme loci was closely related to Dent and Flint types of maize.     

RFLP loci associated with soybean seed protein and oil content across populations and locations

Molecular markers provide the opportunity to identify marker-quantitative trait locus (QTL) associations in different environments and populations. Two soybean [Glycine max (L.) Merr.] populations, Young x PI 416 937 and PI 97100 x Coker 237, were evaluated with restriction fragment length polymorphism (RFLP) markers to identify additional QTLs related to seed protein and oil. For the Young x PI 416937 population, 120 F₄-derived lines were secored for segregation at 155 RFLP loci. The F₄-derived lines and two parents were grown at Plains, G.a., and Windblow and Plymouth, N.C. in 1994, and evaluated for seed protein and oil. For the PI 97100 x Coker 237 population, 111 F₂-derived lines were evaluated for segregation at 153 RFLP loci. Phenotypic data for seed protein and oil were obtained in two different locations (Athens, G.a., and Blackville, S.C.) in 1994. Based on single-factor analysis of variance (ANOVA) for the Young x PI 416937 population, five of seven independent markers associated with seed protein, and all four independent markers associated with seed oil in the combined analysis over locations were detected at all three locations. For the PI 97 100 x Coker 237 population, both single-factor ANOVA and interval mapping were used to detect QTLs. Using single-factor ANOVA, three of four independent markers for seed protein and two of three independent markers for seed oil were detected at both locations. In both populations, singlefactor ANOVA, revealed the consistency of QTLs across locations, which might be due to the high heritability and the relatively few QTLs with large effects conditioning these traits. However, interval mapping of the PI 97100 x Coker 237 population indicated that QTLs identified at Athens for seed protein and oil were different from those at Blackville. This might result from the power of QTL mapping being dependent on the level of saturation of the genetic map. Increased seed protein was associated with decreased seed oil in the PI 97100 x Coker 237 population (r = –0.61). There were various common markers (P0.05) on linkage groups (LG) E, G,H,K, and UNK2 identified for both seed protein and oil. One QTL on LG E was associated with seed protein in both populations. The other QTLs for protein and oil were population specific.     

An extended map of the sugar beet genome containing RFLP and RAPD loci

An updated map of sugar beet (Beta vulgaris L. ssp. vulgaris var altissima Doell) is presented. In this genetic map we have combined 248 RFLP and 50 RAPD loci. Including the loci for rhizomania resistance Rr1, hypocotyl colour R and the locus controlling the monogerm character M, 301 loci have now been mapped to the nine linkage groups covering 815 cM. In addition, the karyotype of some of the Beta vulgaris chromosomes has been correlated with existing RFLP and RAPD linkage maps.