Environmental effects on zein chromatograms of maize inbred lines revealed by reversed-phase high-performance liquid chromatography

Summary Alcohol soluble seed storage proteins (zeins and alcohol soluble glutelins) of maize (Zea mays L.) were separated by reversed-phase high-performance liquid chromatography (RP-HPLC). The objectives were to assess the reproducibility of chromatographic profiles using seed of inbred lines that had been produced in different locations and years. Reproducible differences between sources were seen but these were restricted to proteins that contributed 2% or less to an inbred profile. The majority of variation (93% for peak percent area; 99.8% for elution time) was between inbreds. RP-HPLC can therefore provide distinctive phenotypic profiles that are largely characteristic of genotype. Such qualitative and quantitative data will be valuable for studies of taxonomy, evolution, genetics, and germplasm identification.     

Associations among inbred lines of maize using electrophoretic,chromatographic, and pedigree data

Summary Associations among 17 Iowa Stiff Stalk Synthetic derived inbred lines of maize (Zea mays L.) were determined using multivariate and cluster analysis. Objectives were to assess the level of unique characterization among lines afforded by reversed-phase high-performance liquid chromatography (RP-HPLC) of zeins and starch gel electrophoresis of isozymes and to compare associations among lines revealed by biochemical and pedigree data. Isozymic data for 33 loci provided unique discrimination among 88% of the lines; 2 closely related lines were indistinguishable. Seventy-one percent of the lines could be uniquely and unambiguously identified by RP-HPLC. Biochemical data showed associations between lines that would be expected on the basis of pedigree. Nevertheless, different associations were revealed by allozymic and chromatographic data. Although these data permitted a high degree of unique identification, additional markers, covering a larger proportion of the genome, are needed to more adequately monitor similarities among genes that respond to selection during plant breeding.     

Associations among inbred lines of maize using electrophoretic,chromatographic, and pedigree data

Summary Associations among 18 Lancaster Sure Crop derived inbred lines of maize (Zea mays L.) were determined using multivariate and cluster analysis. Objectives were to assess the degree of unique characterization among lines afforded by reversed-phase high-performance liquid chromatography (RP-HPLC) and starch gel electrophoresis of allozymes and to compare associations among lines revealed by biochemical and pedigree data. RP-HPLC revealed 11 different chromatograms that uniquely identified 79% of lines that differed by more than isogenic or near isogenic segments. Allozymic data for 21 loci provided unique discrimination among 93% of non-isogenic lines. Chromatographic and allozymic data together provided unique characterization of all non-isogenic lines. Cluster and multivariate analyses of biochemical data associated lines into three groups that would have been expected on the basis of pedigree breeding records. More detailed associations were dependent upon the data set employed. Multivariate and cluster analysis of chromatographic, electrophoretic, and pedigree data could be useful in revealing more detailed associations among elite germplasm than hitherto available, thus providing data pertinent to line and hybrid development, plant variety protection, and germplasm security.     

Adaptation of sorghum: characterisation of genotypic flowering responses to temperature and photoperiod

Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop grown in a wide range of tropical and temperate environments. This study was conducted to characterise the photothermal flowering responses of sorghum genotypes and to examine relationships between photothermal characteristics and environment of origin in order to better understand the phenological basis of adaptation to environment in sorghum. Twenty-four germplasm accessions and one hybrid from 24 major sorghum-growing areas were grown in a wide range of environments varying in temperature and photoperiod in India, Kenya and Mali between 1992 and 1995. Times from sowing to flowering (f) were recorded, and the responsiveness of 1/f to temperature and photoperiod was quantified using photothermal models. Times from sowing to flowering were accurately predicted in a wide range of environments using a multiplicative rate photothermal model. Significant variation in the minimum time to flower (F_m) and photoperiod sensitivity (critical photoperiod, P_c, and photoperiod-sensitivity slope, P_s) was observed among the genotypes; in contrast there was little variation in base temperature (T_b). Adaptation of sorghum to the diverse environments in which it is grown was largely determined by photoperiod sensitivity and minimum time to flower; photoperiod sensitivity determines broad adaptation to latitude (daylength), while variation in the minimum time to flower determines specific adaptation within smaller ranges of latitude, e.g. within the humid and sub-humid tropics. Received: 16 January 1999 / Accepted: 11 March 1999     

Genetic diversity of elite sweet sorghum genotypes assessed by SSR markers

To determine genetic diversity among 47 elite sweet sorghum (Sorghum bicolor ssp. bicolor L.) genotypes, 46 simple sequence repeat (SSR) markers evenly distributed on all 10 chromosomes were selected. All SSR markers used were polymorphic among the genotypes studied. A total of 228 alleles were identified with an average of 4.96 alleles per marker. Furthermore, the genotypes studied showed medium genetic diversity. Clustering analysis grouped the 47 genotypes into 5 distinct clusters.     

Combined use of colorimetric and microelectrode methods for evaluating rhizosphere pH

Plant control of rhizosphere pH is important for nutrient mobilization and uptake, and also affects microbial activity and pathogens in the vicinity of the root. Limited information is available on the ability of plant species and genotypes within a species to induce pH changes in the rhizosphere. A growth chamber study was conducted to characterize patterns of pH change within the rhizosphere of selected genotypes in an alkaline environment with a balanced nutrient supply. After germination in incubators, seedlings of 32 genotypes of maize (Zea mays L.), soybean (Glycine max. L.), sorghum (Sorghum bicolor L.), sordan [sorghum (Sorghum bicolor L.), sudangrass (Sorghum sudanese L.) hybrid], wheat (Triticum aestivum L.), oats (Avena sativa L.), and barley (Hordeum vulgare L.) were transferred into aseptic agar medium (pH 7.6) with bromocresol purple indicator. Ability of the embedded roots to induce rhizosphere pH change was followed by photographing the color change of the bromocresol purple indicator. The pH for selected genotypes at different root zones (maturation, elongation, meristematic) was also monitored by a microelectrode at 1-, 2-, 3- and 4-mm distances from the root surface. Rhizosphere acidification for selected genotypes within a species were in the order: soybean, Hawkeye>PI-54169; maize, Pioneer-3737>Pioneer-3732>CM-37; sordan, S-757>S-333; sorghum, SC-33-8-9EYSC-118-15E; barley, Bowman>Primus II; oats, Hytest>SD-84104. The pH patterns within the root system varied from species to species. The highest amount of acidification was found at the elongation and meristematic zones for soybean, while the highest amount of acidification was found at the maturation zone for barley under the same experimental conditions. The agar method allowed the determination of a genotype's capability to induce rhizosphere pH changes while the microelectrode method is necessary for quantifying the spatial variation of specific root developmental zones with high resolution.This work is a part of H.T. Gollany's dissertation in partial fulfillment of the requirements for the PhD degree.     

Yield Results and Stability Analysis from the Sorghum Regional Biomass Feedstock Trial

Sorghum [Sorghum bicolor (L.) Moench] is one of four herbaceous dedicated bioenergy crops the U.S. Department of Energy identified as critical to annually produce one billion tons of dry biomass. Of these four crops, sorghum is unique as it is a drought-tolerant, annual crop established from seed that is readily tractable to genetic improvement. The purpose of this study was to assess the yield potential and stability of sorghums grown across diverse production environments in the USA. For this study, six sorghum genotypes (one cultivar, five hybrids) were grown in yield trials in seven locations in six states for 5 years (2008–2012). Variation in dry and fresh yield was attributable to not only genotypes, but also to the effects of year, location, and year × location. Even with the highest yielding genotype, environmental conditions were a major factor in determining the yield in a given year. This variability affects the consistency of the biomass supply for ethanol production. In general, the southeastern USA had the highest mean yields for fresh weight and dry weight, indicating that this area may be the most reliable for biomass production. A significant variation was detected among genotypes for fresh weight, dry weight, moisture content, and brix, revealing that sufficient variation within sorghum exists for continued improvement and that certain hybrids are more tractable for biomass/bioenergy production. With dedicated bioenergy sorghum germplasm and proper production environments, sorghum will be a valuable tool in the goal of the sustainable production of one billion tons of dry biomass each year in the USA.     

Screening sorghum for tolerance to excess manganese in solution culture

This study was conducted to define traits to screen sorghum (Sorghum bicolor L. Moench) genotypes for tolerance to excess Mn. Visual Mn toxicity symptoms, net and total root lengths, shoot and root dry matter yields, and shoot and root Mn concentrations were determined for plants grown in nutrient solutions (pH 4.5) at different levels of Mn (0, 3, 6, 9 and 12 mM above the initial 18 M) to assess plant responses to excess Mn. Dry matter yields showed greatest variability among genotypes, and was an effective trait to evaluate sorghum for tolerance to excess Mn. Reductions in dry matter yields did not occur until Mn levels were above 3 mM. Levels of Mn between 3 and 6 mM could effectively be used to screen sorghum for genotypic differences to excess Mn. Manganese levels above 6 mM were too severe to allow good genotypic differentiation. Of genotypes tested, NB9040 and Wheatland showed good tolerance and SC283 and ICA-Nataima were sensitive to excess Mn.     

Molecular genetic analysis of the soriz genome (<Emphasis Type="Italic">Sorghum oryzoidum</Emphasis>)

A molecular genetic analysis of soriz genotypes (Sorghum oryzoidum), its parental form Sorghum bicolor (L.) Moench (grain sorghum), possible parents (Sorghum sudanense (Piper.) Stapf. (Sudan grass) and Oryza sativa L. (Rice planting), as well as its closest relatives, has been carried out with the use of microsatellite loci of sorghum and rice. Based on the obtained data, the genetic distances were calculated and the examined species were clustered. It was shown that soriz did not carry rice DNA fragments, but its genome contained DNA fragments, which belonged to Sudan grass. This confirms that the origin of soriz is associated with representatives of Sorghum sudanense.     

Comparisons of zein profiles from inbred,F1, and F2 generations of maize as revealed by reversed-phase high-performance liquid chromatography

Summary Chi-square analyses were performed on zein Chromatographic profiles of inbred lines, F₁, F₂, and reciprocal F₁ seed for 10 hybrids of maize (Zea mays L.). The objective was to test the goodness of fit of observed profiles with those expected on the basis that the F₁ and F₂ generations represent a 21 and 11 addition of Femalemale parents of the F₁, respectively. From 40 available comparisons, 39 showed no difference between the observed chromatograms and those that were expected on the basis of four models that were tested. The one exception was due to closely eluting peaks that were revealed as shoulders and not recorded as separate entities. chromatographic profiles of inbreds, F₁, and bulk F₂ seed sources can be accurately simulated. Even though the chromatographic profile of the F₁ closely resembled that of the female parent, profiles of hybrids with common female but different male parents were distinguishable. The lack of novel peaks in both F₁ and F₂ generations compared with the inbred line thereby revealed no unpredictable interaction among zein loci. Zein protein data can be useful in registration, certification, and in the checking of hybrid pedigree especially when used in concert with isozymic data.     

Chloroplast DNA polymorphism in fertile and male-sterile cytoplasms of sorghum (Sorghum bicolor (L.) Moench)

Summary Restriction endonuclease patterns of chloroplast DNA (cpDNA) were consistently distinguishable between fertile and male-sterile cytoplasms of sorghum [Sorghum bicolor (L.) Moench], whereas no differences in restriction patterns of cpDNA among male-sterile (A₁) lines, including six isocytoplasmic strains, were revealed in this study. It is suggested that chloroplast DNA may contribute to the male sterility of A₁ lines used currently in hybrid sorghum production.This research was supported by a research grant from Kansas Grain Sorghum Commission, Kansas Board of Agriculture. Contribution 90-293-J from the Kansas Agricultural Experiment Station     

Soil aluminium effects on uptake,influx, and transport of nutrients in sorghum genotypes

Sorghum [Sorghum bicolor (L.) Moench] is the fifth most important cereal crop of the world. In South America, it is grown mainly on acid soils, and its production on these soils is limited by deficient levels of available P, Ca, Mg, and micronutrients, and toxic levels of Al and Mn. A greenhouse experiment was undertaken to evaluate the genotypic differences in sorghum for uptake (U), inhibition (IH), influx (IN) into roots, and transport (TR) to shoot for nutrients at three levels of soil Al saturation (2, 41, 64%). Overall shoot nutrient U, IN, and TR showed a significant inverse correlation with soil Al saturation and shoot Al concentration, and a significant positive correlation with shoot and root dry weight. The nutrient uptake parameters differentiated genotypes into most and least efficient categories at various levels of soil Al saturation. The nutrient uptake parameters showed significant differences with respect to soil Al saturation, genotypes, and their interactions. In the current study, Al tolerant genotypes recorded higher IN and TR for P, K, Ca, Mg, Zn, and Fe than Al-sensitive genotypes. Therefore, these U, IN, and TR traits could be used in selection of sorghum plants adaptable to acid soils. Sorghum genotypes used in this study showed intraspecific genetic diversity in U, IN, and TR for essential nutrients. It was concluded that selection of acid soil tolerant genotypes and further breeding of acid (Al) tolerant sorghum cultivars are feasible.IICA/EMBRAPA/World BankIICA/EMBRAPA/World BankIICA/EMBRAPA/World Bank     

Efficient genetic transformation of Sorghum using a visual screening marker.

To transform grain sorghum (Sorghum bicolor (L.) Moench) with a visual reporter gene (gfp) and a target gene (tlp), three genotypes (two inbreds, Tx 430 and C401, and a commercial hybrid, Pioneer 8505) were used. We obtained a total of 1011 fertile transgenic plants from 61 independent callus lines, which were produced from 2463 zygotic immature embryos via Agrobacterium-mediated transformation. The reporter gene, gfp, encoding green fluorescent protein (GFP), was used as a visual screening marker, and the target gene, tlp, encoding thaumatin-like protein (TLP), was chosen for enhancing resistance to fungal diseases and drought. Both genes were under the control of the maize ubi 1 promoter in the binary vector pPZP201. A total of 320 plants showing GFP expression, derived from 45 calli, were selected and analyzed by Southern blot analysis. There was a 100% correlation between the GFP expression and the presence of the target gene, tlp, in these plants. Transgenic plants showing strong TLP expression were confirmed by Western blotting with antiserum specific for TLP. The transgene segregated in various ratios among progeny, which was confirmed by examining seedlings showing GFP fluorescence. The progeny also showed different copy numbers of transgenics. This report describes the successful use of GFP screening for efficient production of stably transformed sorghum plants without using antibiotics or herbicides as selection agents.     

Nitrate Reductase Activity: A Biochemical Criterion of Hybrid Vigour in Sorghum bicolor (L.) Moench

F₁ hybrids of Sorghum bicolor (L.) Moench and their inbred parentswere analysed for NADH-nitrate reductase activity during theearly stages of seedling growth. In all the hybrids both mid-parentaland better parental heterosis were discernible in shoots whereasin roots two hybrids out of the three tested, showed heteroticlevels. It is suggested that in sorghum nitrate reductase activityduring seedling stages can be used as a biochemical criterionfor evaluating hybrid vigour. Sorghum bicolor (L.) Moench, sorghum, hybrid vigour, nitrate reductase     

Chemistry and Microbial Functional Diversity Differences in Biofuel Crop and Grassland Soils in Multiple Geographies

We obtained soil samples from geographically diverse switchgrass (Panicum virgatum L.) and sorghum (Sorghum bicolor L.) crop sites and from nearby reference grasslands and compared their edaphic properties, microbial gene diversity and abundance, and active microbial biomass content. We hypothesized that soils under switchgrass, a perennial, would be more similar to reference grassland soils than sorghum, an annual crop. Sorghum crop soils had significantly higher NO₃ ^? -N, NH₄ ⁺ -N, SO₄ ^2? -S, and Cu levels than grassland soils. In contrast, few significant differences in soil chemistry were observed between switchgrass crop and grassland soils. Active bacterial biomass was significantly lower in sorghum soils than switchgrass soils. Using GeoChip 4.0 functional gene arrays, we observed that microbial gene diversity was significantly lower in sorghum soils than grassland soils. Gene diversity at sorghum locations was negatively correlated with NO₃ ^? -N, NH₄ ⁺ -N, and SO₄ ^2? -S in C and N cycling microbial gene categories. Microbial gene diversity at switchgrass sites varied among geographic locations, but crop and grassland sites tended to be similar. Microbial gene abundance did not differ between sorghum crop and grassland soils, but was generally lower in switchgrass crop soils compared to grassland soils. Our results suggest that switchgrass has fewer adverse impacts on microbial soil ecosystem services than cultivation of an annual biofuel crop such as sorghum. Multi-year, multi-disciplinary regional studies comparing these and additional annual and perennial biofuel crop and grassland soils are recommended to help define sustainable crop production and soil ecosystem service practices.     

Isolation and characterization of an anticoagulant from the salivary glands of the tick, Ornithodoros savignyi (Acari: Argasidae)

An inhibitor of activated coagulation factor X (fXa) was isolated from salivary gland extracts prepared from Ornithodoros savignyi using a two-step procedure, involving reversed-phase high-performance liquid chromatography (RP-HPLC) and diethylaminoethyl (DEAE) ion-exchange chromatography. From its behaviour during DEAE chromatography it could be deduced that it possesses an acidic pI (4.6). Capillary zone electrophoresis (CZE) of the purified inhibitor showed it to be homogeneous. The molecular mass was determined as 12 kDa using capillary gel electrophoresis (CGE) and as 7183.4 using laser desorption mass spectrometry (LDMS). The N-terminal amino acid sequence (residues 1–12) was determined and found to share a 66% identity with tick anticoagulant peptide (TAP). The O. savignyi peptide is a slow, tight-binding inhibitor of fXa (K _i=0.83±0.10 nM). The interaction of the fXa-inhibitor was found to be competitive and dependent on ionic strength. Preliminary investigations show that the inhibitor may be specific for fXa.Deceased.     

Endosperm-specific hypomethylation,and meiotic inheritance and variation of DNA methylation level and pattern in sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> L.) inter-strain hybrids

Understanding dynamics and inheritance of DNA methylation represents important facets for elucidating epigenetic paradigms in plant development and evolution. Using four sets of sorghum (Sorghum bicolor L.) inter-strain hybrids and their inbred parents, the developmental stability and inheritance of cytosine methylation in two tissues, leaf and endosperm, by MSAP analysis were investigated. It was found that in all lines (inbred and hybrid) studied, endosperm exhibited a markedly reduced level of full methylation of the external cytosine or both cytosines at the CCGG sites relative to leaf, which caused a variable reduction in the estimated total methylation level in endosperm by 6.89–19.69% (11.47% on average). For both tissues, a great majority of cytosine methylation profiles transmitted to F1 hybrids, however, from 1.69 to 3.22% of the profiles showed altered patterns in hybrids. Both inherited and altered methylation profiles can be divided into distinct groups, and their frequencies are variable among the cross-combinations, and between the two tissues. The variations in methylation level and pattern detected in the hybrids were not caused by parental heterozygosity, and they could be either non-random or stochastic among hybrid individuals. Homology analysis of isolated bands that showed endosperm-specific hypomethylation or variation in hybrids indicated that diverse sequences were involved, including known-function cellular genes and mobile elements. RT-PCR analysis of six genes representing endosperm-specific hypomethylation in MSAP profiles indicated that all showed higher expression in endosperm than in leaf, suggesting involvement of methylation state in regulating tissue-specific or tissue-biased expression in sorghum. Analysis on leaf-RNA from 5-azacytidine-treated plants further corroborated this possibility. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of soil temperature on root and shoot growth traits and iron deficiency chlorosis in sorghum genotypes grown on a low iron calcareous soil

Iron deficiency chlorosis (FeDC) is a common disorder for sorghum [Sorghum bicolor (L.) Moench] grown on alkaline calcareous soils. Four sorghum genotypes were grown in growth chambers on a low Fe (1.3 g/g DTPA-extractable), alkaline (pH 8.0), calcareous (3.87% CaCO₃ equivalent) Aridic Haplustoll to determine effects of different soil temperatures (12, 17, 22 and 27°C at a constant 27°C air temperature) on various root and shoot growth traits and development of FeDC. As soil temperature increased, leaf chlorosis became more severe, and shoot and root dry weights, root lengths, and leaf areas increased markedly. Shoot/root ratios, shoot weight/root length, leaf area/shoot weight and leaf area/root weight and root length also increased while root length/root weight decreased as soil temperature increased. Severe FeDC developed in all genotypes even though genotypes had previously shown different degrees of resistance to FeDC. Genotypes differed in most growth traits, especially dry matter yields, root lengths, and leaf areas, but most traits did not appear to be related to genotype resistance to FeDC. The most FeDC resistant genotype had the slowest growth rate and this may be a mechanism for its greater resistance to FeDC.     

A Diploid,Interspecific, Fertile Hybrid from Cultivated Sorghum, <Emphasis Type="Italic">Sorghum Bicolor</Emphasis>, and the Common Johnsongrass Weed <Emphasis Type="Italic">Sorghum Halepense</Emphasis>

Valuable agronomic traits are often present but inaccessible in the wild relatives of cultivated crop species. Utilization of wild germplasm depends on the production of fertile interspecific hybrids. Several unsuccessful attempts have been made to hybridize cultivated sorghum with its wild relatives to broaden its genetic base and enhance agronomic value. The successful approach used in this study employed the nuclear male sterility gene ms3 to generate a diploid fertile hybrid between the diploid cultivated sorghum (Sorghum bicolor (L) Pers.) and its weedy tetraploid wild relative Johnsongrass (Sorghum halepense (L.) Pers.). Eight sorghum plants were selected from a Nebraska stiff stalk collection that contains the male sterility gene ms3 and were used as the female parent. About 36,000 florets of male sterile sorghum were pollinated with Johnsongrass pollen to produce an average of one well-developed and 180 severely shriveled seed/18,000 crosses. The well-developed seed gave rise to a self-fertile diploid, while none of the shriveled seed were able to germinate. The F₁ hybrid was confirmed by using cultivated sorghum SSR markers and was selfed to produce an F₂ population. A sub-sample of 96 segregating F₂ plants was examined with 36 sorghum polymorphic SSR markers. Thirty-four markers showed a normal 1:2:1 segregation ratio, evidence of normal recombination across the genome. Preliminary results showed that several desirable traits from Johnsongrass, including resistance to greenbug and chinch bug and adaptability to cold temperatures, were expressed in the resulting progenies. These observations suggest that speciation within the genus Sorghum, giving rise to widely divergent phenotypes, is effected largely by ploidy-maintained crossing barriers but apparently not by extensive genomic divergence.