SRAP Polymorphisms Associated to Cell Wall Degradability in Lignified Stems of Alfalfa

The identification of DNA polymorphisms associated to increased cell wall (CW) degradability could accelerate the development of alfalfa (Medicago sativa L.) cultivars with superior ethanol conversion yields. Genotypes with high (D+) or low (D?) CW degradability were recently identified within a biomass-type and three winter-hardy-type populations using near-infrared reflectance spectroscopy (NIRS) prediction of CW glucose released by enzyme saccharification. In this report, we used sequence-related amplified polymorphism to search for DNA variations associated to differences in enzyme-released glucose. A bulk segregant analysis (BSA) of pooled DNA samples (20 plants/bulk) from D+, D?, and randomly chosen genotypes uncovered polymorphisms associated to CW degradability. Polymorphisms that increase or decrease in intensity between D+ and D? bulks indicated the presence of genomic regions with either positive or negative effects on CW degradability. A primer pair (Me4-R14) generated a fragment, which increased in intensity in the D+ bulk of the biomass-type population. Conversely, the amplification of that fragment declined in the D+ bulks of the winter-hardy-type populations. Interestingly, these populations differ in their degradability. Assessment of the genotypic occurrence of this fragment confirmed that polymorphism detected with BSA reflects changes in the frequency of occurrence within populations. Sequence analysis of the Me4-R14 fragment revealed homologies with sequences from Medicago truncatula, a model species for legumes with documented synteny with M. sativa. Our results show that genomic regions associated to CW degradability can be identified using the combination of BSA of genotypes with contrasted degradability and a PCR-based amplification technique.     

Improving Saccharification Efficiency of Alfalfa Stems Through Modification of the Terminal Stages of Monolignol Biosynthesis

A series of transgenic lines of alfalfa (Medicago sativa) were generated in which either one of the two potentially terminal enzymes of the monolignol pathway, cinnamoyl CoA reductase (CCR) or cinnamyl alcohol dehydrogenase (CAD) was down-regulated by expression of antisense transgenes. Levels of CCR enzymatic activity were reduced to between 10% to 65% of the control level, and levels of CAD activity were similarly reduced to between 5% to 40% of the control. Biomass yields were reduced in the most strongly down-regulated lines for both transgenes, but many of the lines exhibited reduced lignin levels but normal biomass and flowering time. In vitro dry matter digestibility was increased for most transgenic lines compared to controls. Saccharification efficiency was determined by measuring the release of sugars from cell walls directly, or after sulfuric acid pre-treatment and subsequent digestion with a mixture of cellulase and cellobiase. Several CCR down-regulated lines had significantly enhanced saccharification efficiency with both pre-treated and untreated tissues, whereas CAD down-regulation had less impact on sugar release when compared to that from CCR lines with similar lignin contents. One CCR line with a 50–60% improvement in saccharification efficiency exhibited normal biomass production, indicating the potential for producing high yielding, improved feedstocks for bioethanol production through genetic modification of the monolignol pathway.     

Effect of Alfalfa Wilts on the Hydroxyproline Content in Cell Wall

The Hyp content was studied in cell wall of alfalfa susceptible and resistant strains on the 3rd, the 7th and on the 14th day after inoculation with Verticillium albo-atrum or Corynebacterium michiganense pv. insidiosum. The changes of Hyp content after inoculation with both pathogens were markedly expressed in alfalfa roots. Resistant plants of R 337 strain responded to inoculation with V. albo-atrum or C. michiganense pv. insidiosum by the decrease of Hyp content mainly on the 3rd and on the 7th day. On the 14th day after inoculation Hyp content practically did not differ from that of the control. Susceptible plants of S 354 and S 321 srains responded to inoculation with wilt pathogens by the slight decrease of Hyp content at the 3rd day after inoculation. A significant increase of Hyp content was found on the 7th and mainly on the 14th day after inoculation in comparison with control plants. The cell wall Hyp content was also determined with 7 R-strains and 7 S-strains at 120 days after inoculation with both pathogens. In each R and S strain two categories of plants were used for chemical analyses: Wilt-free plants (0 to 1 classes) and diseased, wilted plants (2 to 6 classes). In the resistant alfalfa strains no differences in Hyp content between the wilt-free and diseased plants were found. In the susceptible alfalfa strains the Hyp content was significantly higher in roots of diseased plants comparing with the wilt-free ones. Only negligible changes in Hyp content were registered in the overground parts of all inoculated alfalfa strains.     

Dynamics of Genetic Variability in Two-Locus Models of Stabilizing Selection

We study a two locus model, with additive contributions to the phenotype, to explore the dynamics of different phenotypic characteristics under stabilizing selection and recombination. We demonstrate that the interaction of selection and recombination results in constraints on the mode of phenotypic evolution. Let V(g) be the genic variance of the trait and C(L) be the contribution of linkage disequilibrium to the genotypic variance. We demonstrate that, independent of the initial conditions, the dynamics of the system on the plane (V(g), C(L)) are typically characterized by a quick approach to a straight line with slow evolution along this line afterward. We analyze how the mode and the rate of phenotypic evolution depend on the strength of selection relative to recombination, on the form of fitness function, and the difference in allelic effect. We argue that if selection is not extremely weak relative to recombination, linkage disequilibrium generated by stabilizing selection influences the dynamics significantly. We demonstrate that under these conditions, which are plausible in nature and certainly the case in artificial stabilizing selection experiments, the model can have a polymorphic equilibrium with positive linkage disequilibrium that is stable simultaneously with monomorphic equilibria.     

Downregulation of Cinnamyl Alcohol Dehydrogenase (CAD) Leads to Improved Saccharification Efficiency in Switchgrass

The bioconversion of carbohydrates in the herbaceous bioenergy crop, switchgrass (Panicum virgatum L.), is limited by the associated lignins in the biomass. The cinnamyl alcohol dehydrogenase (CAD) gene encodes a key enzyme which catalyzes the last step of lignin monomer biosynthesis. Transgenic switchgrass plants were produced with a CAD RNAi gene construct under the control of the maize ubiquitin promoter. The transgenic lines showed reduced CAD expression levels, reduced enzyme activities, reduced lignin content, and altered lignin composition. The modification of lignin biosynthesis resulted in improved sugar release and forage digestibility. Significant increases of saccharification efficiency were obtained in most of the transgenic lines with or without acid pretreatment. A negative correlation between lignin content and sugar release was found among these transgenic switchgrass lines. The transgenic materials have the potential to allow for improved efficiency of cellulosic ethanol production.     

The Hydroxyproline Content in Cell Wall of Resistant and Susceptible Alfalfa Plants Inoculated with Wilt Pathogens

The changes of hydroxyproline (Hyp) content in cell wall after inoculation with Corynebacterium michiganense pv. insidiosum or Verticillium albo-atrum in 8 cultivars of alfalfa (Medicago sativa) were studied. In our experiments cultivars ‘Trek’ and ‘Vernal’– resistant, ‘Du Puits’ and ‘Vertus’– susceptible to C. michiganense;‘Vela’ and ‘Maris Kabul’– resistant to V. albo-atrum, ‘Sabilt’ and ‘Lahontan’– susceptible to V. albo-atrum were used. In resistant plants inoculated with C. michiganense none or a small increase of Hyp content in comparison with control plants was registered. On the other hand, inoculated susceptible plants showed markedly increase of Hyp content in comparison with the control plants. The differences in Hyp content between control and inoculated resistant and susceptible plants with V. albo-atrum were not markedly expressed in comparison with the alfalfa –C. michiganense pair. The changes had similar tendency both in C. michiganense and V. albo-atrum.–alfalfa pairs.     

The Maintenance of Genetic Variability in Two-Locus Models of Stabilizing Selection

The maintenance of genetic variability at two diallelic loci under stabilizing selection is investigated. Generations are discrete and nonoverlapping; mating is random; mutation and random genetic drift are absent; selection operates only through viability differences. The determination of the genotypic values is purely additive. The fitness function has its optimum at the value of the double heterozygote and decreases monotonically and symmetrically from its optimum, but is otherwise arbitrary. The resulting fitness scheme is identical to the symmetric viability model. Linkage disequilibrium is neglected, but the results are otherwise exact. Explicit formulas are found for all the equilibria, and explicit conditions are derived fro their existence and stability. A complete classification of the six possible global convergence patterns is presented. In addition to the symmetric equilibrium (with gene frequency 1/2 at both loci), a pair of unsymmetric equilibria may exist; the latter are usually, but not always, unstable. If the ratio of the effect of the major locus to that of the minor one exceeds a critical value, both loci will be stably polymorphic. If selection is weak at the minor locus, the more rapidly the fitness function decreases near the optimum, the lower is this critical value; for rapidly decreasing fitness functions, the critical value is close to one. If the fitness function is smooth at the optimum, then a stable polymorphism exists at both loci only if selection is strong at the major locus.     

Comparison of Cell Wall Polysaccharide Hydrolysis by a Dilute Acid/Enzymatic Saccharification Process and Rumen Microorganisms

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance in the bioenergy conversion process. Cell wall polysaccharide hydrolysis was compared for a dilute sulfuric acid pretreatment at 121°C followed with cellulase hydrolysis for 72?h conversion assay (CONV) with in vitro rumen microflora incubation for 72?h (RUMEN) for a set of maize (Zea mays L.) stover samples with a wide range in cell wall composition. Residual polysaccharides from the assays were analyzed for sugar components and extent of hydrolysis calculated. Cell wall polysaccharide hydrolysis was different for all sugar components between the CONV and RUMEN assays. The CONV assay hydrolyzed xylose-, arabinose-, galactose-, and uronic acid-containing polysaccharides to a greater degree than did the RUMEN assay, whereas the RUMEN assay was more effective at hydrolyzing glucose- and mannose-containing polysaccharides. Greater hydrolysis of hemicelluloses and pectins by CONV can be attributed to the acid hydrolysis mechanism of the CONV assay for noncellulosic polysaccharides, whereas the RUMEN assay was dependent on enzymatic hydrolysis. While CONV and RUMEN hydrolysis were correlated for most polysaccharide components, the greatest correlation was only r?=?0.70 for glucose-containing polysaccharides. Linear correlations and multiple regressions indicated that polysaccharide hydrolysis by the RUMEN assay was negatively associated with lignin concentration and ferulate ether cross linking as expected. Corresponding correlations and regressions for CONV were less consistent and occasionally positive. Use of rumen microbial hydrolysis to characterize biomass performance in a conversion process may have some limited usefulness for genetic evaluations, but such assays would be unreliable for biomass pricing.     

Maintenance of Genetic Variability under Strong Stabilizing Selection: A Two-Locus Model

We study a two locus model with additive contributions to the phenotype to explore the relationship between stabilizing selection and recombination. We show that if the double heterozygote has the optimum phenotype and the contributions of the loci to the trait are different, then any symmetric stabilizing selection fitness function can maintain genetic variability provided selection is sufficiently strong relative to linkage. We present results of a detailed analysis of the quadratic fitness function which show that selection need not be extremely strong relative to recombination for the polymorphic equilibria to be stable. At these polymorphic equilibria the mean value of the trait, in general, is not equal to the optimum phenotype, there exists a large level of negative linkage disequilibrium which ``hides' additive genetic variance, and different equilibria can be stable simultaneously. We analyze dependence of different characteristics of these equilibria on the location of optimum phenotype, on the difference in allelic effect, and on the strength of selection relative to recombination. Our overall result that stabilizing selection does not necessarily eliminate genetic variability is compatible with some experimental results where the lines subject to strong stabilizing selection did not have significant reductions in genetic variability.     

供体细胞的选择与动物克隆效率研究进展

哺乳动物体细胞克隆经过几年的发展,已取得了很大进展.已有多种细胞被采用并得到了克隆后代.但动物克隆效率仍然很低,供体细胞的选择是动物克隆中的重要步骤.供体细胞的细胞周期、细胞类型、细胞来源和分化程度等方面都可能影响动物克隆的效率.     

Improved Colorimetric Determination of Cell Wall Chitin in Wood Decay Fungi

The Svennerholm modification of the Elson-Morgan method for glucosamine analysis was evaluated for its applicability to the rapid determination of chitin in wood decay fungi. The evaluation included extent of chromogen interference, sensitivity, color stability, and hydrolysis conditions for maximum release of glucosamine from fungal cell walls. With our further modification, the Svennerholm method was shown to be suitable for rapid quantitative determination of fungal chitin without chromatographic separation of hydrolysate chromogens.     

High Efficiency Selection of Full-length cDNA by Improved Biotinylated Cap Trapper

We report here an improved protocol for the preparation of full-lengthcDNA libraries that improves the previously reported method(Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics,137, 327–336), that allows long cDNAs to be cloned moreefficiently. One potential disadvantage of the original biotinylatedCAP trapper protocol is the exposure of mRNA to chemical andenzymatic attacks during the biotinylation of the cap structure,before the first-strand cDNA synthesis (and selection of full-lengthcDNA by biotinylated cap). Here, we show that the biotinylationof the cap structure is very specific and effective even ifbiotinylation is performed on the mRNA/cDNA hybrid producedby the first-strand cDNA synthesis reaction. Consequently, mRNAremains protected from chemical and enzymatic degradation duringthe overnight biotinylation step, thus making it possible toselect full-length cDNAs of longer average size. We herein reportthe efficiency and specificity of the new version of the protocolfor cap structure biotinylation and capture of full-length cDNA.     

Maintenance of Genetic Variability under the Joint Effect of Mutation, Selection and Random Drift

Formulae are developed for the distribution of allele frequencies (the frequency spectrum), the mean number of alleles in a sample, and the mean and variance of heterozygosity under mutation pressure and under either genic or recessive selection. Numerical computations are carried out by using these formulae and Watterson's (1977) formula for the distribution of allele frequencies under overdominant selection. The following properties are observed: (1) The effect of selection on the distribution of allele frequencies is slight when 4Ns 相似文献   

杂花和紫花苜蓿原生质体分离培养条件的筛选

以杂花苜蓿‘甘农1号’和紫花苜蓿‘甘农4号’、‘阿尔冈金’及‘清水’4个适宜西北内陆黄土高原地区栽培的苜蓿愈伤组织为材料,研究酶解时间、酶液组合、酶液渗透压、愈伤组织继代培养时间、预处理措施及不同培养方法等对原生质体分离和培养效果的影响,并对培养条件进行优化。结果表明:(1)适宜4个苜蓿品种愈伤组织酶解的最佳预处理措施为0.55mol/L蔗糖或CPW溶液中预质壁分离1h,最佳继代时间均为12d。(2)‘甘农1号’、‘甘农4号’和‘清水’的最佳酶液组合均为2%纤维素酶+0.5%果胶酶+0.3%崩溃酶;‘阿尔冈金’的最佳酶液组合为2%纤维素酶+0.5%果胶酶+0.3%半纤维素酶+0.3%离析酶+0.3%崩溃酶;‘甘农1号’和‘阿尔冈金’的最佳酶解时间为12h,‘甘农4号’和‘清水’分别为14h和10h。(3)适宜4个品种酶解的甘露醇浓度分别为‘甘农1号’0.75mol/L,‘甘农4号’0.65mol/L,‘阿尔冈金’0.6mol/L,‘清水’0.55~0.6mol/L。(4)经液体浅层培养和固液培养方式均可获得4个苜蓿品种的再生愈伤组织,且固液培养法较液体浅层培养法更有利于苜蓿原生质体早期的培养和再生。     

Phase Separation of Plant Cell Wall Polysaccharides and Its Implications for Cell Wall Assembly

Concentrated binary mixtures of polymers in solution commonly exhibit immiscibility, resolving into two separate phases each of which is enriched in one polymer. The plant cell wall is a concentrated polymer assembly, and phase separation of the constituent polymers could make an important contribution to its structural organization and functional properties. However, to our knowledge, there have been no published reports of the phase behavior of cell wall polymers, and this phenomenon is not included in current cell wall models. We fractionated cell walls purified from the pericarp of unripe tomatoes (Lycopersicon esculentum) by extraction with cyclohexane diamine tetraacetic acid (CDTA), Na2CO3, and KOH and examined the behavior of concentrated mixtures. Several different combinations of fractions exhibited phase separation. Analysis of coexisting phases demonstrated the immiscibility of the esterified, relatively unbranched pectic polysaccharide extracted by CDTA and a highly branched, de-esterified pectic polysaccharide present in the 0.5 N KOH extract. Some evidence for phase separation of the CDTA extract and hemicellulosic polymers was also found. We believe that phase separation is likely to be a factor in the assembly of pectic polysaccharides in the cell wall and could, for example, provide the basis for explaining the formation of the middle lamella.     

Genetic Variability of Adult Body Mass Index: A Longitudinal Assessment in Framingham Families

Objective: To explore the contribution of genetics to the mean, SD, maximum value, maximum less the mean, and change over time in body mass index (BMI) and the residual of body weight after adjustment for height. BMI is frequently used as a general indicator of obesity because of its ease and reliability in ascertainment. Cross‐sectional twin and family studies have shown a moderate‐to‐substantial genetic component for BMI. However, the contribution of genetics to the long‐term average, variability, or change over time in BMI is less clear. Research Methods and Procedures: Longitudinal data from the Framingham heart study were used to create pedigrees of age‐matched individuals. Heritability estimates were derived using variance‐decomposition methods on a total of 1051 individuals from 380 extended pedigrees followed for a period of 20 years. All subjects were followed from approximately age 35 to 55 years. Results: Moderate heritability estimates were found for the mean BMI (h² = 0.37), maximum BMI (h² = 0.40), and the mean residual of body weight (h² = 0.36). Low heritability estimates (h² ? 0.20) were found for the maximum less the mean in BMI and the SDs of BMI and residual of body weight. No additive genetic contribution was found for the average change over time in BMI or the residual of body weight. Discussion: These findings suggest that there is a significant genetic component for the magnitude of BMI throughout an individual's middle‐adult years; however, little evidence was found for a genetic contribution to the variability or rate of change in an individual's BMI.     

Understanding the Physicochemical Characteristics and the Improved Enzymatic Saccharification of Corn Stover Pretreated with Aqueous and Gaseous Ammonia

Physicochemical characteristics of corn stover pretreated by soaking in aqueous ammonia (SAA) and low-moisture anhydrous ammonia (LMAA) were compared and investigated. The glucan digestibility of the treated biomass reached 90 % (SAA) and 84 % (LMAA). The LMAA pretreatment enhanced the digestibility by cleaving cross-linkages between cell wall components, whereas the SAA pretreatment additionally improved the digestibility by efficiently removing a major portion of the lignin under mild reaction conditions without significant loss of carbohydrates. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and gel permeation chromatography (GPC) revealed the structural and chemical transformations of lignin during the pretreatments. Both pretreatments effectively cleaved ferulate cell wall cross-linking that is associated with the recalcitrance of grass lignocellulosics toward enzymatic saccharification. Extracted lignin from SAA pretreatment was extensively depolymerized but retained “native” character, as evidenced by the retention of β-ether linkages.