Wheat Sequences Research Articles

Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation

Detailed molecular characterisation of transgene loci is a requirement for gaining regulatory approval for environmental release of genetically modified crops. In cereals, it is generally accepted that Agrobacterium-mediated transformation generates cleaner transgene loci with lower copy number and fewer rearrangements than those generated by biolistics. However, in wheat there has been little detailed analysis of T-DNA insertions at genetic and molecular level. Wheat lines transformed using Agrobacterium tumefaciens with bar and gusA (GUS) genes were subjected to genetic and molecular analysis. Unlike previous studies of transgene loci in wheat, we used functional assays for PAT and GUS proteins, combined with PCR and Southern analysis to detect the presence, copy number, linkage and transmission of two transgenes inserted in the same T-DNA. Thirty-four independent transgenic lines were categorised into three types: type I events (38% of total) where the gusA and bar genes displayed complete genetic linkage, segregating together as a single functional locus at the expected ratio of 3:1; type II events (18%), which possessed two or more transgene loci each containing gusA and bar; and type III events (44%), containing an incomplete T-DNA in which either the gusA or bar gene was lost. Most lines in this last category had lost the bar gene situated near the left T-DNA border. Southern analysis indicated that 30% of all lines possessed a single T-DNA copy containing gusA and bar. However, when data on expression and molecular analysis are combined, only 23% of all lines have single copy T-DNAs in which both gene cassettes are functioning. We also report on the presence of plasmid backbone DNA sequence in transgene loci detected using primer pairs outside the left and right T-DNA borders and within the plasmid selectable marker (NptI) gene. Approximately two thirds of the lines contained some vector backbone DNA, more frequently adjacent to the left border. Taken together, these data imply unstable left border function causing premature T-strand termination or read-through into vector backbone. As far as we are aware, this is the first report revealing near border T-DNA truncation and vector backbone integration in wheat transgenic lines produced by Agrobacterium-mediated transformation.

Resistance gene analogues of wheat: molecular genetic analysis of ESTs

Using two divergent nucleotide binding site (NBS) regions from wheat sequences of the NBS-LRR (leucine rich repeat) class, we retrieved 211 wheat and barley NBS-containing resistance gene analogue (RGA) expressed sequence tags (ESTs). These ESTs were grouped into 129 gene sequence groups that contained ESTs that were at least 70% identical at the DNA level over at least 200 bp. Probes were obtained for 89 of these RGA families and chromosome locations were determined for 72 of these probes using nullitetrasomic Chinese Spring wheat lines. RFLP analysis of 49 of these RGA probes revealed 65 mappable polymorphic bands in the doubled haploid Cranbrook x Halberd wheat population (C x H). These bands mapped to 49 loci in C x H. RGA loci were detected on all 21 chromosomes using the nullitetrasomic lines and on 18 chromosomes (linkage groups) in the C x H map. This identified a set of potential markers that could be developed further for use in mapping and ultimately cloning NBS-LRR-type disease resistance genes in wheat.

Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5' flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety 'Chinese Spring' was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5' flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5' flanking regions of LMW-GS genes are likely to have evolved in concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.

Effect of Nitrogen Management on Yield and Water Use Efficiency of Rainfed Wheat and Maize in Northwest China

A field experiment with four treatments and four replicates in a randomized complete block design was conducted at the Changwu Experimental Station in Changwu County, Shaanxi Province, of Northwest China from 1998 to 2002. The local cropping sequence of wheat, wheat-beans, maize, and wheat over the 4-year period was adopted. A micro-plot study using N-15-labelled fertilizer was carried out to determine the fate of applied N fertilizer in the first year. When N fertilizer was applied wheat (years 1, 2 and 4) and maize (year 3) grain yield increased significantly (P 30%), with no significant yield differences in normal rainfall years (Years 1, 2 and 3) for N application at the commonly application rate and at 2/3 of this rate. Grain yield of wheat varied greatly between years, mainly due to variation in annual rainfall. Results of N-15 studies on wheat showed that plants recovered 36.6%-38.4% of the N applied, the N remained in soil (0-40 cm) ranged from 29.2% to 33.6%, and unaccounted-for N was 29.5%-34.2%. The following crop (wheat) recovered 2.1%-2.8% of the residual N from N applied to the previous wheat crop with recovery generally decreasing in the subsequent three crops (beans, maize and wheat).

The turnover of carbohydrate carbon in a cultivated soil estimated by 13C natural abundances

SummaryUnderstanding the chemical composition of soil organic matter (SOM) requires the determination of the dynamics of each class of compounds. We measured the dynamics of carbon in neutral carbohydrates by use of natural 13C labelling in an experimental wheat and maize sequence extending over 23 years. The isotopic composition of individual neutral monosaccharides was determined in hydrolysed particle‐size fractions by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) of trimethylsilyl (TMS) derivatives. The sensitivity in terms of 13C/12C ratios ranged between 1 and 2‰ depending on the monosaccharide. The age distribution of neutral sugar carbon was very similar to that of total soil carbon. Particulate organic matter (POM) was characterized by the predominance of glucose and xylose of vegetal origin. In POM > 200 µm, the mean age of sugar‐C (5 years) was slightly less than that of total carbon (7 years). Xylose was younger than glucose. The fine fraction 0–50 µm contained mainly glucose, arabinose, galactose, xylose, fucose and mannose, which had predominantly microbial origins. The mean age of carbohydrate carbon in the fraction 0–50 µm was between 60 and 100 years and was similar to that of total organic carbon (OC). No difference in the age of carbon between the individual monosaccharides was found. The POM fraction 50–200 µm had an intermediate signature and turnover. Considering the typical lability of carbohydrates, the relatively great age of carbohydrate carbon may be explained by physical or chemical protection from degradation, as well as by recycling of soil organic matter carbon by soil microbes.

Mitochondrial microsatellite variability in common wheat and its ancestral species

On the basis of the entire mitochondrial DNA sequence of common wheat, Triticum aestivum, 21 mitochondrial microsatellite loci having more than ten mononucleotide repeats were identified. The mitochondrial microsatellite variability at all loci was examined with 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploidy evolution. Polymorphic banding patterns were obtained at 15 out of 21 mitochondrial microsatellite loci. The number of alleles per polymorphic microsatellite ranged from 2 to 5 with an average of 3.07, and the diversity values (H) ranged from 0.09 to 0.50 with an average of 0.29. These values are almost two third of wheat chloroplast microsatellite values, indicating that variability of mitochondrial microsatellite is much less than that of chloroplast microsatellite. Based on the allele variation at all loci, a total of seven mitochondrial haplotypes were identified among common wheat and its ancestral species. Three diploid species showed their own specific haplotypes and timopheevi group (11 accessions) had three types, whereas 29 accessions of emmer and common wheat groups shared the same haplotype. These results indicate that a single mitochondrial haplotype determined by microsatellite analysis has conservatively been maintained in the evolutionary lineage from wild tetraploid to cultivated hexaploid species.

Is photosynthetic transcriptional regulation in Triticum aestivum L. cv. ‘TugelaDN’ a contributing factor for tolerance to Diuraphis noxia (Homoptera: Aphididae)?

Diuraphis noxia (Russian wheat aphid, RWA) is a major pest on wheat in South Africa and most other wheat growing countries. Being a probing-sucking insect, RWAs insert their stylets into the phloem sieve elements and feed on the phloem sap. This feeding causes necrotic lesions in resistant varieties, or decoloration of leaves and death in susceptible varieties. In an effort to broaden our understanding on the response of the plant to RWA feeding, we synthesized and analyzed expressed sequence tags (ESTs) from suppression subtractive hybridization (SSH) libraries. These libraries were constructed using near isogenic wheat lines susceptible "Tugela" and resistant "TugelaDN" (Dn1) to RWA, as well as accession lines PI137739 (Dn1) and PI294994 (Dn5). Analysis of 200 ESTs from the libraries revealed the involvement of transcripts encoding genes involved in cell maintenance, growth and regulation, plant defense and signaling, photosynthesis and energy production, and of unknown function. A selection of these ESTs, in combination with clones obtained from other sources, were used on a custom array to study the expression profiles of 256 candidate wheat sequences putatively involved in plant defense against RWA. The selected sequences included wheat genomic clones with putative nucleotide binding site (NBS) motifs, rapid amplification of cDNA ends PCR (RACE-PCR), and cDNA clones from RWA induced libraries. Genomic banana and flax clones that were obtained using representative difference analysis (RDA), and suspected to be involved in abiotic stress responses, were also spotted onto the microarray slides. The spotted custom arrays were then hybridized against cDNA isolated from a resistant cultivar "TugelaDN" on 0, 2, 5, and 8 days after infestation, post-labeled with Cy3- or Cy5-fluorescent dyes. The subsequent expression profiling using DNA microarray, RT-PCR, and Northern Blot analysis identified 29 transcripts associated with the feeding response. These transcripts encoded proteins functioning in direct defense and signaling, oxidative burst, cell wall degradation, cell maintenance, photosynthesis, and energy production. Results indicate that plants co-ordinately regulate gene expression when attacked by RWA. It is hypothesized that the NBS-LRR proteins are important in receptor recognition and signaling, which enable the plant to overcome the stresses inflicted by RWA feeding. It is further suggested that the ability to maintain photosynthetic function with resultant energy production is one of the determining factors ensuring the survival of the resistant varieties when coping with the RWA feeding.

Cloning and characterization of wheat PDI (protein disulfide isomerase) homoeologous genes and promoter sequences

The genomic and cDNA sequences of three PDI homoeologous genes located on chromosomes 4A, 4B and 4D of bread wheat and their promoters were cloned and sequenced. The three sequences showed a very high conservation of the coding region and of the exon/intron structure, which consisted of ten exons. The comparison of wheat sequences with those of rice and Arabidopsis showed a significant conservation of the exon/intron structure across the three species. The expression of each gene was analysed by RT-PCR in different plant tissues (roots, coleoptiles, spikelets, leaves and developing caryopses). All the genes showed a higher expression in developing caryopses than in other analysed tissues, wherein some differences were detected. The promoter sequences of the three genes possessed some regulatory motifs typical of endosperm specific expression.

Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon–intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Management of declining groundwater in the Trans Indo-Gangetic Plain (India): Some options

The average productivity of rice–wheat sequence is quite impressive in the Trans Indo-Gangetic Plain (India) but these gains are over-shadowed due to declining groundwater, particularly in the areas, where groundwater quality is either good or marginal. The groundwater decline can be reversed through artificial groundwater recharge and by adopting suitable land and water management practices. Groundwater recharge is found technically feasible through vertical shafts conducting water from the ground surface directly to aquifers, after it has been passed through a sand-gravel filter. The recharge rate through this system is almost equal to a shallow cavity/filter well yield (about 11 l/s) and its cost is estimated at about INR 10/100 m 3 (1 US$ = 45 INR). Further study in the Kaithal and Karnal districts of Haryana for stabilizing watertable within 6–7 m, which permits continuous use of shallow tubewell technology, indicated that the rice area could be supported at 60% of cultivable command area (CCA) and wheat between 65 and 80% of CCA with the existing management practices. The cultivation of wheat crop is sustainable in larger area, mainly due to its medium water requirement, salt resistance characteristics and consistent market demand resulting in assured returns. There is a possibility of supporting rice at a higher level, if part of the area (up to 10%) is left fallow and used for rainwater conservation and recharge. The fallow area may be subsequently put under early rabi (winter) crops like mustard, gram and other pulses. The effect of varying irrigation and fallowing would increase 23% equivalent wheat yield by changing land and water management practices. The analysis further indicated that the adoption of proposed irrigation management practices might stabilize watertable at desired level of 6–7 m in 10–15 years in high (3–4 m), 5 years in medium (5–10 m) and 40 years in deep (>10 m) watertable areas.

Updating of transposable element annotations from large wheat genomic sequences reveals diverse activities and gene associations

Triticeae species (including wheat, barley and rye) have huge and complex genomes due to polyploidization and a high content of transposable elements (TEs). TEs are known to play a major role in the structure and evolutionary dynamics of Triticeae genomes. During the last 5 years, substantial stretches of contiguous genomic sequence from various species of Triticeae have been generated, making it necessary to update and standardize TE annotations and nomenclature. In this study we propose standard procedures for these tasks, based on structure, nucleic acid and protein sequence homologies. We report statistical analyses of TE composition and distribution in large blocks of genomic sequences from wheat and barley. Altogether, 3.8 Mb of wheat sequence available in the databases was analyzed or re-analyzed, and compared with 1.3 Mb of re-annotated genomic sequences from barley. The wheat sequences were relatively gene-rich (one gene per 23.9 kb), although wheat gene-derived sequences represented only 7.8% (159 elements) of the total, while the remainder mainly comprised coding sequences found in TEs (54.7%, 751 elements). Class I elements [mainly long terminal repeat (LTR) retrotransposons] accounted for the major proportion of TEs, in terms of sequence length as well as element number (83.6% and 498, respectively). In addition, we show that the gene-rich sequences of wheat genome A seem to have a higher TE content than those of genomes B and D, or of barley gene-rich sequences. Moreover, among the various TE groups, MITEs were most often associated with genes: 43.1% of MITEs fell into this category. Finally, the TRIM and copia elements were shown to be the most active TEs in the wheat genome. The implications of these results for the evolution of diploid and polyploid wheat species are discussed.

Development of Taxon-Specific Sequences of Common Wheat for the Detection of Genetically Modified Wheat

Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.

Wheat EST sequence assembly facilitates comparison of gene contents among plant species and discovery of novel genes

Using a strategy requiring only modest computational resources, wheat expressed sequence tag (EST) sequences from various sources were assembled into contigs and compared with a nonredundant barley sequence assembly, with ESTs, with complete draft genome sequences of rice and Arabidopsis thaliana, and with ESTs from other plant species. These comparisons indicate that (i) wheat sequences available from public sources represent a substantial proportion of the diversity of wheat coding sequences, (ii) prediction of open reading frames in the whole genome sequence improves when supplemented with EST information from other species, (iii) a substantial number of candidates for novel genes that are unique to wheat or related species can be identified, and (iv) a smaller number of genes can be identified that are common to monocots and dicots but absent from Arabidopsis. The sequences in the last group may have been lost from Arabidopsis after descendance from a common ancestor. Examples of potential novel wheat genes and Triticeae-specific genes are presented.

Ancient haplotypes resulting from extensive molecular rearrangements in the wheat A genome have been maintained in species of three different ploidy levels

Plant genomes, in particular grass genomes, evolve very rapidly. The closely related A genomes of diploid, tetraploid, and hexaploid wheat are derived from a common ancestor that lived <3 million years ago and represent a good model to study molecular mechanisms involved in such rapid evolution. We have sequenced and compared physical contigs at the Lr10 locus on chromosome 1AS from diploid (211 kb), tetraploid (187 kb), and hexaploid wheat (154 kb). A maximum of 33% of the sequences were conserved between two species. The sequences from diploid and tetraploid wheat shared all of the genes, including Lr10 and RGA2 and define a first haplotype (H1). The 130-kb intergenic region between Lr10 and RGA2 was conserved in size despite its activity as a hot spot for transposon insertion, which resulted in >70% of sequence divergence. The hexaploid wheat sequence lacks both Lr10 and RGA2 genes and defines a second haplotype, H2, which originated from ancient and extensive rearrangements. These rearrangements included insertions of retroelements and transposons deletions, as well as unequal recombination within elements. Gene disruption in haplotype H2 was caused by a deletion and subsequent large inversion. Gene conservation between H1 haplotypes, as well as conservation of rearrangements at the origin of the H2 haplotype at three different ploidy levels indicate that the two haplotypes are ancient and had a stable gene content during evolution, whereas the intergenic regions evolved rapidly. Polyploidization during wheat evolution had no detectable consequences on the structure and evolution of the two haplotypes.

Phosphorus fractions in an acid soil continuously fertilized with mineral and organic fertilizers

The effect of different treatments on the fate of applied P was investigated in a long-term field experiment started in 1972–1973 following a maize–wheat sequence. The soil samples were collected after 29 years of continuous addition of mineral fertilizers and amendments such as farmyard manure (FYM) and lime. The total P content of all the treatments increased compared to the original soil; NaOH-inorganic P (Pi) (NaOH-Pi) representing Fe and Al-bound P was the dominant Pi fraction. At the beginning of the experiment (1972–1973), the various P pools could be quantitatively ranked in the following order: residual P>NaOH-organic P (Po)>NaOH-Pi>NaHCO3-Po>NaHCO3-Pi>HCl-P>H2O-P. As a result of continued P fertilization and cropping, the order changed as follows: residual P>NaOH-Pi>NaOH-Po>NaHCO3-Pi>NaHCO3-Po>HCl-P>H2O-P. Compared to the imbalanced mineral fertilizer application, the balanced as well as integrated application of nutrients resulted in significantly lower P adsorption capacity of soils. The Olsen extractable-P fraction (plant-available P) increased from about 12 mg kg−1 soil in 1972 to about 81 mg kg−1 soil in the treatments receiving P for the last 29 years.

Alien DNA introgression and wheat DNA rearrangements in a stable wheat line derived from the early generation of distant hybridization

Polyploidy has been found to be common in plants. Bread or common wheat (Triticum aestivum L., 2n=42) is a good example of allopolyploid made up of three diploid genomes A, B and D. In recent years, by the study of mimicking the origination of common wheat, it was found that changes of DNA sequence and gene expression occurred at the early stages of artificial allohexaploid between tetraploid wheat and Aegilops tauschii, which was probably favorable to genetic diploidization of new synthetic hexaploid wheat. Common wheat 99L2 is a new line stable in genetic, which was derived from the early self-pollinated generation of wide hybrids between common wheat and rye. In this study, it was found that at least two rye DNA segments had been introgressed into 99L2. This result suggested that a mechanism of alien DNA introgression may exist, which was different from the traditional mechanism of chromosome pairing and DNA recombination between wheat and alien species. Meanwhile, during the introgression process of alien rye DNA segments, the changes in DNA sequences of wheat itself occurred.

Take‐all and grain yields in sequences of winter wheat crops testing fluquinconazole seed treatment applied in different combinations of years

SummaryA seed treatment containing fluquinconazole as the only active ingredient was tested in sequences of up to six consecutive crops of winter wheat. It was applied or not applied in each year, and was tested in all possible combinations with treatments applied in previous years. Take‐all was controlled effectively, and grain yield usually increased, when the disease intensity was moderate or severe in non‐treated crops, but control of the most severe take‐all did not result in acceptable yields or grain quality. Treatment of a first wheat or second wheat with little take‐all did not usually benefit the subsequent crop. Non‐treatment of a crop grown after a treated, diseased crop usually resulted in a marked increase in disease, indicating that treatment had delayed progress of the epidemic. Take‐all was controlled by treatment of a crop grown after a treated, diseased crop but the amount of control and of increased yield was often less than that in a treated crop grown after a non‐treated crop in the same crop sequence. Similar effects of seed treatment were apparent in crops grown on a site with take‐all decline. The alternative fungicide, silthiofam, applied as a seed treatment in the later years of some experiments, was usually as effective as fluquinconazole. From these experiments, it is recommended that: a) fluquinconazole seed treatment should be applied to a second or third wheat crop, grown after a first wheat crop that was managed to avoid rapid take‐all development (e.g. by avoiding very early sowing); b) a break crop should follow the treated crop; c) the seed treatment should not normally be used in longer sequences of wheat or on take‐all decline soil unless it is planned to follow the treated crop with a non‐cereal break.

Effect of tillage on growth, yield and nutrient uptake in wheat after rice in the Indo-Gangetic Plains of India

A 3-year field study was conducted during 1998/99–2000/01 on the effect of tillage on crop growth, yield and nutrient use in wheat (Triticum aestivum L.) grown after different methods of rice (Oryza sativa L.) seeding. Treatments comprised three methods of rice seeding, viz. direct seeding (unpuddled), manual transplanting and mechanical transplanting by self-propelled rice transplanter as main plots and three tillage levels in wheat (conventional tillage (CT), reduced tillage (RT), zero tillage (ZT)) as subplots. Results indicated that tillage significantly decreased soil bulk density (1·59 Mg/m3) over the zero tillage system (1·69 Mg/m3). Greater root density in terms of root dry weight (7·50 Mg/20 cm row length) was recorded in CT and the lowest root dry weight (5·80 Mg/20 cm row length) was obtained in ZT during 2000/01. Significantly higher dry matter accumulation (254 g/m row) and leaf area index of wheat (3·04) were consistently recorded under direct seeding of rice, which was statistically different from the other methods of seeding adopted in the preceding rice crop. CT resulted in significantly higher dry matter (253 g/m row) and leaf area index of wheat (3·02) than RT and ZT respectively, during 2000/01. The highest mean yield of wheat (6·02 Mg/ha) was obtained in direct seeding of rice, followed by mechanical and manual transplanting. Among different tillage levels, CT recorded the highest mean yield of wheat (5·90 Mg/ha) followed by RT (5·82 Mg/ha) and ZT (5·40 Mg/ha). The yield reduction was in the order of 11·28 and 6·31% under ZT and RT, respectively. Soil chemical analysis showed that available soil N, P and K contents were affected significantly due to seeding method and tillage after each cycle of rice–wheat sequence. Significantly greater available soil N, P and K were recorded under direct seeding of rice followed by manual and mechanical transplanting. CT recorded significantly lower values of available soil N and higher values of soil P and K whereas ZT recorded higher values of available soil N and lower values of available soil P and K during the 3 years of study.

Biologically Defined Soil Organic Matter Pools as Affected by Rotation and Tillage

The importance of soil organic matter is well recognized; however, changes in C and N fractions are inadequately quantified. The objective of this study was to determine tillage and crop rotation effects on soil organic C and N fractions from a long-term (27-year) study in eastern Kansas. Cropping systems included continuous and rotation sequences of wheat (Triticum aestivum L.), grain sorghum (Sorghum bicolor (L.) Moench), and soybean (Glycine max (L.) Merrill) on a Muir silt loam (fine-silty, mixed, mesic Cumulic Haplustolls). Tillage included conventional (CT), reduced (RT), and no-till (NT). Total C and N (CT and NT) were determined on all treatments. Mineralizable C and N (Co and No) and microbial biomass C and N were determined for the NT and CT soybean and sorghum rotations. Cropping systems that included wheat contained the greatest amount of CT and NT. Continuous wheat contained 2910 g C m−2 and 287 g N m−2, compared to 2225 g C m−2 and 222 g N m−2 (0–15 cm) for continuous soybean. No-tillage contained 1128 g C m−2 and 109 g N m−2 at 0–5 cm compared to 918 g C m−2 and 87 g N m−2 for CT. Sorghum contained 51% more Co than soybean, and NT accounted for 59% more Co than CT. More crop residue was produced and retained in rotations that included sorghum. No-tillage increased C 2440 kg ha−1, while CT increased C 340 kg ha−1 across all soybean/sorghum rotations. The highest sequestration rate (122 kg C ha−1 y−1) was observed with NT sorghum and was equivalent to ∼3.2% of the plant material (root and shoot, less gain harvest) remaining in the soil annually.

Universal primers for the amplification of chloroplast microsatellites in grasses (Poaceae)

AbstractAttempts to design truly universal primers to amplify chloroplast microsatellites have met with limited success due to nonconservation of repeat loci across widely divergent taxa. We have used the complete chloroplast genome sequences of rice, maize and wheat to design five pairs of primers that amplify homologous mononucleotide repeats across the Poaceae (grasses). Sequencing confirmed conservation of repeat motifs across subfamilies and a preliminary study in Anthoxanthum odoratum revealed polymorphism at two loci with a haplotype diversity value of 0.495. These primers provide a valuable tool to study cytoplasmic diversity in this extensively studied and economically important range of taxa.