Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis.

BACKGROUND: Alfalfa (Medicago sativa L.) is a widely adapted perennial forage crop that has high biomass production potential. Enhanced cellulose content in alfalfa stems would increase the value of the crop as a bioenergy feedstock. We examined if increased expression of sucrose synthase (SUS; EC 2.4.1.13) would increase cellulose in stem cell walls. RESULTS: Alfalfa plants were transformed with a truncated alfalfa phosphoenolpyruvate carboxylase gene promoter (PEPC7-P4) fused to an alfalfa nodule-enhanced SUS cDNA (MsSUS1) or the ß-glucuronidase (GUS) gene. Strong GUS expression was detected in xylem and phloem indicating that the PEPC7-P4 promoter was active in stem vascular tissue. In contrast to expectations, MsSUS1 transcript accumulation was reduced 75-90 % in alfalfa plants containing the PEPC7-P4::MsSUS1 transgene compared to controls. Enzyme assays indicated that SUS activity in stems of selected down-regulated transformants was reduced by greater than 95 % compared to the controls. Although SUS activity was detected in xylem and phloem of control plants by in situ enzyme assays, plants with the PEPC7-P4::MsSUS1 transgene lacked detectable SUS activity in post-elongation stem (PES) internodes and had very low SUS activity in elongating stem (ES) internodes. Loss of SUS protein in PES internodes of down-regulated lines was confirmed by immunoblots. Down-regulation of SUS expression and activity in stem tissue resulted in no obvious phenotype or significant change in cell wall sugar composition. However, alkaline/neutral (A/N) invertase activity increased in SUS down-regulated lines and high levels of acid invertase activity were observed. In situ enzyme assays of stem tissue showed localization of neutral invertase in vascular tissues of ES and PES internodes. CONCLUSIONS: These results suggest that invertases play a primary role in providing glucose for cellulose biosynthesis or compensate for the loss of SUS1 activity in stem vascular tissue.

A Long-Read Transcriptome Assembly of Cotton (Gossypium hirsutum L.) and Intraspecific Single Nucleotide Polymorphism Discovery.

Upland cotton (Gossypium hirsutum L.) has a narrow germplasm base, which constrains marker development and hampers intraspecific breeding. A pressing need exists for high-throughput single nucleotide polymorphism (SNP) markers that can be readily applied to germplasm in breeding and breeding-related research programs. Despite progress made in developing new sequencing technologies during the past decade, the cost of sequencing remains substantial when one is dealing with numerous samples and large genomes. Several strategies have been proposed to lower the cost of sequencing for multiple genotypes of large-genome species like cotton, such as transcriptome sequencing and reduced-representation DNA sequencing. This paper reports the development of a transcriptome assembly of the inbred line Texas Marker-1 (TM-1), a genetic standard for cotton, its usefulness as a reference for RNA sequencing (RNA-seq)-based SNP identification, and the availability of transcriptome sequences of four other cotton cultivars. An assembly of TM-1 was made using Roche 454 transcriptome reads combined with an assembly of all available public expressed sequence tag (EST) sequences of TM-1. The TM-1 assembly consists of 72,450 contigs with a total of 70 million bp. Functional predictions of the transcripts were estimated by alignment to selected protein databases. Transcriptome sequences of the five lines, including TM-1, were obtained using an Illumina Genome Analyzer-II, and the short reads were mapped to the TM-1 assembly to discover SNPs among the five lines. We identified >14,000 unfiltered allelic SNPs, of which ∼3,700 SNPs were retained for assay development after applying several rigorous filters. This paper reports availability of the reference transcriptome assembly and shows its utility in developing intraspecific SNP markers in upland cotton.

Development and bin mapping of gene-associated interspecific SNPs for cotton (Gossypium hirsutum L.) introgression breeding efforts.

BACKGROUND: Cotton (Gossypium spp.) is the largest producer of natural fibers for textile and is an important crop worldwide. Crop production is comprised primarily of G. hirsutum L., an allotetraploid. However, elite cultivars express very small amounts of variation due to the species monophyletic origin, domestication and further bottlenecks due to selection. Conversely, wild cotton species harbor extensive genetic diversity of prospective utility to improve many beneficial agronomic traits, fiber characteristics, and resistance to disease and drought. Introgression of traits from wild species can provide a natural way to incorporate advantageous traits through breeding to generate higher-producing cotton cultivars and more sustainable production systems. Interspecific introgression efforts by conventional methods are very time-consuming and costly, but can be expedited using marker-assisted selection. RESULTS: Using transcriptome sequencing we have developed the first gene-associated single nucleotide polymorphism (SNP) markers for wild cotton species G. tomentosum, G. mustelinum, G. armourianum and G. longicalyx. Markers were also developed for a secondary cultivated species G. barbadense cv. 3-79. A total of 62,832 non-redundant SNP markers were developed from the five wild species which can be utilized for interspecific germplasm introgression into cultivated G. hirsutum and are directly associated with genes. Over 500 of the G. barbadense markers have been validated by whole-genome radiation hybrid mapping. Overall 1,060 SNPs from the five different species have been screened and shown to produce acceptable genotyping assays. CONCLUSIONS: This large set of 62,832 SNPs relative to cultivated G. hirsutum will allow for the first high-density mapping of genes from five wild species that affect traits of interest, including beneficial agronomic and fiber characteristics. Upon mapping, the markers can be utilized for marker-assisted introgression of new germplasm into cultivated cotton and in subsequent breeding of agronomically adapted types, including cultivar development.

An RNA-Seq transcriptome analysis of orthophosphate-deficient white lupin reveals novel insights into phosphorus acclimation in plants.

Phosphorus, in its orthophosphate form (P(i)), is one of the most limiting macronutrients in soils for plant growth and development. However, the whole-genome molecular mechanisms contributing to plant acclimation to P(i) deficiency remain largely unknown. White lupin (Lupinus albus) has evolved unique adaptations for growth in P(i)-deficient soils, including the development of cluster roots to increase root surface area. In this study, we utilized RNA-Seq technology to assess global gene expression in white lupin cluster roots, normal roots, and leaves in response to P(i) supply. We de novo assembled 277,224,180 Illumina reads from 12 complementary DNA libraries to build what is to our knowledge the first white lupin gene index (LAGI 1.0). This index contains 125,821 unique sequences with an average length of 1,155 bp. Of these sequences, 50,734 were transcriptionally active (reads per kilobase per million reads ≥ 3), representing approximately 7.8% of the white lupin genome, using the predicted genome size of Lupinus angustifolius as a reference. We identified a total of 2,128 sequences differentially expressed in response to P(i) deficiency with a 2-fold or greater change and P ≤ 0.05. Twelve sequences were consistently differentially expressed due to P(i) deficiency stress in three species, Arabidopsis (Arabidopsis thaliana), potato (Solanum tuberosum), and white lupin, making them ideal candidates to monitor the P(i) status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in P(i) deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to P(i) deficiency.

Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems.

BACKGROUND: Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling. RESULTS: Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes. CONCLUSIONS: Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.

MicroRNA expression profile in common bean (Phaseolus vulgaris) under nutrient deficiency stresses and manganese toxicity.

*MicroRNAs (miRNAs) play a pivotal role in post-transcriptional regulation of gene expression in plants. Information on miRNAs in legumes is as yet scarce. This work investigates miRNAs in an agronomically important legume, common bean (Phaseolus vulgaris). *A hybridization approach employing miRNA macroarrays - printed with oligonucleotides complementary to 68 known miRNAs - was used to detect miRNAs in the leaves, roots and nodules of control and nutrient-stressed (phosphorus, nitrogen, or iron deficiency; acidic pH; and manganese toxicity) common bean plants. *Thirty-three miRNAs were expressed in control plants and another five were only expressed under stress conditions. The miRNA expression ratios (stress:control) were evaluated using principal component and hierarchical cluster analyses. A group of miRNAs responded to nearly all stresses in the three organs analyzed. Other miRNAs showed organ-specific responses. Most of the nodule-responsive miRNAs showed up-regulation. miRNA blot expression analysis confirmed the macroarray results. Novel miRNA target genes were proposed for common bean and the expression of selected targets was evaluated by quantitative reverse transcriptase-polymerase chain reaction. *In addition to the detection of previously reported stress-responsive miRNAs, we discovered novel common bean stress-responsive miRNAs, for manganese toxicity. Our data provide a foundation for evaluating the individual roles of miRNAs in common bean.

Transcript profiling of common bean (Phaseolus vulgaris L.) using the GeneChip Soybean Genome Array: optimizing analysis by masking biased probes.

BACKGROUND: Common bean (Phaseolus vulgaris L.) and soybean (Glycine max) both belong to the Phaseoleae tribe and share significant coding sequence homology. This suggests that the GeneChip(R) Soybean Genome Array (soybean GeneChip) may be used for gene expression studies using common bean. RESULTS: To evaluate the utility of the soybean GeneChip for transcript profiling of common bean, we hybridized cRNAs purified from nodule, leaf, and root of common bean and soybean in triplicate to the soybean GeneChip. Initial data analysis showed a decreased sensitivity and accuracy of measuring differential gene expression in common bean cross-species hybridization (CSH) GeneChip data compared to that of soybean. We employed a method that masked putative probes targeting inter-species variable (ISV) regions between common bean and soybean. A masking signal intensity threshold was selected that optimized both sensitivity and accuracy of measuring differential gene expression. After masking for ISV regions, the number of differentially-expressed genes identified in common bean was increased by 2.8-fold reflecting increased sensitivity. Quantitative RT-PCR (qRT-PCR) analysis of 20 randomly selected genes and purine-ureide pathway genes demonstrated an increased accuracy of measuring differential gene expression after masking for ISV regions. We also evaluated masked probe frequency per probe set to gain insight into the sequence divergence pattern between common bean and soybean. The sequence divergence pattern analysis suggested that the genes for basic cellular functions and metabolism were highly conserved between soybean and common bean. Additionally, our results show that some classes of genes, particularly those associated with environmental adaptation, are highly divergent. CONCLUSIONS: The soybean GeneChip is a suitable cross-species platform for transcript profiling in common bean when used in combination with the masking protocol described. In addition to transcript profiling, CSH of the GeneChip in combination with masking probes in the ISV regions can be used for comparative ecological and/or evolutionary genomics studies.

Transcript profiling of two alfalfa genotypes with contrasting cell wall composition in stems using a cross-species platform: optimizing analysis by masking biased probes.

BACKGROUND: The GeneChip(R) Medicago Genome Array, developed for Medicago truncatula, is a suitable platform for transcript profiling in tetraploid alfalfa [Medicago sativa (L.) subsp. sativa]. However, previous research involving cross-species hybridization (CSH) has shown that sequence variation between two species can bias transcript profiling by decreasing sensitivity (number of expressed genes detected) and the accuracy of measuring fold-differences in gene expression. RESULTS: Transcript profiling using the Medicago GeneChip(R) was conducted with elongating stem (ES) and post-elongation stem (PES) internodes from alfalfa genotypes 252 and 1283 that differ in stem cell wall concentrations of cellulose and lignin. A protocol was developed that masked probes targeting inter-species variable (ISV) regions of alfalfa transcripts. A probe signal intensity threshold was selected that optimized both sensitivity and accuracy. After masking for both ISV regions and previously identified single-feature polymorphisms (SFPs), the number of differentially expressed genes between the two genotypes in both ES and PES internodes was approximately 2-fold greater than the number detected prior to masking. Regulatory genes, including transcription factor and receptor kinase genes that may play a role in development of secondary xylem, were significantly over-represented among genes up-regulated in 252 PES internodes compared to 1283 PES internodes. Several cell wall-related genes were also up-regulated in genotype 252 PES internodes. Real-time quantitative RT-PCR of differentially expressed regulatory and cell wall-related genes demonstrated increased sensitivity and accuracy after masking for both ISV regions and SFPs. Over 1,000 genes that were differentially expressed in ES and PES internodes of genotypes 252 and 1283 were mapped onto putative orthologous loci on M. truncatula chromosomes. Clustering simulation analysis of the differentially expressed genes suggested co-expression of some neighbouring genes on Medicago chromosomes. CONCLUSIONS: The problems associated with transcript profiling in alfalfa stems using the Medicago GeneChip as a CSH platform were mitigated by masking probes targeting ISV regions and SFPs. Using this masking protocol resulted in the identification of numerous candidate genes that may contribute to differences in cell wall concentration and composition of stems of two alfalfa genotypes.

Single-feature polymorphism discovery by computing probe affinity shape powers.

BACKGROUND: Single-feature polymorphism (SFP) discovery is a rapid and cost-effective approach to identify DNA polymorphisms. However, high false positive rates and/or low sensitivity are prevalent in previously described SFP detection methods. This work presents a new computing method for SFP discovery. RESULTS: The probe affinity differences and affinity shape powers formed by the neighboring probes in each probe set were computed into SFP weight scores. This method was validated by known sequence information and was comprehensively compared with previously-reported methods using the same datasets. A web application using this algorithm has been implemented for SFP detection. Using this method, we identified 364 SFPs in a barley near-isogenic line pair carrying either the wild type or the mutant uniculm2 (cul2) allele. Most of the SFP polymorphisms were identified on chromosome 6H in the vicinity of the Cul2 locus. CONCLUSION: This SFP discovery method exhibits better performance in specificity and sensitivity over previously-reported methods. It can be used for other organisms for which GeneChip technology is available. The web-based tool will facilitate SFP discovery. The 364 SFPs discovered in a barley near-isogenic line pair provide a set of genetic markers for fine mapping and future map-based cloning of the Cul2 locus.

Transgenic maize endosperm containing a milk protein has improved amino acid balance.

In order to meet the protein nutrition needs of the world population, greater reliance on plant protein sources will become necessary. The amino acid balance of most plant protein sources does not match the nutritional requirements of monogastric animals, limiting their nutritional value. In cereals, the essential amino acid lysine is deficient. Maize is a major component of human and animal diets worldwide and especially where sources of plant protein are in critical need such as sub-Saharan Africa. To improve the amino acid balance of maize, we developed transgenic maize lines that produce the milk protein alpha-lactalbumin in the endosperm. Lines in which the transgene was inherited as a single dominant genetic locus were identified. Sibling kernels with or without the transgene were compared to determine the effect of the transgene on kernel traits in lines selected for their high content of alpha-lactalbumin. Total protein content in endosperm from transgene positive kernels was not significantly different from total protein content in endosperm from transgene negative kernels in three out of four comparisons, whereas the lysine content of the lines examined was 29-47% greater in endosperm from transgene positive kernels. The content of some other amino acids was changed to a lesser extent. Taken together, these changes resulted in the transgenic endosperms having an improved amino acid balance relative to non-transgenic endosperms produced on the same ear. Kernel appearance, weight, density and zein content did not exhibit substantial differences in kernels expressing the transgene when compared to non-expressing siblings. Assessment of the antigenicity and impacts on animal health will be required in order to determine the overall value of this technology.