Siva P. Kumpatla

Transgene silencing in monocots

Plant gene silencing was originally thought to be a quirk of transformation procedures, but is now recognized to be a facet of vitally important gene regulatory systems, present in all organisms. Monocot plants, especially the grasses, play a foremost role in the agricultural economy of all nations, and their biotechnological manipulation offers great potential for both developed and developing countries. Here, we review reported instances of transgene silencing in monocots and relate the processes of transcriptional and post-transcriptional gene silencing (TGS, PTGS) in perspective to the rapidly burgeoning knowledge of these phenomena in many organisms. Recent findings include the involvement of an RNA-dependent RNA polymerase and a nuclease in PTGS systems and the close relationship between methylation and chromatin structure in TGS events.

SNP Markers and Their Impact on Plant Breeding

The use of molecular markers has revolutionized the pace and precision of plant genetic analysis which in turn facilitated the implementation of molecular breeding of crops. The last three decades have seen tremendous advances in the evolution of marker systems and the respective detection platforms. Markers based on single nucleotide polymorphisms (SNPs) have rapidly gained the center stage of molecular genetics during the recent years due to their abundance in the genomes and their amenability for high-throughput detection formats and platforms. Computational approaches dominate SNP discovery methods due to the ever-increasing sequence information in public databases; however, complex genomes pose special challenges in the identification of informative SNPs warranting alternative strategies in those crops. Many genotyping platforms and chemistries have become available making the use of SNPs even more attractive and efficient. This paper provides a review of historical and current efforts in the development, validation, and application of SNP markers in QTL/gene discovery and plant breeding by discussing key experimental strategies and cases exemplifying their impact.

Genome intruder scanning and modulation systems and transgene silencing

The widespread occurrence of transgene inactivation in plants and classical cases of silencing of duplicated sequences in fungi suggest that all genomes contain defense systems that are capable of monitoring and manipulating intrusive DNA. Such DNA might be recognized by its structure, its sequence composition relative to that of its genomic environment and possibly by its disruption of normal biochemical functions. Although methylation, especially of repeated sequences, is widely associated with gene inactivation, other attributes, including chromatin modification, may be involved. Elimination of inactivated intrusive DNA (presently best documented for filamentous fungi) may also contribute to genomic defense mechanisms in plants. Stable integration and expression of introduced genes are essential for genetically engineered crops, and thus transformation constructs must be designed to avoid host surveillance processes.

Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice.

Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btt cryIIIA and bar transgenes in rice (Oryza sativa L.) that displayed aberrant segregation in R1 progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R1 plants had a DNA hybridization profile identical to that of the resistant R0 parent and R1 siblings. Genomic DNA analysis revealed substantial methylation of the Ubi1/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R2 progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubi1 promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences.

CMD: a Cotton Microsatellite Database resource for Gossypium genomics

BackgroundThe Cotton Microsatellite Database (CMD) http://www.cottonssr.org is a curated and integrated web-based relational database providing centralized access to publicly available cotton microsatellites, an invaluable resource for basic and applied research in cotton breeding.DescriptionAt present CMD contains publication, sequence, primer, mapping and homology data for nine major cotton microsatellite projects, collectively representing 5,484 microsatellites. In addition, CMD displays data for three of the microsatellite projects that have been screened against a panel of core germplasm. The standardized panel consists of 12 diverse genotypes including genetic standards, mapping parents, BAC donors, subgenome representatives, unique breeding lines, exotic introgression sources, and contemporary Upland cottons with significant acreage. A suite of online microsatellite data mining tools are accessible at CMD. These include an SSR server which identifies microsatellites, primers, open reading frames, and GC-content of uploaded sequences; BLAST and FASTA servers providing sequence similarity searches against the existing cotton SSR sequences and primers, a CAP3 server to assemble EST sequences into longer transcripts prior to mining for SSRs, and CMap, a viewer for comparing cotton SSR maps.ConclusionThe collection of publicly available cotton SSR markers in a centralized, readily accessible and curated web-enabled database provides a more efficient utilization of microsatellite resources and will help accelerate basic and applied research in molecular breeding and genetic mapping in Gossypium spp.

A High-Density Simple Sequence Repeat and Single Nucleotide Polymorphism Genetic Map of the Tetraploid Cotton Genome

Genetic linkage maps play fundamental roles in understanding genome structure, explaining genome formation events during evolution, and discovering the genetic bases of important traits. A high-density cotton (Gossypium spp.) genetic map was developed using representative sets of simple sequence repeat (SSR) and the first public set of single nucleotide polymorphism (SNP) markers to genotype 186 recombinant inbred lines (RILs) derived from an interspecific cross between Gossypium hirsutum L. (TM-1) and G. barbadense L. (3-79). The genetic map comprised 2072 loci (1825 SSRs and 247 SNPs) and covered 3380 centiMorgan (cM) of the cotton genome (AD) with an average marker interval of 1.63 cM. The allotetraploid cotton genome produced equivalent recombination frequencies in its two subgenomes (At and Dt). Of the 2072 loci, 1138 (54.9%) were mapped to 13 At-subgenome chromosomes, covering 1726.8 cM (51.1%), and 934 (45.1%) mapped to 13 Dt-subgenome chromosomes, covering 1653.1 cM (48.9%). The genetically smallest homeologous chromosome pair was Chr. 04 (A04) and 22 (D04), and the largest was Chr. 05 (A05) and 19 (D05). Duplicate loci between and within homeologous chromosomes were identified that facilitate investigations of chromosome translocations. The map augments evidence of reciprocal rearrangement between ancestral forms of Chr. 02 and 03 versus segmental homeologs 14 and 17 as centromeric regions show homeologous between Chr. 02 (A02) and 17 (D02), as well as between Chr. 03 (A03) and 14 (D03). This research represents an important foundation for studies on polyploid cottons, including germplasm characterization, gene discovery, and genome sequence assembly.

Sugarcane DIRIGENT and O-METHYLTRANSFERASE promoters confer stem-regulated gene expression in diverse monocots.

Transcription profiling analysis identified Saccharum hybrid DIRIGENT (SHDIR16) and Ο-METHYLTRANSFERASE (SHOMT), putative defense and fiber biosynthesis-related genes that are highly expressed in the stem of sugarcane, a major sucrose accumulator and biomass producer. Promoters (Pro) of these genes were isolated and fused to the β-glucuronidase (GUS) reporter gene. Transient and stable transgene expression analyses showed that both ProDIR16:GUS and ProOMT:GUS retain the expression characteristics of their respective endogenous genes in sugarcane and function in orthologous monocot species, including rice, maize and sorghum. Furthermore, both promoters conferred stem-regulated expression, which was further enhanced in the stem and induced in the leaf and root by salicylic acid, jasmonic acid and methyl jasmonate, key regulators of biotic and abiotic stresses. ProDIR16 and ProOMT will enable functional gene analysis in monocots, and will facilitate engineering monocots for improved carbon metabolism, enhanced stress tolerance and bioenergy production.

Development of highly polymorphic SNP markers from the complexity reduced portion of maize [Zea mays L.] genome for use in marker-assisted breeding

The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.

Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity

The extraction of high-quality genomic DNA for PCR amplification from sunflower (Helianthus annuus) and cotton (Gossypium spp.) is challenging because of the presence of polysaccharides, secondary metabolites, and polyphenolics in the tissues. A high-throughput DNA extraction protocol was needed in our laboratory for simple sequence repeats (SSR)-marker screening and other molecular analyses that do not require organic extraction steps of phenol or chloroform. Here we describe 2 improved highthroughput protocols for DNA extraction and in-PCR modification that result in successful PCR amplification of sunflower and cotton. While the sunflower DNA extraction protocol uses reducing agents such as sodium metabisulfite and dithiothreitol (DTT), the cotton protocol uses polyvinylpyrrolidone (PVP) in PCR reactions and reducing agents in the DNA extraction procedure.

Longevity of 5-azacytidine-mediated gene expression and re-establishment of silencing in transgenic rice.

Epigenetic silencing of a bialaphos resistance (bar) gene in R1 progeny of a transgenic rice line was found to be meiotically stable since selfed (R2) progeny were also susceptible and the bar locus highly methylated. A high proportion of R2 seedlings germinated in the presence of 5-azacytidine (AzaC) were herbicide-resistant and also contained at least one unmethylated copy of the bar gene, further establishing the relationship between silencing and methylation. Restored bar gene expression was typically maintained for 20–50 days, but eventual methylation and silencing of the bar locus underscores the ability of the recipient genome to recognize and inactivate intrusive DNA.