Amitabh Mohanty

The sequence of rice chromosomes 11 and 12, rich in disease resistance genes and recent gene duplications

Rice is an important staple food and, with the smallest cereal genome, serves as a reference species for studies on the evolution of cereals and other grasses. Therefore, decoding its entire genome will be a prerequisite for applied and basic research on this species and all other cereals. We have determined and analyzed the complete sequences of two of its chromosomes, 11 and 12, which total 55.9 Mb (14.3% of the entire genome length), based on a set of overlapping clones. A total of 5,993 non-transposable element related genes are present on these chromosomes. Among them are 289 disease resistance-like and 28 defense-response genes, a higher proportion of these categories than on any other rice chromosome. A three-Mb segment on both chromosomes resulted from a duplication 7.7 million years ago (mya), the most recent large-scale duplication in the rice genome. Paralogous gene copies within this segmental duplication can be aligned with genomic assemblies from sorghum and maize. Although these gene copies are preserved on both chromosomes, their expression patterns have diverged. When the gene order of rice chromosomes 11 and 12 was compared to wheat gene loci, significant synteny between these orthologous regions was detected, illustrating the presence of conserved genes alternating with recently evolved genes. Because the resistance and defense response genes, enriched on these chromosomes relative to the whole genome, also occur in clusters, they provide a preferred target for breeding durable disease resistance in rice and the isolation of their allelic variants. The recent duplication of a large chromosomal segment coupled with the high density of disease resistance gene clusters makes this the most recently evolved part of the rice genome. Based on syntenic alignments of these chromosomes, rice chromosome 11 and 12 do not appear to have resulted from a single whole-genome duplication event as previously suggested.BackgroundRice is an important staple food and, with the smallest cereal genome, serves as a reference species for studies on the evolution of cereals and other grasses. Therefore, decoding its entire genome will be a prerequisite for applied and basic research on this species and all other cereals.ResultsWe have determined and analyzed the complete sequences of two of its chromosomes, 11 and 12, which total 55.9 Mb (14.3% of the entire genome length), based on a set of overlapping clones. A total of 5,993 non-transposable element related genes are present on these chromosomes. Among them are 289 disease resistance-like and 28 defense-response genes, a higher proportion of these categories than on any other rice chromosome. A three-Mb segment on both chromosomes resulted from a duplication 7.7 million years ago (mya), the most recent large-scale duplication in the rice genome. Paralogous gene copies within this segmental duplication can be aligned with genomic assemblies from sorghum and maize. Although these gene copies are preserved on both chromosomes, their expression patterns have diverged. When the gene order of rice chromosomes 11 and 12 was compared to wheat gene loci, significant synteny between these orthologous regions was detected, illustrating the presence of conserved genes alternating with recently evolved genes.ConclusionBecause the resistance and defense response genes, enriched on these chromosomes relative to the whole genome, also occur in clusters, they provide a preferred target for breeding durable disease resistance in rice and the isolation of their allelic variants. The recent duplication of a large chromosomal segment coupled with the high density of disease resistance gene clusters makes this the most recently evolved part of the rice genome. Based on syntenic alignments of these chromosomes, rice chromosome 11 and 12 do not appear to have resulted from a single whole-genome duplication event as previously suggested.

Rice transformation for crop improvement and functional genomics.

Although several japonica and some indica varieties of rice have already been transformed, there is significant scope for improvement in the technology for transformation of economically important indica varieties. Successful transformation of rice employing Agrobacterium and recent advances in direct gene transfer by biolistics, evidenced by transfer of multiple genes, have removed some of the serious impediments in the area of gene engineering. The transfer of genes for nutritionally important biosynthetic pathway has provided many opportunities for performing metabolic engineering. Other useful genes for resistance against pests, diseases and abiotic stresses have also been transferred to rice. But the limited knowledge about important target genes requires rapid progress in the field of functional genomics. Transgenic rice system can be applied to isolate new genes, promoters, and enhancers and their functions could be unravelled. The combination of novel regulatory systems for targeted expression and useful new genes should pave the way for improvement of rice and other cereals.

Agrobacterium-mediated high frequency transformation of an elite indica rice variety Pusa Basmati 1 and transmission of the transgenes to R2 progeny

Among rice, indica varieties are considered recalcitrant to tissue culture and genetic manipulation. We report here Agrobacterium-mediated transformation of an economically important, elite indica variety Pusa Basmati 1. A large number of morphologically normal and fertile transgenic plants have been obtained. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgenes in the progeny of these plants. Twenty seven percent plants contain single copy gene insertion and copy number of transgenes has been found to vary from 1-4 in transgenic plants. Mendelian as well as non-Mendelian inheritance patterns of the introduced genes have been obtained in the R1 progeny.

Transgenic Rice: A Valuable Monocot System for Crop Improvement and Gene Research

ABSTRACT: Since the first fertile transgenic rice was obtained during the late 1980s, studies on rice transformation have undergone rapid strides. Several physical methods of gene delivery, including Agrobacterium, have been employed to produce transgenic rice. Up to now, about 50 rice cultivars have been transformed that include many japonica and also a few indica cultivars. Consequent to the availability of an efficient transformation system in rice, the expression of monocot genes is better understood because expression of several genes and regulatory elements from rice and other related cereals such as wheat has been studied in rice. Genes of agronomic importance for herbicide, insect, virus, and fungal resistance have been introduced in rice, and some of the transgenics have already completed a few years of field trials. In this context, rice is being looked on as a model monocot plant to study gene expression and to introduce agronomically useful genes. While this progress is expected to supplement ...

Analysis of Inheritability and Expression Profile of Single and Multi-copy Transgene(s) in Rice Over Generations

Transformation of Oryza sativa subsp indica variety Pusa Basmati 1 with Agrobacterium tumefaciens strain LBA4404(pTOK233) carrying genes coding for neomycin phosphotransferase (nptII), β-glucuronidase (gus) and hygromycin phosphotransferase (hph) under the control of plant-specific promoters (pnos and pCaMV35S) within its T-DNA region produced transgenics with single and multiple copies of T-DNA. Simple Mendelian as well as complex patterns of the inheritance for hygromycin resistance trait were observed in R1 and R2 generations. Non-segregating lines selected in R2 generation did not show further segregation of the resistance trait in R3 and R4 generations accompanied by stabilization of integrated transgenes. One of these lines showed the presence of truncated T-DNA in R1 generation. The single copy transgenics showed high stability of expression of gus gene, whereas multi-copy transgenics were prone to silencing up to R3 generation after which no further reduction in gene expression was observed.

Genome-wide Molecular Approaches in Plants: From Structure to Function

Advent of technologies capable of creating ‘genome map’ even without knowledge of the phenotype or a product of a gene has tremendously improved our capability to comprehend genomes. Large fragments of DNA (> 100 kb) can be cloned in YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome) and PAC (PI-derived artificial chromosome) vectors, which can be aligned to chromosomes with the help of DNA markers. Clones of contigs can be used to prepare shotgun sub-clones to generate sequence of the entire genome of organisms. Such an approach has been successfully used for Arabidopsis genome sequencing, and the International Rice Genome Sequencing Programme has also adopted the same strategy. Another approach involves sequencing the shotgun clones of the entire genome, which can then be assembled by computation. Simultaneously, progress is being made in the functional genomics of plants with more than a million ESTs (expressed sequence tags) available in databases and expression chips representing thousands of genes being put to test. The challenge for the future would be to define the function of genes and give them a place in the regulatory network of the cell. Gene knockouts and gene tags would prove to be of significant value as also computational biology. These developments should eventually pave the way not only for discovery of novel genes but also help in precision breeding.

Mechanism of Regulation of Gene Expression for Chloroplast Proteins

Both plastid- and nuclear-encoded proteins are involved in photosynthetic reactions carried out by chloroplasts. While the structure of various plastome-encoded photosynthesis-related genes has been worked out, little is known about the mechanism of regulation of gene expression which is responsible for differentiation of chloroplasts. At the same time, several signal cascades have been worked out in animals and plants for regulation of nuclear gene expression. The aim of the present investigation was to study regulation of plastid and nuclear-encoded photosynthesis-related genes at steady-state mRNA level as influenced by light and development. Earlier, we have reported an interaction of light and developmental (temporal and spatial) cues in establishing steady-state transcript levels of photosynthesis-related genes in rice (Grover et al 1998, 1999a, Kapoor et al 1993, 1994) and Arabidopsis (Grover et al 1999b, Jain et al 1998, Kochhar et al 1996). In this article, a summary of these results along with the new information about the signal transduction, the level of gene regulation and promoter analysis, and the progress made towards developing a plastid transformation system for rice has been presented.