Mu Insertions Are Repaired by the Double-Strand Break Repair Pathway of Escherichia coli

Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5′ flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli—the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps

FLP Recombinase-Mediated Site-Specific Recombination in Silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species

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Not AvailableSamba Mahsuri (BPT5204) is a medium slender grain indica rice variety that is very popular with farmers and consumers across India because of its high yield and excellent cooking quality. However, the variety is susceptible to several diseases and pests, including bacterial blight (BB). We have used PCR based molecular markers in a backcross-breeding program to introgress three major BB resistance genes (Xa21, xa13 and xa5) into Samba Mahsuri from a donor line (SS1113) in which all the three genes are present in a homozygous condition. At each backcross generation, markers closely linked to the three genes were used to select plants possessing these resistance genes (foreground selection) and microsatellite markers polymorphic between donor and recurrent parent were used to select plants that have maximum contribution from the recurrent parent genome (background selection). A selected BC4F1 plant was selfed to generate homozygous BC4F2 plants with different combinations of BB resistance genes. The three-gene pyramid and two-gene pyramid lines exhibited high levels of resistance against the BB pathogen. Under conditions of BB infection, the three-gene pyramid lines exhibited a significant yield advantage over Samba Mahsuri. Most importantly, these lines retain the excellent grain and cooking qualities of Samba Mahsuri without compromising the yield as determined in multi-location trials. This work demonstrates the successful application of marker-assisted selection for targeted introgression of multiple resistance genes into a premium quality rice variety.Not Availabl

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Not AvailableXanthomonas oryzae pv. oryzae ( Xoo) is a serious pathogen of rice causing bacterial leaf blight disease. Resistant varieties and breeding programs are being hampered by the emergence of highly virulent strains. Herein we report population based whole genome sequencing and analysis of 100 Xoo strains from India. Phylogenomic analysis revealed the clustering of Xoo strains from India along with other Asian strains, distinct from African and US Xo strains. The Indian Xoo population consists of a major clonal lineage and four minor but highly diverse lineages. Interestingly, the variant alleles, gene clusters and highly pathogenic strains are primarily restricted to minor lineages L-II to L-V and in particularly to lineage L-III. We could also find the association of an expanded CRISPR cassette and a highly variant LPS gene cluster with the dominant lineage. Molecular dating revealed that the major lineage, L-I is youngest and of recent origin compared to remaining minor lineages that seems to have originated much earlier in the past. Further, we were also able to identify core effector genes that may be helpful in efforts towards building durable resistance against this pathogen.Council of Scientific and Industrial Researc

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Not AvailableRice is an important staple food crop and primary diet source for majority of the world’s population. However, biotic stress such as Sheath blight (ShB) is one of major disease which effects 50-60% yield loss. ShB disease is mainly caused by Rhizoctonia solani, however, no rice cultivar has been found to be completely tolerance. We developed the BPT5204 mutant lines through EMS method. The mutant lines (BPT5204) were screened through detached leaf method (Dath 1987) under standard glass house conditions and the protocol was standardized in ICAR-IIRR. After 72hrs of infection, the lesion length of each cut leaf was measured and according to tolerance we scored 0-9 scale. We observed that 13 out of 40 were showed tolerance against sheath blight. ShB-1, ShB-5, ShB-11, ShB-12, ShB-13 (score-0) lines were showed highly tolerance, ShB-2 and ShB-8 (score 1-8) were moderately tolerant, wild type (BPT5204) was complete susceptible (scores up to 9). Therefore, this standardized detached leaf assay can be used to assess against sheath blight disease.Council for scientific and industrial research, Government of India, New Delh

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Not AvailableSamba Mahsuri (BPT5204) is a medium slender rice variety highly popular among the farmers in South and Eastern India. It is one of the best Rice varieties with good cooking quality. The yield of BPT 5204 is 6-6.5 tons/ha, even though it is showing maximum susceptibility to many biotic stresses and exhibits incomplete panicle emergence, this make it an ideal genotype for identifying mutational changes in traits of agronomic importance. To obtain agronomical important traits mutation breeding plays an important role. Mutagenesis plays key role among these genetic resources, mutant stocks with discrete genetic lesions are essential to determining gene function and dissecting biochemical and metabolic pathways. Chemical mutagenesis has been routinely used to generate genetic variability for breeding research and genetic studies. In rice, there are several advantages of using chemical mutagenesis to produce mutant populations suitable for both forward and reverse genetics. First, mutant populations can be produced using any genotypes. Second, because of the high density of mutations, genome-wide saturation mutagenesis can be achieved using a relatively small mutant population [1, 2]. Third, it provides a large allelic series as a complement to the knockout mutants produced by insertional mutagenesis or transformation methods (over- and under-expression) [3-6]. Morphological variations including grain types, maturity and traits contributing to yield are observed in every generation. In present study, observed the agro-morphological variations in the BPT-5204 sheath blight tolerant mutants which developed through chemical mutagen EMS (Ethyl Methane Sulfonate) [7].o Council for scientific and industrial research, Government of India, New Delh

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Bacterial blight (BB) is a serious disease of rice in India. We have used molecular marker-assisted selection in a backcross breeding program to introgress three genes (Xa21, xa13, and xa5) for BB resistance into Triguna, a mid-early duration, high yielding rice variety that is susceptible to BB. At each generation in the backcross program, molecular markers were used to select plants possessing these resistance genes and to select plants that have maximum contribution from the Triguna genome. A selected BC3F1 plant was selfed to generate homozygous BC(3)F(2) plants with different combinations of BB resistance genes. Plants containing the two-gene combination, Xa21 and xa13, were found to exhibit excellent resistance against BB. Single plant selections for superior agronomic characteristics were performed on the progeny of these plants, from BC(3)F(3) generation onwards. The selected plants were subjected to yield trials at the BC(3)F(8) generation and were found to have a significant yield advantage over Triguna. The newly developed lines are being entered into national multi-location field trials. This work represents a successful example of the application of molecular marker-assisted selection for BB resistance breeding in rice.Department of Biotechnology, Government of Indi

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Not AvailableBackground: Rice, a major food crop of the world, endures many major biotic stresses like bacterial blight (BB), fungal blast (BL) and the insect Asian rice gall midge (GM) that cause significant yield losses. Progress in tagging, mapping and cloning of several resistance (R) genes against aforesaid stresses has led to marker assisted multigene introgression into elite cultivars for multiple and durable resistance. However, no detailed study has been made on possible interactions among these genes when expressed simultaneously under combined stresses. Results: Our studies monitored expression profiles of 14 defense related genes in 11 rice breeding lines derived from an elite cultivar with different combination of R genes against BB, BL and GM under single and multiple challenge. Four of the genes found implicated earlier under combined GM and BB stress were confirmed to be induced (≥ 2 fold) in stem tissue following GM infestation; while one of these, cytochrome P450 family protein, was also induced in leaf in plants challenged by either BB or BL but not together. Three of the genes highlighted earlier in plants challenged by both BB and BL were also found induced in stem under GM challenge. Pi54 the target R gene against BL was also found induced when challenged by GM. Though expression of some genes was noted to be inhibited under combined pest challenge, such effects did not result in compromise in resistance against any of the target pests. Conclusion: While R genes generally tended to respond to specific pest challenge, several of the downstream defense genes responded to multiple pest challenge either single, sequential or simultaneous, without any distinct antagonism in expression of resistance to the target pests in two of the pyramided lines RPNF05 and RPNF08.Indian Council of Agricultural Research – National Agricultural Science grant No. NASF/ABP-5009/2015-1