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Cantos C.,International Rice Research Institute | Francisco P.,International Rice Research Institute | Trijatmiko K.R.,Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development | Slamet-Loedin I.,International Rice Research Institute | Chadha-Mohanty P.K.,International Rice Research Institute
Frontiers in Plant Science | Year: 2014

Zinc-finger nucleases (ZFNs) have proved to be successful tools for targeted genome manipulation in several organisms. Their main property is the induction of double-strand breaks (DSBs) at specific sites, which are further repaired through homologous recombination (HR) or non-homologous end joining (NHEJ). However, for the appropriate integration of genes at specific chromosomal locations, proper sites for gene integration need to be identified. These regions, hereby named safe harbor loci, must be localized in non-coding regions and possess high gene expression. In the present study, three different ZFN constructs (pZFN1, pZFN2, pZFN3), harboring β-glucuronidase (GUS) as a reporter gene, were used to identify safe harbor loci on rice chromosomes. The constructs were delivered into IR64 rice by using an improved Agrobacterium-mediated transformation protocol, based on the use of immature embryos. Gene expression was measured by histochemical GUS activity and the flanking regions were determined through thermal-asymmetric interlaced polymerase chain reaction (TAIL PCR). Following sequencing, 28 regions were identified as putative sites for safe integration, but only one was localized in a non-coding region and also possessed high GUS expression. These findings have significant applicability to create crops with new and valuable traits, since the site can be subsequently used to stably introduce one or more genes in a targeted manner. © 2014 Cantos, Francisco, Trijatmiko, Slamet-Loedin and Chadha-Mohanty.

Indrasumunar A.,University of Queensland | Indrasumunar A.,Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development | Kereszt A.,University of Queensland | Kereszt A.,Institute for Plant Genomics | And 8 more authors.
Plant and Cell Physiology | Year: 2010

Chemically induced non-nodulating nod139 and nn5 mutants of soybean (Glycine max) show no visible symptoms in response to rhizobial inoculation. Both exhibit recessive Mendelian inheritance suggesting loss of function. By allele determination and genetic complementation in nod139 and nn5, two highly related lipo-oligochitin LysM-type receptor kinase genes in Glycine max were cloned; they are presumed to be the critical nodulation-inducing (Nod) factor receptor similar to those of Lotus japonicus, pea and Medicago truncatula. These duplicated receptor genes were called GmNFR5α and GmNFR5β. Nonsense mutations in GmNFR5α and GmNFR5β were genetically complemented by both wild-type GmNFR5α and GmNFR5β in transgenic roots, indicating that both genes are functional. Both genes lack introns. In cultivar Williams82 GmNFR5α is located in chromosome 11 and in tandem with GmLYK7 (a related LysM receptor kinase gene), while GmNFR5β is in tandem with GmLYK4 in homologous chromosome 1, suggesting ancient synteny and regional segmental duplication. Both genes are wild type in G. soja CPI100070 and Harosoy63; however, a non-functional NFR5β allele (NFR5β (*)) was discovered in parental lines Bragg and Williams, which harbored an identical 1,407 bp retroelement-type insertion. This retroelement (GmRE-1) and related sequences are located in several soybean genome positions. Paradoxically, putatively unrelated soybean cultivars shared the same insertion, suggesting a smaller than anticipated genetic base in this crop. GmNFR5α but not GmNFR5β (*) was expressed in inoculated and uninoculated tap and lateral root portions at about 1025 of GmATS1 (ATP synthase subunit 1), but not in trifoliate leaves and shoot tips.

Koehorst-van Putten H.J.J.,Wageningen University | Sudarmonowati E.,Indonesian Institute of Sciences | Herman M.,Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development | Pereira-Bertram I.J.,Wageningen University | And 6 more authors.
Transgenic Research | Year: 2012

The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of genetically modified low-amylose transformants of commercial cassava variety Adira4 in Indonesia. Twenty four transformants were produced and selected in the Netherlands based on phenotypic and molecular analyses. Nodal cuttings of these plants were sent to Indonesia where they were grown under biosafety conditions. After two screenhouse tests 15 transformants remained for a field trial. The tuberous root yield of 10 transformants was not significantly different from the control. Starch from transformants in which amylose was very low or absent showed all physical and rheological properties as expected from amylose-free cassava starch. The improved functionality of the starch was shown for an adipate acetate starch which was made into a tomato sauce. This is the first account of a field trial with transgenic cassava which shows that by using genetic modification it is possible to obtain low-amylose cassava plants with commercial potential with good root yield and starch quality. © 2011 The Author(s).

Ha J.,Seoul National University | Lestari P.,Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development | Lee S.-H.,Seoul National University
Genes and Genomics | Year: 2013

MADS-box genes are involved in plant reproductive development. However, the role of gene nucleotide diversity in soybean flowering and maturity remains unknown. Therefore, in this study, the distribution of DNA polymorphisms in the putative MADS-box gene located near the quantitative trait loci (QTL) for flowering time and maturity was targeted for association analysis using Glycine max (cultivated soybean) and Glycine soja (wild soybean). Sixteen single nucleotide polymorphisms identified in the upstream region of the putative MADS-box gene around QTL Pod mat 13-7 and Fflr 4-2 on chromosome 7 were found to be highly associated with maturity in soybean. The genetic diversity between cultivated soybeans and the wild relative was comparable, although the early maturity group (EMG) was less diverse than the late maturity group (LMG) of the cultivated soybean. Population size changes of the MADS-box gene in this soybean germplasm appeared to result from non-random selection. A selective pressure seemed to act on this gene in the EMG, while the LMG and G. soja were in genetic equilibrium. Neutrality tests and the constructed neighborjoining tree indicate that the EMG of G. max has experienced strong artificial selection for its domestication and genetic improvement. © The Genetics Society of Korea 2013.

Yan Y.-F.,Seoul National University | Yan Y.-F.,Jilin Agricultural University | Lestari P.,Seoul National University | Lestari P.,Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development | And 4 more authors.
Genome | Year: 2013

Cadmium (Cd) poses a serious risk to human health due to its biological concentration through the food chain. To date, information on genetic and molecular mechanisms of Cd accumulation and distribution in rice remains to be elucidated. We developed an independent F7 RIL population derived from a cross between two japonica cultivars with contrasting Cd levels, 'Suwon490' and 'SNU-SG1', for QTLs identification of Cd accumulation and distribution. 'Suwon490' accumulated five times higher Cd in grain than 'SNU-SG1'. Large genotypic variations in Cd accumulation (17-fold) and concentration (12-fold) in grain were found among RILs. Significant positive correlations between Cd accumulation in grain with shoot Cd accumulation and shoot to grain distribution ratio of Cd signify that both shoot Cd accumulation and shoot to grain Cd distribution regulate Cd accumulation in japonica rice grain. A total of five main effect QTLs (scc10 for shoot Cd accumulation; gcc3, gcc9, gcc11 for grain Cd accumulation; and sgr5 for shoot to grain distribution ratio) were detected in chromosomes 10, 3, 9, 11, and 5, respectively. Of these, the novel potential QTL sgr5 has the strongest effect on shoot to grain Cd distribution. In addition, two digenic epistatic interaction QTLs were identified, suggesting the substantial contribution of nonallelic genes in genetic control of these Cd-related traits. © 2013 Published by NRC Research Press.

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