Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach

Background: Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening. [br/] Methodology/Principal Findings: To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect. [br/] Conclusions/Significance: We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community

TILLING - a shortcut in functional genomics

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING

Genomics-assisted breeding in four major pulse crops of developing countries: present status and prospects

The global population is continuously increasing and is expected to reach nine billion by 2050. This huge population pressure will lead to severe shortage of food, natural resources and arable land. Such an alarming situation is most likely to arise in developing countries due to increase in the proportion of people suffering from protein and micronutrient malnutrition. Pulses being a primary and affordable source of proteins and minerals play a key role in alleviating the protein calorie malnutrition, micronutrient deficiencies and other undernourishment-related issues. Additionally, pulses are a vital source of livelihood generation for millions of resource-poor farmers practising agriculture in the semi-arid and sub-tropical regions. Limited success achieved through conventional breeding so far in most of the pulse crops will not be enough to feed the ever increasing population. In this context, genomics-assisted breeding (GAB) holds promise in enhancing the genetic gains. Though pulses have long been considered as orphan crops, recent advances in the area of pulse genomics are noteworthy, e.g. discovery of genome-wide genetic markers, high-throughput genotyping and sequencing platforms, high-density genetic linkage/QTL maps and, more importantly, the availability of whole-genome sequence. With genome sequence in hand, there is a great scope to apply genome-wide methods for trait mapping using association studies and to choose desirable genotypes via genomic selection. It is anticipated that GAB will speed up the progress of genetic improvement of pulses, leading to the rapid development of cultivars with higher yield, enhanced stress tolerance and wider adaptability

Hans Urs von Balthasar. Liturgie cosmique, Maxime le Confesseur

Dalmais J.-H. Hans Urs von Balthasar. Liturgie cosmique, Maxime le Confesseur. In: Revue de l'histoire des religions, tome 138, n°2, 1950. pp. 237-241

Hans Urs von Balthasar. Liturgie cosmique, Maxime le Confesseur

Dalmais J.-H. Hans Urs von Balthasar. Liturgie cosmique, Maxime le Confesseur. In: Revue de l'histoire des religions, tome 138, n°2, 1950. pp. 237-241

J. Darrouzès. Documents inédits d'ecclésiologie byzantine

Dalmais J.-H. J. Darrouzès. Documents inédits d'ecclésiologie byzantine. In: Revue de l'histoire des religions, tome 174, n°1, 1968. pp. 95-96

J. Darrouzès. Documents inédits d'ecclésiologie byzantine

Dalmais J.-H. J. Darrouzès. Documents inédits d'ecclésiologie byzantine. In: Revue de l'histoire des religions, tome 174, n°1, 1968. pp. 95-96

The Sesquiterpene Synthase from the Botrydial Biosynthetic Gene Cluster of the Phytopathogen Botrytis cinerea

The fungus Botrytis cinerea is the causal agent of the economically important gray mold disease that affects more than 200 ornamental and agriculturally important plant species. B. cinerea is a necrotrophic plant pathogen that secretes nonspecific phytotoxins, including the sesquiterpene botrydial and the polyketide botcinic acid. The region surrounding the previously characterized BcBOT1 gene has now been identified as the botrydial biosynthetic gene cluster. Five genes including BcBOT1 and BcBOT2 were shown by quantitative reverse transcription-PCR to be co-regulated through the calcineurin signaling pathway. Inactivation of the BcBOT2 gene, encoding a putative sesquiterpene cyclase, abolished botrydial biosynthesis, which could be restored by in trans complementation. Inactivation of BcBOT2 also resulted in overproduction of botcinic acid that was observed to be strain-dependent. Recombinant BcBOT2 protein converted farnesyl diphosphate to the parent sesquiterpene of the botrydial biosynthetic pathway, the tricyclic alcohol presilphiperfolan-8 -ol