Nashr ma`lumotlari: // Ўзбекистонда озиқ-овқат хавфсизлигини таъминлашда мева-сабзавот ҳамда узумчилик соҳасининг роли ва аҳамияти // мавзусидахалқаро илмий-амалий анжуман. –Тошкент - 2017
To‘plamni yuklash uchun havola:


Schafleitner Roland
The World Vegetable Center, Shanhua, Tainan, 74151, Taiwan

Future vegetable varieties need to give high yields of quality products, be resistant to pests and diseases and cope with increased climate variability. Access to genetic resources harboringrequired traits for breeding is essential for developing improved varieties. While genebanks conserve large germplasm collections, establishing smaller core collections displaying a maximum of the crop diversity facilitates the identification of promising material for breeding. Molecular markers are efficient tools for defining core collections and for marker-assisted breeding of new varieties. In the last years, genome editing technologies emerged that cancomplement breeding with traits that are difficult to obtain by conventional breeding. These technologies could significantly contribute to future food and nutrition security.
Keywords: vegetable biodiversity, genebank, core collection, quantitative trait loci, marker-assisted breeding, genome editing.
Introduction. Fruit and vegetable supply in Uzbekistan currently exceeds the per capita daily intake of 400 grams recommended by the World Health Organization- recommended (WHO) by more than two times. Further production increases are desirable to satisfy increasing local demand and cater to growing export markets. Increasing vegetable production through intensification and diversification requires varieties with good yield and qualitythat are adapted to the local conditionsand carry resistances to common diseases and abiotic stresses. Environmental stresses such as heat and more intense droughts as well as soil salinization are among the key problems of agriculture in Uzbekistan. Farmers need varieties that can cope with these problems.
Breeding of improved varieties is a long-term effort and requires access to germplasm that provides the traits needed by farmers for successful production. Often these traits have to be sourced from landraces or closely related wild species conserved in genebanks. Screening of large germplasm collections for traits of interest is laborious and costly. Establishing subset of collections, so-called core collections, which represent the diversity of the whole collection, can make identification of suitable material for breeding more practical. Phenotypic selection is a major cost factor in breeding. Molecular markers tightly linked to traits of interest lower the costs for selecting favorable genotypes and accelerate genetic gain. By now, marker-assisted selection is widely used for introgressing and maintaining single and oligogenic disease resistances in breeding lines and thus speeds up the development of new improved varieties.
Some traits required for crop improvement may be difficult to introgress from wild into cultivated species. During the last years, genome editing technologies have emerged which facilitate the introduction of new alleles into plants without leaving selective markers or other foreign DNA sequences in the product. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) became the method of choice for genome editing and has proven potential for crop improvement.
Materials and Methods. The World Vegetable Center Genebank maintains the world’s largest public vegetable germplasm collection with more than 61,000 accessions of around 450 species (https://avrdc.org/our- work/managing-germplasm). Core collections comprising about 20% of the accessions of the whole collection, but maintaining a large proportion of the available diversity have been established for several species. The general approach for defining core collections is combining information on geographical origin and phenotype with genotypic data to identify a set of accessions samples that display a maximum of phenotypic and genetic variation (Schafleitner et al., 2015).
Molecular markers associated with traits of interest, e.g. disease and pest resistance, are identified by quantitative trait locus (QTL) analyses in segregating populations (F3 or recombinant inbred lines) or by genome­wide association studies (GWAS) on germplasm samples. Marker analysis of experimental populations wasdone by genotyping by sequencing according to Elishire et al., (2011). Publically available software tools such as IciMapping and Tassel were used for QTL analysis and GWAS (Meng et al., 2015, Glaubitz et al., 2014). Population structure was analyzed by fast Structure soft ware (https://rajanil.github.io/fastStructure/). Experimental and breeding populations were phenotyped according to publically available protocols.
Genome editing in tomato was performed using CRISPR/Cas9 construct pKSE401 obtained from the Addgene non-profit plasmid depository (https://www.addgene.org/). DNA oligonucleotides for the upper and lower strand of the target-specific section of the guide RNA were designed, synthesized and cloned into the BsaI site of the vector. The guide RNA was designed to target exon sequences of the genes AdhI (Solyc04g064710, Solyc06g059740), eIF(iso)4E (Solyc09g090580), eIF4E1 (Solyc03g005870) and eIF4E2 (Solyc02g021550). The constructs were transformed into explants of tomato line CLN1621L through Agrobacterium-mediated transformation. Shoots were regenerated and tested by restriction enzyme analysis, PCR and sequencing for mutations in the target genes.
Research Results. The large size of genebank collections can deter their use. The World Vegetable Center Genebank contains 6,768 Vigna radiata and 8,565 wild and cultivated tomato accessions. Screening large collections like those is economically and logistically challenging. Smaller core collections that represent the diversity available in larger collections improve the access for breeders and scientists to the crop diversity to source traits of interest for breeding. The World Vegetable Center has established core collections for wild tomato (Solanum pimpinellifolium), okra (Abelmoschus sp.) and mungbean (Vigna radiata).The mungbean core collection has been further reduced to a minicore of 297 accessions and was distributed to scientists and breeders in six Asian countries, including Uzbekistan, and was evaluated for a range of agronomical traits. Various accessions with favorable traits for breeding were identified and two accessions were selected for variety trials.
Core collections for the tomato and eggplant (Solanum sp.) and pepper (Capsicum sp.) germplasm are under construction in the “G2P-SOL” project funded by the Horizon 2020 European Union framework program with the aim to promote the use of new genetic resources of Solanaceous crops in breeding programs.
Core collections are highly suitable to map traits of interest. For example, resistance to Mungbean yellow mosaic disease and tolerance to saline soils have been identified in the mungbean minicore collection. The collection was densely genotyped with 8,000 single nucleotide polymorphism markers and statistical analyses revealed genomic locations associated with these traits. In addition, resistance to Bruchid beetles, a serious storage pest of mungbean, has been mapped in segregating populations andmarkers for a resistance allele on chromosome 5 of mungbean were highly suitable for marker-assisted selection of bruchid-resistant breeding lines.
Some traits may be difficult to be obtained from genebank material. Crossing barriers and strong linkage drag may hinder the introgression of traits from wild relatives into elite lines. Genome-editing via site-directed mutagenesis using CRISPR/Cas9 is likely to become an important breeding tool to overcome these obstacles. The technology is based on nuclease Cas9 derived from Streptococcus pyogenes that forms a complex with a 100 bases RNA molecule, called guide RNA, which directs the nuclease to a DNA target sequence through base pairing with complementary nucleotides of over 20 bases. When these components are expressed in a cell, the guide RNA will direct the Cas9 nuclease to the specified site of the host DNA to cut the DNA double strand. Cellular DNA repair mechanisms will close the gap. Because DNA repair is error prone, deletions, insertions, and point mutations are introduced at the cutting site, leading to site directed nucleotide sequence changes. A range of variants of this method for different purposes have been developed (Puchta, 2017).
We have tested the CRISPR/Cas9 system in tomato, first on a model gene (alcohol dehydrogenase), and then on eukaryotic initiation factors eIF(iso)4E, elF4E1 and elF4E2 genes known to be associated with recessive resistance against potyviruses. In up to 20% of the regenerated plants mutations were found in the target genes. Short deletions were the most frequently observed, followed by single base pair insertions. Single base exchanges were least frequent. The predominant outcome of mutagenesis was a change of the amino acid sequence and the introduction of a premature stop codon, presumably leading to truncation of the target protein. The method was highly suitable to knock out genes and thus is an ideal tool to verify gene functions and to generate recessive mutants for breeding. Variants of the method can mediate site-specific integration of DNA sequences and thus precisely change or add DNA sequences at a locus.
Conclusions and Recommendations. Collecting, preserving, characterizing and distributing plant genetic resources iskey for future food and nutrition security. Without access to crop genetic diversity, generating new improved varieties would soon come to a halt. Genetic resource conservation and characterization is a long term effort and is best achieved through multidisciplinary partnerships that combineexpertise in botany, physiology, agronomy, biochemistry, genetics, and informatics. Mobilization of new genetic resources for breeding can be improved by generating core collections that display a maximum of the crop diversity in a limited number of accessions. Smaller collections are accessible to multi-location analyses that facilitate the discovery of useful traits for breeding. Trait introgression from non-adapted germplasm into elite material can be enhanced through marker-assisted selection, making variety development cheaper and faster. Traits that are difficult to obtain from genetic resources may be generated by genome editing. Governments should help clarifying the legal and intellectual property situation around genome editing to provide a framework for the use of this technology in plant breeding to contribute to food and nutrition security.
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