Targeting Induced Local Lesions In Genomes Research Articles

Phenomics: A Challenge for Crop Improvement in Genomic Era

The last two decades have observed tremendous progress in the genomics for plant breeding research, especially the availability of a large number of high-throughput cost-effective molecular markers and genotyping platforms, advances in sequencing technologies leading the reduction in sequencing costs and making available genome sequences of major crop genomes, etc. But the advance in phenomics is lagging far behind our present day capacity to generate high-throughput molecular genotyping data, thus creating phenotypic bottleneck”. The accurate and precise phenotypic data is an essential component in the discovery of genes/QTLs of important agronomic traits using modern genomic approaches; otherwise it leads to false positives and false negatives. Therefore, it necessitates the development of high-throughput phenotyping facilities. Although, several phenotyping facilities have been developed around the world that can scan and record precise and accurate data for thousands of plants quickly by making use of non-invasive imaging, spectroscopy, image analysis, robotics and high-performance computing facilities, but more efforts and funds are required to be allocated in this field to achieve fruitful results from genomics/molecular breeding approaches like QTL interval mapping, association mapping, genome-wide association studies (GWAS), QTL cloning, QTL validation, marker-assisted selection (MAS), marker-assisted recurrent selection (MARS), TILLING (Targeting Induced Local Lesions in Genomes) and genomic selection (GS) or genome-wide selection (GWS).

Development and evaluation of a cucumber TILLING population.

BackgroundOrdered collections of mutants serve as invaluable tools in biological research. TILLING (Targeting Induced Local Lesions IN Genomes) provides an efficient method to discover, in mutagenized populations, the possible phenotypes controlled by gene sequences whose function is unknown. This method can replace transgenic techniques for the functional validation of cloned genes, especially in the case of transformation-recalcitrant plants such as cucumber.ResultsWe report the development of a TILLING cucumber population, generated by EMS mutagenesis in the Poinsett76 genetic background. The population was evaluated by screening for morphological mutations, and a range of developmental, pigmentation and spontaneous lesion mutants were observed. Suitability for detecting single nucleotide polymorphism in selected genes has been tested by screening a sample of amplicons, with detection rate of 1 SNP in ~1 Mbp.ConclusionThe population described in this Research Note represents a useful asset in cucumber research, to be exploited for forward genetic screens and functional genomics purposes.

First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement.

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding.

BrMYB4, a suppressor of genes for phenylpropanoid and anthocyanin biosynthesis, is down-regulated by UV-B but not by pigment-inducing sunlight in turnip cv. Tsuda.

The regulation of light-dependent anthocyanin biosynthesis in Brassica rapa subsp. rapa cv. Tsuda turnip was investigated using an ethyl methanesulfonate (EMS)-induced mutant R30 with light-independent pigmentation. TILLING (targeting induced local lesions in genomes) and subsequent analysis showed that a stop codon was inserted in the R2R3-MYB transcription factor gene BrMYB4 and that the encoded protein (BrMYB4mu) had lost its C-terminal region. In R30, anthocyanin accumulated in the below-ground portion of the storage root of 2-month-old plants. In 4-day-old seedlings and 2-month-old plants, expression of BrMYB4 was similar between R30 and the wild type (WT), but the expression of the cinnamate 4-hydroxylase gene (BrC4H) was markedly enhanced in R30 in the dark. In turnip seedlings, BrMYB4 expression was suppressed by UV-B irradiation in the WT, but this negative regulation was absent in R30. Concomitantly, BrC4H was repressed by UV-B irradiation in the WT, but stayed at high levels in R30. A gel-shift assay revealed that BrMYB4 could directly bind to the promoter region of BrC4H, but BrMYB4mu could not. The BrMYB4-enhanced green fluorescent protein (eGFP) protein could enter the nucleus in the presence of BrSAD2 (an importin β-like protein) nuclear transporter, but BrMYB4mu-eGFP could not. These results showed that BrMYB4 functions as a negative transcriptional regulator of BrC4H and mediates UV-B-dependent phenylpropanoid biosynthesis, while BrMYB4mu has lost this function. In the storage roots, the expression of anthocyanin biosynthesis genes was enhanced in R30 in the dark and in sunlight in both the WT and R30. However, in the WT, anthocyanin-inducing sunlight did not suppress BrMYB4 expression. Therefore, sunlight-induced anthocyanin biosynthesis does not seem to be regulated by BrMYB4.

Screening of mutations by TILLING in plants.

TILLING (Targeting Induced Local Lesions IN Genomes) is a well-known reverse genetics technique designed to detect unknown SNPs (single nucleotide polymorphisms) in genes of interest using an enzymatic digestion and is widely employed in plant and animal genomics. The main advantage of this technique is that it allows for the high-throughput identification of an allelic series of mutants with a range of modified functions for a particular gene. In this chapter, we aim to give a detailed introduction of how to establish a TILLING platform for identifying mutants in plants, including generation of a large mutant population, DNA and seed library preparation, mutation identification based on a LI-COR4300 DNA analyzer, and confirmation of functions of the mutated genes.

New starch phenotypes produced by TILLING in barley.

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.

Molecular nature of chemically and physically induced mutants in plants: a review

More than 3200 new mutant varieties have been bred and used by millions of farmers, which has significantly contributed to world food security. A lot more mutants have also served as tools for gene discovery and functional analysis. Recent genomic approaches including TILLING (Targeting Induced Local Lesions In Genome) have enabled screening of mutations at the molecular level. This review describes the molecular nature of chemically and physically induced mutations and their repercussions. Analyses of TILLING reports indicate that chemically induced mutations are mostly nucleotide substitutions, but that mutation frequencies fluctuate among plant species ranging from one DNA lesion per 300 kbp inArabidopsisto one DNA lesion per 30 kbp in bread wheat, which reciprocate with an increase in ploidy levels. The majority (>95%) of chemically induced DNA lesions are silent or missense mutations. Mutations induced by physical mutagens seem to be more diverse, including single-nucleotide substitutions, insertions, inversions and translocations, although short deletions ( < 15 bp) are relatively more predominant. The proportion of complex mutations (translocation, inversions, etc.) may increase with an increase in the linear energy transfer of radiations. In addition, the implications of these findings for the roles of induced mutants in breeding and gene function analysis are briefly discussed.

Pea (Pisum sativum L.) in biology prior and after Mendel's discovery

Pea (Pisum sativum L.) has been extensively used in early hybridization studies and it was the model organism of choice for Mendel’s discovery of the laws of inheritance, making pea part of the foundation of modern genetics. Pea has also been used as model for experimental morphology and physiology. However, subsequent progress in pea genomics has lagged behind many other plant species, largely as a consequence of its genome size and low economic significance. The availability of the genome sequences of five legume species (Medicago truncatula, Lotus japonicus, Glycine max, Cajanus cajan and Cicer aerietinum) offers opportunities for genome wide comparison. The combination of a candidate gene and synteny approach has allowed the identification of genes underlying agronomically important traits such as virus resistances and plant architecture. Useful genomic resources already exist and include several types of molecular marker sets as well as both transcriptome and proteome datasets. The advent of greater computational power and access to diverse germplasm collections enable the use of association mapping to identify genetic variation related to desirable agronomic traits. Current genomic knowledge and technologies can facilitate the allele mining for novel traits and their incorporation from wild Pisum sp. into elite domestic backgrounds. Fast neutron and targeting-induced local lesions in genomes (TILLING) pea mutant populations are available for reverse genetics approaches, BAC libraries for positional gene cloning as well as transgenic and in vitro regeneration for proof of function through gene silencing or over-expression. Finally, recently formed International Pea Genome Sequencing Consortium, holds promise to provide the pea genome sequence by 2015, a year of 150 anniversary of Mendel’s work.

The Rice Endosperm ADP-Glucose Pyrophosphorylase Large Subunit is Essential for Optimal Catalysis and Allosteric Regulation of the Heterotetrameric Enzyme

Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells and by TILLING (Targeting Induced Local Lesions in Genomes). Interestingly, seeds from three of the missense mutants (two containing T139I and A171V) were severely shriveled and had seed weight and starch content comparable with the shriveled seeds from OsAGPL2 null mutants. Results from kinetic analysis of the purified recombinant enzymes revealed that the catalytic and allosteric regulatory properties of these mutant enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower specific (catalytic) activities and affinities for the activator 3-phosphoglycerate (3-PGA). The missense heterotetramer enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase, while the S2b homotetramer was profoundly tolerant to Pi inhibition. Thus, our results provide definitive evidence that starch biosynthesis during rice endosperm development is controlled predominantly by the catalytic activity of the cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme.

Progress in TILLING as a tool for functional genomics and improvement of crops

Food security is a global concern and substantial yield increases in crops are required to feed the growing world population. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. Targeting Induced Local Lesions in Genomes (TILLING), which combines traditional chemical mutagenesis with high-throughput genome-wide screening for point mutations in desired genes, offers a powerful way to create novel mutant alleles for both functional genomics and improvement of crops. TILLING is generally applicable to genomes whether small or large, diploid or even allohexaploid, and shows great potential to address the major challenge of linking sequence information to the function of genes and to modulate key traits for plant breeding. TILLING has been successfully applied in many crop species and recent progress in TILLING is summarized below, especially on the developments in mutation detection technology, application of TILLING in gene functional studies and crop breeding. The potential of TILLING/EcoTILLING for functional genetics and crop improvement is also discussed. Furthermore, a small-scale forward strategy including backcross and selfing was conducted to release the potential mutant phenotypes masked in M2 (or M3) plants.

Phenotyping and other breeding approaches for a New Green Revolution

Phenotyping and other breeding approaches for a New Green Revolution

Genome-wide DNA polymorphism and transcriptome analysis of an early-maturing rice mutant

In order to develop a rice population with improved important traits such as flowering time, we developed 2,911 M2 targeting-induced local lesions in genomes (TILLING) lines by irradiating rice seeds with γ-rays. In all, 15 M3 lines were obtained from 3 different M2 lines that exhibited an early-maturing phenotype: these plants matured approximately 25 days faster than wild-type (WT) plants. To identify genome-wide DNA polymorphisms, we performed whole-genome resequencing of both the plant types, i.e., WT and early-maturing TILLING 1 (EMT1), and obtained mapped reads of 118,488,245 bp (99.53 %) and 128,489,860 bp (99.72 %), respectively; Nipponbare was used as the reference genome. We obtained 63,648 and 147,728 single nucleotide polymorphisms (SNPs) and 33,474 and 31,082 insertions and deletions (InDels) for the WT and EMT1, respectively. Interestingly, there was a higher number of SNPs (2.6-fold) and slightly lower number of InDels (0.9-fold) in EMT1 than in WT. The expression of at least 202 structurally altered genes was changed in EMT1, and functional enrichment analysis of these genes revealed that their molecular functions were related to flower development. These results might provide a critical insight into the regulatory pathways of rice flowering.

Development of a TILLING resource in durum wheat for reverse- and forward-genetic analyses

A durum wheat TILLING (targeting induced local lesions in genomes) population of 2601 M3 families was developed from cv. Svevo using ethyl methanesulfonate as a chemical mutagen. The entire M3 population was field-grown for phenotypic evaluations. Despite the polyploid nature of the wheat genome, a preliminarily phenotypic screening showed a high frequency of morphological alterations (~22%); specific phenotyping for seed morphology was undertaken. Furthermore, a reverse-genetics experiment was performed on DNA collected from M2 leaves for the homoeologous genes SBEIIa-A and SBEIIa-B involved in starch metabolism. One non-sense mutation for both genes was identified; specific crosses are planned in order to pyramid the two mutations.

A novel GmFAD3-2a mutant allele developed through TILLING reduces α-linolenic acid content in soybean seed oil.

Soybean (Glycine max (L.) Merr.) oil typically contains 8% α-linolenic acid that is highly unstable and easily oxidized. This property is undesirable in many food and industrial applications. Genetic strategies for reducing α-linolenic acid content would enhance the commercial value. However, genetic resources for low α-linolenic acid content are limited among natural soybean variations. Microsomal omega-3-fatty acid desaturase (FAD3) is responsible for the synthesis of α-linolenic acid in the polyunsaturated fatty acid pathway. There are four FAD3 homologs (Glyma02g39230, Glyma11g27190, Glyma14g37350 and Glyma18g06950) in the soybean genome. While non-functional alleles have been reported for Glyma02g39230 (GmFAD3-1a) and Glyma14g37350 (GmFAD3-1b), little variation is seen in Glyma18g06950 (GmFAD3-2a). We isolated seven mutant GmFAD3-2a alleles, each containing a single-nucleotide substitution, from 39,100 independent mutant lines by using targeting induced local lesions in genomes (TILLING). Analysis of GmFAD3-2a transcripts and enzyme activities revealed that one missense mutant, ‘P1-A9’, contains a non-functional allele of GmFAD3-2a. By combining three non-functional alleles (GmFAD3-1a, GmFAD3-1b, and GmFAD3-2a), we generated soybean lines containing <2% α-linolenic acid in their seeds. The reverse-genetics-based development of novel mutant alleles in the fatty acid metabolic pathway will allow the improvement of soybean with better oil quality through conventional breeding.

Exploring the Pathogenetic Mechanisms underlying Parkinson's Disease in Medaka Fish

Teleost fish have recently been employed as a model for human neurodegenerative diseases. We used toxin exposure and genetic engineering to develop models of Parkinson's disease (PD) in the teleost fish, medaka. Among the toxins examined, 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors, and tunicamycin all induced important features of PD in medaka. Specifically, these agents induced dopaminergic cell loss and reduced spontaneous movement, and the latter three toxins produced inclusion bodies that were ubiquitously distributed in the medaka brain. Despite the extensive distribution of these inclusion bodies, the middle diencephalic dopaminergic neurons were particularly susceptible to the effect of the toxins, suggesting that this cluster of dopaminergic neurons is analogous to the human substantia nigra. We have also created a variety of different genetic models using the Targeting Induced Local Lesions in Genomes (TILLING) method, and found that neither PTEN-induced putative kinase 1 (PINK1) mutants nor Parkin mutants disclosed significant dopaminergic cell loss. Surprisingly however, PINK1 and Parkin double mutants exhibited selective dopamine cell loss, as well as aggregation and deficit of mitochondrial activity. Another mutant, the ATP13A2 mutant, also expressed a PD phenotype, exhibiting marked cathepsin D reduction and fingerprint-like structures that are generally found in lysosome storage diseases. Taken together, these data indicate that medaka fish can serve as a new animal model for PD. In this review, we summarize our data and discuss the potential for future application of this animal model.

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis

BackgroundIn fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions INGenomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported.ResultsIn this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries.ConclusionsThese results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

Validation of doubled haploid plants by enzymatic mismatch cleavage

BackgroundDoubled haploidy is a fundamental tool in plant breeding as it provides the fastest way to generate populations of meiotic recombinants in a genetically fixed state. A wide range of methods has been developed to produce doubled haploid (DH) plants and recent advances promise efficient DH production in otherwise recalcitrant species. Since the cellular origin of the plants produced is not always certain, rapid screening techniques are needed to validate that the produced individuals are indeed homozygous and genetically distinct from each other. Ideal methods are easily implemented across species and in crops where whole genome sequence and marker resources are limited.ResultsWe have adapted enzymatic mismatch cleavage techniques commonly used for TILLING (Targeting Induced Local Lesions IN Genomes) for the evaluation of heterozygosity in parental, F1 and putative DH plants. We used barley as a model crop and tested 26 amplicons previously developed for TILLING. Experiments were performed using self-extracted single-strand-specific nuclease and standard native agarose gels. Eleven of the twenty-six tested primers allowed unambiguous assignment of heterozygosity in material from F1 crosses and loss of heterozygosity in the DH plants. Through parallel testing of previously developed Simple Sequence Repeat (SSR) markers, we show that 3/32 SSR markers were suitable for screening. This suggests that enzymatic mismatch cleavage approaches can be more efficient than SSR based screening, even in species with well-developed markers.ConclusionsEnzymatic mismatch cleavage has been applied for mutation discovery in many plant species, including those with little or no available genomic DNA sequence information. Here, we show that the same methods provide an efficient system to screen for the production of DH material without the need of specialized equipment. This gene target based approach further allows discovery of novel nucleotide polymorphisms in candidate genes in the parental lines.

PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimumL.) mutant population and TILLinG platform for forward and reverse genetics

BackgroundFlax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics.ResultsA population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species.ConclusionsWe have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax.

An MSH4 Homolog, stpp1 , from Pleurotus pulmonarius Is a “Silver Bullet” for Resolving Problems Caused by Spores in Cultivated Mushrooms

The enormous number of spores produced by fruiting bodies during cultivation of mushrooms can lead to allergic reactions of workers, reduction of commercial value, spread of mushroom disease, pollution of facilities, and depletion of genetic diversity in natural populations. A cultivar harboring a sporulation-deficient (sporeless) mutation would be very useful for preventing these problems, but sporeless commercial cultivars are very limited in usefulness because sporeless traits are often linked with traits that are unfavorable for commercial cultivation. Thus, identifying a causal gene of a sporeless phenotype not linked to the adverse traits in breeding and cultivation is crucial for the establishment of sporeless breeding using a strategy employing targeting induced local lesions in genomes (TILLING) in cultivated mushrooms. We used a Pleurotus pulmonarius (Fr.) Quél. sporeless strain to identify and characterize the single recessive gene controlling the mutation. The 3,853-bp stpp1 gene encodes a protein of 854 amino acids and belongs to the MutS homolog (MSH) family associated with mismatch repair in DNA synthesis or recombination in meiosis. Gene expression analysis of the fruiting body showed that this gene is strongly expressed in the gills. Phenotypic analysis of disruptants formed by gene targeting suggested a reproducible sporeless phenotype. Mutants deficient in a functional copy of this gene have no unfavorable traits for sporeless cultivar breeding, so this gene will be an extremely useful target for efficient and versatile sporeless breeding in P. pulmonarius and various other cultivated mushrooms.

Induced mutagenesis in Sugar Beet (Beta vulgaris L.): Obtained results and prospects for use in development of TILLING project

At the Research Institute of Sugar Beets, induced mutagenesis is used for sugar beets as one of the lines in plant selection that have been in development since the 1940s. The following significant scientific and practical results have been obtained: the mutability of the initial material of different genetic origin was tested; the activity of the used physical and chemical mutagens and biologically active substances during various modes of processing was assessed; optimal variants for obtaining mutants with desirable traits were chosen; M2 populations and subsequent generations were created; and the need to follow a definite subsequence and recurrence in work was substantiated. Beet mutants with high root mass, sugarance, and resistance have been obtained that are used in hybridization. The widespread use of methods of the molecular biology in the 1990s decreased the interest in induced mutagenesis. In the 21st century, this interest was revived due to the new method of targeting-induced local lesions in genomes (TILLING), which combines the use of induced mutagenesis with molecular methods of searching for mutations. TILLING is based on the creation of a population treated with mutagens that leads to single nucleotide polymorphism (SNP). This process makes it possible to obtain a set of allele variants in genes that is very important for selection. In different countries, TILLING projects have been developed for many agricultural plants, including sugar beets in Germany. The results obtained by the induced mutagenesis of sugar beets in Russia are of great scientific and practical importance.