Prospects and methods of polyploid selection of new high-yielding miscanthus clones in the natural climatic conditions of Ukraine

Goal. Expanding the breeding trait collection of breeding genotypes of the genus Miscanthus (Anderson) and the genetic basis for new allotriploid clones by transferring components for hybridization of natural wild species Miscanthus sinensis and Miscanthus sacchariflorus to the tetraploid level. Methods. Cytological, biotechnological, fluorescent cytphotometry, field, laboratory. Results. The efficiency of polyploidisation for induction of new tetraploid forms of miscanthus in liquid nutrient media supplemented with colchicine (0.05% mass) and for stabilization of myxoploid shoots (0.005% mass) for 6 h of cultivation is examined. The period of exposure for Miscanthus sinensis (2x=2x= 38) and Miscanthus sacchariflorus (2x = 2x = 38) varied from 2 hours to 3 days depending on the genetic origin of the material, with transfer to a hormonal environment. The best indicators of tetraploid induction for Miscanthus sinensis were observed for the exposure period of 2 days with polyploidization efficiency (Db%) of 31.25% and 21.42%, and for Miscanthus sacchariflorus 2 and 6 hours with 35.0% and 27.3%, respectively. The technology of transfering rooted shoots into the soil on the Yaltushkiv Eperimental Breeding Farm was improved and the composition of the soil mixture that provided 99% rooting of culture seedlings at a humidity of 60−70% and air temperature of 35−55°С was found. Conclusions. New biotechnological tetraploid lines of Miscanthus sinensis (2xn=4x=76) and Miscanthus sacchariflorus (2xn=4x=76) were created in the conditions of liquid nutrient media supplemented with colchicine (0.05 % mass). It is investigated that flowering of new tetraploid clones in the conditions of Ukraine for the second year of vegetation occurs from late September to early October with formation of fertile pollen grains.

PECULIARITIES OF CREATION OF MISCANTHUS SINENSIS AND MISCANTHUS SACCHARIFLORUS TETRAPLOID LINES

The work aimed at creating tetraploid lines of Miscanthus sinensis and Miscanthus sacchariflorus species. To achieve this goal, we used methods of microclonal propagation, fluorescence cytophotometry, and genomic status differentiation using computer software of AP ‘Partec’ (Germany). It was found that the percentage of cultivated shoots of Miscanthus sacchariflorus for an exposure period of 1 day was 18.86 ± 5.37%. To compare, it was51.78 ± 6.51% in Miscanthus sinensis. The best indicators of tetraploid induction in Miscanthus sinensis were observed for the exposure to colchicine for 2 days with polyploidization efficiency of 31.25% and 21.42%, and in Miscanthus sacchariflorusf or 2 hours and 6 hours with rates of 35.0% and 27.3%, respectively. To stabilize the tetraploid level of genome ploidy, we used Murashige and Skoog liquid media (1962) supplemented with 0.005% colchicine and an exposure period of myxoploids for 6 hours. The flowering of new tetraploid clones in the conditions of Ukraine was observed on the second year of vegetation in late September and the beginning of October with the formation of fertile pollen grains. However, development of a microgametophyte depends on temperature conditions, both for Miscanthus sinensis (4x) and Miscanthus sacchariflorus (4x). Breeding schemes for the formation of anisoploid populations have also been developed: M sinensis (4x) x M sinensis (2x); M. sa?chariflorus (4x) x M sinensis (2x); M sinensis (4x) x M. sachariflorus (2x)

Marker-assisted pyramiding of potato late blight resistance genes Rpi-rzc1 and Rpi-phu1 on di- and tetraploid levels

Late blight is a disease with the biggest economic impact on potato cultivation worldwide. Pyramiding of the resistance genes originating from potato wild relatives is a breeding strategy that has a potential to produce potato cultivars durably resistant to late blight. Growing such cultivars would allow limiting the intensive chemical control of the disease. The goal of this work was to transfer the late blight resistance gene Rpi-rzc1 from Solanum ruiz-ceballosii to the tetraploid level of cultivated potato and to pyramid it with the Rpi-phu1 gene. We obtained two diploid and, through 4x-2x cross, a tetraploid potato population segregating for the Rpi-rzc1 presence, as well as one diploid and one tetraploid population where both genes were introgressed. In total, 754 progeny clones were tested for resistance to late blight in detached leaflet assays. Pathogen isolates avirulent on plants with both genes and virulent on plants with the Rpi-phu1 were used. The selection was assisted by two PCR markers flanking the Rpi-rzc1 gene and a newly designed, highly specific intragenic marker indicating the Rpi-phu1 gene presence. We obtained 26 diploid and 49 tetraploid potato clones with pyramid of both genes that should enhance the durability and spectrum of their late blight resistance and that can be exploited in potato breeding. The specificity of the marker for the Rpi-phu1 gene and the precision of the Rpi-rzc1 mapping were improved in this work.

UPLC-DAD Assisted Phytochemical Quantitation Reveals a Sex, Ploidy and Ecogeography Specificity in the Expression Levels of Selected Secondary Metabolites in Medicinal Tinospora cordifolia: Implications for Elites’ Identification Program

Background: Medicinal phytochemistry involving UPLC-DAD in an exhaustive analysis involving quantification of eight commercially important phytochemicals viz. syringin, cordifolioside A, magnoflorine, tinocordiside, palmatine, 20β-hydroxyecdysone, L-tetrahydropalmatine and berberine has been done in 143 accessions from eight states and the union territories of Delhi and Jammu & Kashmir of India representing three different ploidy levels viz. diploid (2x), triploid (3x) and synthetic tetraploid (4x). The study was done to assess the effect of sex, ploidy level and ecogeography on the expression level of secondary metabolites in stems of dioecious, medicinally important shrub Tinospora cordifolia. Methods: Two different UPLC-DAD methods were used for the quantification of eight selected phytochemicals from the alcoholic stem extracts of T. cordifolia accessions. The Waters Acquity UPLC system hyphenated to the QTOF micromass system, equipped with PDA and ESI-Q-TOF detectors was utilized for the quantitative analysis, Mass Lynx v 4.0 software was used for data analysis. Results: Significant quantitative changes were observed in the analysed secondary metabolites among different accessions of T. cordifolia. The triploid (3x) cytotypes revealed higher amounts of seven out of eight analysed secondary metabolites than diploids and only 20β-hydroxyecdysone was observed to be present in significantly higher amount in diploid cytotypes. Further, at the tetraploid level, novel induced colchiploid (synthetic 4x) genotypes revealed increase in the yield of all of the analysed eight phytochemicals than their respective diploid counterparts. The quantity of active principles in tetraploid cytotypes were also higher than the average triploid levels at multiple locations in five out of eight tested phytochemicals, indicating the influence of ploidy on expression levels of secondary metabolites in T. cordifolia. Additionally, at each of the three ploidy levels (2x, 3x and synthetic 4x), a significant sex specificity could be observed in the expression levels of active principles, with female sex outperforming the male in the content of some phytochemicals, while others getting overexpressed in the male sex. The manifestation of diverse ecogeographies on secondary metabolism was observed in the form of identification of high yielding accessions from the states of Madhya Pradesh, Delhi and Himachal Pradesh and the Union territory of Jammu & Kashmir. Two triploid female accessions that contained approximately two- to eight fold higher amounts of five out of the eight analysed phytochemicals have been identified as superior elites from the wild from the states of Delhi and Madhya Pradesh. Conclusion: The paper shows the first observations of ploidy specificity along with subtle sex and ecogeography influence on the expression levels of secondary metabolome in T. cordifolia.

Production of hybrid potatoes: Are heterozygosity and ploidy levels important?

It has been proposed that maximizing heterosis for yield in potato may be achieved by maximizing heterozygosity and associated intra and interlocus interactions. Tetraploids offer more opportunities to create such interactions than diploids hence the general observations that tetraploids are higher yielding than diploids. Consequently, efforts have been made to increase heterozygosity in tetraploids by introgressing allelic diversity from other Solanum species into cultivated potato. However, conventional potato breeding is difficult because the cultivated potato is an autotetraploid with tetrasomic inheritance and it comprises highly heterozygous individuals which suffer inbreeding depression upon selfing; breeding at the tetraploid level is slow and less efficient than at diploid level. At the diploid level, it is possible to breed for and fix traits under recessive genetic control; it is nearly impossible to do so at the tetraploid level. There is also rapid response to selection due to greater variation in diploids than tetraploids. Consequently, there have been efforts to convert potato from an asexually propagated tetraploid crop into an inbred seed-propagated diploid; this is by production of inbred lines through selfing of the tetraploids to assemble desirable combinations of genes in the inbreds. These efforts are at the experimental stages and a lot of research needs to be done before they are confirmed. Because currently there is little experimental evidence to support superiority of the inbred seed-propagated diploid strategy, it appears the theory that heterosis for yield in potato may be achieved by maximizing heterozygosity and associated intra and interlocus interactions remain unchallenged; these interactions are more in tetraplods than in diploids. This paper therefore looks at genetic basis of yield heterosis in cultivated potato and the role of heterozygosity and ploidy level in production of hybrid potatoes.

RYEGRASS PERENNIAL VARIETY VORONEZHSKIY FOR THE CONDITIONS OF CENTRAL BLACK EARTH REGION

A brief review of the efficiency of cultivation of perennial ryegrass in the areas of steppe and forest-steppe zones is given. The necessity of breeding varieties of perennial ryegrass adapted for use in the conditions of the Central Black Earth region is shown. A brief characteristic of the variety of ryegrass perennial Voronezhskiy (selection of Voronezh Experimental Station for perennial grasses) is presented. The variety is included in the state register in all regions of the Russian Federation. It is characterized by high winter hardiness, drought resistance, longevity. Recommended for sowing as part of grass mixtures when creating hayfields and pastures on various soil types (except sandy), including when reclaiming de-graded land, as well as creating cultivated irrigated pastures in the steppe zone and as part of grass mix-tures when creating meadow-type lawns. The main technological parameters of cultivating the variety for seeds are included, including optimal sowing time and seeding rates, a rational system of fertilizers, the use of herbicides, harvesting, autumn cutting of grass stands, sorting features that allow maintaining tetraploid level of sorting population during reproduction.

SSR Based Genetic Diversity Analysis in Diploid Algaroba (Prosopis spp.) Population

Algaroba (Prosopis palida and Prosopis juliflora species) provides important environmental and economic benefits for semi-arid regions of the world. These are resistant to drought, and its fruits are used in the manufacture of flour and algarobina syrup. In the present study, the chromosome number, the ploidy level, and the genetic diversity based on 40 microsatellite loci of Prosopis spp. were determined in samples of a Brazilian algaroba population. The cytogenetic analysis in the metaphase showed only diploid individuals (2n = 28), with multiple cells featuring two CMA3/DAPI- heterochromatic blocks suggesting diploid level. However, polysomatism was found by the presence of some cells with four CMA3/DAPI- blocks, showing the tetraploid level just in some somatic cells. Among all of the primers tested for cross-amplification in algaroba, 22 were selected to characterize the samples. Thirteen loci revealed allele polymorphisms in the population and are recommended for future population studies and genetic improvement. The mean values of the analyzes showed low genetic diversity (two alleles per locus and HE = 0.181), reflecting the history of the introduction of algaroba in this sampled locality, and suggesting the genetic bottleneck and probable events of founders, as well as the characteristics of the species of this genera. However, amplified loci indicated low inbreeding (allelic fixation index of -0.007), although heterozygosis was higher than expected by the Hardy-Weinberg equilibrium. Therefore, this algaroba population is formed by diploid individuals and adjusts to the tendency of low number of alleles per locus SSR commonly observed in different species of Prosopis.

Hybridity of a plant created in a combination of crossing of (Vaccinium uliginosum L. × V. vitis-idaea L.) × Oxycoccus macrocarpus (Aiton) Pursh at the tetraploid level

The aim of the study was to determine the hybridity of the F1 generation of Vaccinium cf. uliginosum × V. vitis-idaea × Oxycoccus macrocarpus created through the consecutive crossing of some common berry species of the family Ericaceae (bog whortleberry, cowberry and marsh cranberry) at the tetraploid level. Certain aspects of phenology and morphometric parameters of vegetative organs of the supposed hybrid and parent plants were analysed using traditional methods of comparative analysis. Molecular genetic assay, including random amplification of polymorphic DNA, simple sequence repeat and sequencing, were also used. Comparison of the phenological and morphometric features of the experimental plant and its parents allows suggesting that in the combination of crossing of (V. uliginosum × V. vitis-idaea) × O. macrocarpus (the cultivar Searles), a three-species hybrid was created. The allelic variants, specific for V. uliginosum, V. vitis-idaea and O. macrocarpus, were detected in the V. cf. uliginosum × V. vitis-idaea × O. macrocarpus genotype. A next-generation sequencing approach is suggested for estimating the share of the genomes of Vaccinium spp. in the formation of the interspecies hybrid.

Allium monophyllum (Amaryllidaceae) is a diploid species

In the last decade there was a remarkable progress in karyological analyses among members of Allium Linnaeus (1753: 294) subg. Melanocrommyum (Webb & Berthelot 1848: 347) Rouy (1910: 378) (Gurushidze et al. 2012, Genç et al. 2013, Akhavan et al. 2015, Genç & Firat 2016, Fritsch 2016), confirming that most species are diploid based on x = 8 (x = 9 and x = 10 are only rare exceptions, Fritsch & Astanova 1998). Triploid plants were rarely found in some members of A. sect. Melanocrommyum Webb & Berthel. (Tzanoudakis 1999, Genç & Özhatay 2014). The tetraploid level was repeatedly reported by several authors for A. cyrilli Tenore (1827: 364) and sporadically for some other species, but higher ploidy levels were only exceptionally reported: 2n = 48 for A. cyrilli by Khoshoo et al. (1966) and for A. giganteum Regel (1883: 97) by Mensinkai (1939), and even 2n = 64 for A. monophyllum Vved. in Czerniakowska (1930: 266) by Kurita (1956). Unfortunately, these hexa- and octoploid counts were based on plants from botanical collections, and the taxonomic identity cannot be proofed because herbarium vouchers of these counts are not known to exist.

Breeding for nutritional quality and pest resistance: potential of a set of non-commercial tetraploid potato cultivars with purple and red flesh

Potatoes are the most widely cultivated tubers and also a valuable source of nutrients and non-nutrient health-promoting compounds. Cultivars with pigmented skin and flesh are of especial interest due to the presence of high concentrations of carotenoids, anthocyanins, flavonoids and phenolic acids. Several investigations have focused on the characterization of native and wild potato accessions, particularly highly pigmented genotypes in order to broaden the overall genetic variation in terms of commercial and processing aptitude, pest resistance and nutritional quality. However, the incorporation of some of these traits into new breeding lines at the tetraploid level is hampered by the presence of detrimental traits as well as varying ploidy levels. Therefore, a set of ten highly-pigmented tetraploid cultivars (Solanum tuberosum L.) was selected and characterized on the basis of tuber morphology, pest resistance and nutritional related traits in order to select parental lines for breeding. Attractive colors and color patterns with potential for the potato snack industry have been identified. Varying degrees of resistance to Globodera spp. and Phythophora infestans, which are considered major threats for the crop, have been found among the collection of pigmented cultivars. Nutritional quality related parameters including significantly higher levels of phenolic compounds, anthocyanins, carotenoids, vitamin C and micronutrients (Zn and Fe) were found when compared with the commercial cultivars used as checks. These findings could assist in the selection of parents (progenitors) towards the introduction of interesting traits into new potato advanced clones at the tetraploid level, without the problems derived from ploidy levels and non-commercial characteristics.