Role of kura clover (Trifolium Ambiguum bieb.) of Vys variety in the growth of the forage base of the Tambov region

In the conditions of the forest-steppe of the Central Black Earth region, the issue of the sustainable development of forage production, which can be increased through the expansion of the range of cultivated species of perennial legumes, has become relevant. The need to increase the share of areas of perennial grasses is connected with the fact that natural lands and annual forage crops have low productivity. Forage grasses are characterized by a number of unique economically valuable properties and characteristics. In this regard, there is an urgent need to increase their sown areas, as well as to improve natural lands, create hayfields and pastures. One of the limiting factors is the insufficient provision of seeds for a new fodder crop - Vys clover of a kura variety. It is kura clover that plays an important role in the creation of a solid forage base for animal husbandry in the Tambov region. Kura clover (Trifolium ambiguum Bieb) is characterized by a number of valuable traits and properties. In this regard, for the first time in the Central Black Earth Region, its biology and morphological development began to be studied and the optimal terms and methods of sowing this perennial legume crop are being developed. It will allow obtaining stable seed yields. The technology for obtaining seeds of this forage crop of the Vys variety in the forest-steppe conditions of the Central Black Earth Region is currently in the process of development. Therefore, the ongoing research on agrotechnical methods of the cultivation of kura clover seeds is very relevant and the development of individual agricultural methods of its cultivation technology of seeds is one of the important tasks of forage production.

Comprehensive evaluation of candidate reference genes for quantitative real-time PCR-based analysis in Caucasian clover

The forage species Caucasian clover (Trifolium ambiguum M. Bieb.), a groundcover plant, is resistant to both cold and drought. However, reference genes for qRT-PCR-based analysis of Caucasian clover are lacking. In this study, 12 reference genes were selected on the basis of transcriptomic data. These genes were used to determine the most stably expressed genes in various organs of Caucasian clover under cold, salt and drought stress for qRT-PCR-based analysis. Reference gene stability was analyzed by geNorm, NormFinder, BestKeeper, the ∆Ct method and RefFinder. Under salt stress, RCD1 and PPIL3 were the most stable reference genes in the leaves, and NLI1 and RCD1 were the most stable references genes in the roots. Under low-temperature stress, APA and EFTu-GTP were the most stable reference genes in the leaves, and the RCD1 and NLI2 genes were highly stable in the roots. Under 10% PEG-6000 stress, NLI1 and NLI2 were highly stable in the leaves, and RCD1 and PPIL3 were the most stable in the roots. Overall, RCD1 and NLI2 were the most stable reference genes in organs under normal conditions and across all samples. The most and least stable reference genes were validated by assessing their appropriateness for normalization via WRKY genes.

Study of the genetic organization of the strain Rhizobium leguminosarum bv. trifolii forming a symbiosis with clover Trifolium ambiguum

R. leguminosarum bv. trifolii strains are characterized by narrow host specificity. We have identified a strain that forms nodules on several types of clover and studied the genetic organization of its symbiotic region.

Revealing the full-length transcriptome of caucasian clover rhizome development

Background Caucasian clover (Trifolium ambiguum M. Bieb.) is a strongly rhizomatous, low-crowned perennial leguminous and ground-covering grass. The species may be used as an ornamental plant and is resistant to cold, arid temperatures and grazing due to a well-developed underground rhizome system and a strong clonal reproduction capacity. However, the posttranscriptional mechanism of the development of the rhizome system in caucasian clover has not been comprehensively studied. Additionally, a reference genome for this species has not yet been published, which limits further exploration of many important biological processes in this plant. Result We adopted PacBio sequencing and Illumina sequencing to identify differentially expressed genes (DEGs) in five tissues, including taproot (T1), horizontal rhizome (T2), swelling of taproot (T3), rhizome bud (T4) and rhizome bud tip (T5) tissues, in the caucasian clover rhizome. In total, we obtained 19.82 GB clean data and 80,654 nonredundant transcripts were analysed. Additionally, we identified 78,209 open reading frames (ORFs), 65,227 coding sequences (CDSs), 58,276 simple sequence repeats (SSRs), 6821 alternative splicing (AS) events, 2429 long noncoding RNAs (lncRNAs) and 4501 putative transcription factors (TFs) from 64 different families. Compared with other tissues, T5 exhibited more DEGs, and co-upregulated genes in T5 are mainly annotated as involved in phenylpropanoid biosynthesis. We also identified betaine aldehyde dehydrogenase (BADH) as a highly expressed gene-specific to T5. A weighted gene co-expression network analysis (WGCNA) of transcription factors and physiological indicators were combined to reveal 11 hub genes (MEgreen-GA3), three of which belong to the HB-KNOX family, that are up-regulated in T3. We analysed 276 DEGs involved in hormone signalling and transduction, and the largest number of genes are associated with the auxin (IAA) signalling pathway, with significant up-regulation in T2 and T5. Conclusions This study contributes to our understanding of gene expression across five different tissues and provides preliminary insight into rhizome growth and development in caucasian clover.

Pedigree analysis of pre-breeding efforts in Trifolium spp. germplasm in New Zealand

Background Prebreeding in plants is the activity designed to identify useful characteristics from wild germplasm and its integration in breeding programs. Prebreeding aims to introduce new variation into the populations of a species of interest. Pedigree analysis is a valuable tool for evaluation of variation in genebanks where pedigree maps are used to visualize and describe population structure and variation within these populations. Margot Forde Germplasm Centre (MFGC) is New Zealand’s national forage genebank and holds a collection of ~ 75 species of the genus Trifolium, of which only a dozen have been taken through prebreeding programs. The main objective of this study was to construct pedigree maps and analyse patterns of relatedness for seven minor Trifolium species accessions contained at the MFGC. These species are Trifolium ambiguum, Trifolium arvense, Trifolium dubium, Trifolium hybridum, Trifolium medium, Trifolium subterraneum and the Trifolium repens x Trifolium occidentale interspecific hybrids. We present a history of Trifolium spp. prebreeding in New Zealand and inform breeders of possible alternative forage species to use. Results Pedigree data from accessions introduced between 1950 and 2016 were used and filtered based on breeding activity. Kinship levels among Trifolium spp. remained below 8% and no inbreeding was found. Influential ancestors that contributed largely to populations structure were identified. The Australian cultivar ‘Monaro’ had a strong influence over the whole population of accessions in T. ambiguum. T. subterraneum and T. repens x T. occidentale had the largest number of generations (3). T. ambiguum and T. medium had the highest cumulative kinship across the decades. Conclusions We conclude that there are high levels of diversity in the seven Trifolium spp. studied. However, collection and prebreeding efforts must be strengthened to maximize utilization and bring useful genetic variation.

Living Mulch Management Spatially Localizes Nutrient Cycling in Organic Corn Production

Kura clover (Trifolium ambiguum) is a perennial living mulch species that can be used in conjunction with zone tillage to reduce nitrogen pollution, maintain ground cover, and provide nitrogen to crops. In such systems, kura clover is maintained between crop rows by limiting tillage only to within-row areas. However, the effect of zone-tilled living mulches on soil quality and nutrient cycling in these distinct regions is relatively unexplored. We examined three pools of labile soil organic matter (SOM): microbial biomass, particulate organic matter (POM), and permanganate oxidizable carbon (POXC). Soil samples were collected from both within-row and between-row locations of a zone-tilled kura clover living mulch at three time points per year: before spring zone tillage, approximately ten days after spring zone tillage and corn (Zea mays) planting, and at corn harvest in 2015 and 2016. In 2016, POM and POXC decreased within rows relative to between-row regions after tillage, suggesting that zone till management stimulated decomposition of readily available SOM to effectively localize nutrient cycling in this region and slow mineralization between rows where living kura clover remained. This work shows that zone-tilled living mulches may be a promising avenue for enhancing the synchrony of nutrient mineralization specifically within crop rows, while maintaining year-round ground cover between rows.

MELITTOPHILIC COMPLEX OF PLANTS PROVIDING FORAGE BASE FOR HONEY BEES (APIS MELLIFERA) IN THE MOUNTAIN-FOREST ZONE OF CRIMEA

For the first time, in the mountain-forest ecosystems of Crimea of parcel organization level adjacent to the beehives of honey bees (Apis mellifera L.), a species composition of plants representing the melittophilic complex has been identified which provides honey bees with pollen and nectar during the entire warm season. The complex includes 57 species of plants where the honey bees that collect pollen or nectar have been registered. The core of the melittophilic complex consisted of 35 species of plants, the proportion of simultaneously flowering (in any period of the season) flowers of each one exceeded 2.5 % of the total number of flowers of all flowering species at this time. In early spring, the list of such species included 15 plant species belonging to 14 genera and 12 families. In this period, the decisive forage value (species whose flower share exceeded 10 % of all flowering flowers) was represented by 4 plant species: Cornus mas L., Corylus avellana L., Dentaria quinquefolia M. B. and Prunus divaricata Ledeb., the important value (the flowers share made <10 %, but >2.5 %) had 4 species: Ficaria verna Huds., Galanthus plicatus M. Bieb., Primula vulgaris Huds. and Scilla bifolia L. In spring, the forage base made up 20 plant species belonging to 18 genera and 7 families. Four species had decisive forage value: Cerasus avium L., Malus sylvestris L., Prunus spinosa L. and Pyrus elaeagrifolia Jacq., 7 species had important value: Crataegus monogyna Jacq., Fragaria vesca L., Lamium purpureum L., Crepis pulchra L., Prunus domestica L., Thlaspi arvense L. and Trifolium ambiguum M. Most of the species belonged to the Rosaceae family (10 species). In summer period, the forage basis comprised 20 species of melittophilic plants belonging to 19 genera and 9 families. 5 species had decisive value: Cichorium intybus L., Cirsium arvense (L.) Scop., Echium vulgare L., Medicago sativa L. and Trifolium ambiguum M., the important value had 7 species: Achillea millefolium L., Betonica officinalis L., Ballota nigra L., Centaurea diffusa Lam., Clematis vitalba L., Linaria vulgaris Mill. and Onobrychis sativa Lam. The representatives of the Asteraceae family (7 species) dominated; the representatives of Fabaceae (5 species) and Lamiaceae (4 species) also played a significant role. In autumn, there were the following food sources for honey bees: Diplotaxis tenuifolia (L.) DC., Centaurea diffusa Lam., Erýngium campéstre L., Carduus crispus L., and on the steppe slopes of the mountains: Scilla autumnalis L. and Crocus speciosus M. B. It has been established that the greatest number of melittophilic plant species in the studied mountain-forest biogeocenoses grow on parcels of forest edges and glades, steppe slopes of the mountains in their natural state. Oak-hornbeam and floodplain tree-shrub parcels have a smaller variety of species, but are essential for bees in early spring and spring..

A Eurasia-wide polyploid species complex involving 6x Trifolium ambiguum, 2x T. occidentale and 4x T. repens produces interspecific hybrids with significance for clover breeding

Background Trifolium ambiguum occurs as a 2x, 4x, 6x polyploid series in W Asia. The 6x form is the most agronomically desirable, having strong rhizomatous spread and drought tolerance. These traits would be potentially very valuable if they could be transferred to white clover (T. repens) which is the most important agronomic clover species. However, to-date, no fertile interspecific hybrids with 6x T. ambiguum are available. Previously, 2x T. occidentale from W Europe has produced synthetic fertile hybrids with both 2x and 4x T. ambiguum and these were inter-fertile with white clover. Here we ask whether 2x T. occidentale can form fertile hybrids with 6x T. ambiguum and act as a genetic bridge to white clover and bring these species together as part of a common gene pool. Results Ten verified F1 (6x T. ambiguum x 2x T. occidentale) hybrids were produced by embryo rescue and seven were studied further. All four investigated for chromosome number were 2n = 4x = 32 and FISH confirmed the expected 21 T. ambiguum and 8 T. occidentale chromosomes. Hybrid fertility was extremely low but 2n female gametes functioned with white clover pollen to produce seeds. Derived plants were confirmed using FISH and were successfully backcrossed to white clover to produce partially fertile breeding populations. Conclusions Although T. occidentale and 6x T. ambiguum are widely separated by geography and ecological adaptation they have maintained enough genomic affinity to produce partially fertile hybrids. Inter-fertility of the hybrids with allotetraploid T. repens showed that T. occidentale can provide a genetic bridge between 6x T. ambiguum and white clover to produce plants with new phenotypes combining the traits of all three species. Use of this information should enable potentially valuable stress tolerance traits from 6x T. ambiguum to be used in white clover breeding for the first time.

A Eurasia-wide polyploid species complex involving 6x Trifolium ambiguum, 2x T. occidentale and 4x T. repens produces interspecific hybrids with significance for clover breeding

Background Trifolium ambiguum occurs as a 2x, 4x, 6x polyploid series in W Asia, The 6x form is the most agronomically desirable, having strong rhizomatous spread and drought tolerance. These traits would be potentially very valuable if they could be transferred to white clover (T. repens) which is the most important agronomic clover species. However, to-date, no fertile interspecific hybrids with 6x T. ambiguum are available. Previously, 2x T. occidentale from W Europe has produced synthetic fertile hybrids with both 2x and 4x T. ambiguum and these were inter-fertile with white clover. Here we ask whether 2x T. occidentale can form fertile hybrids with 6x T. ambiguum and act as a genetic bridge to white clover and bring these species together as part of a common gene pool. Results Ten verified F1 (6x T. ambiguum x 2x T. occidentale) hybrids were produced by embryo rescue and seven were studied further. All four investigated for chromosome number were 2n=4x=32 and FISH confirmed the expected 21 T. ambiguum and 8 T. occidentale chromosomes. Hybrid fertility was extremely low but 2n female gametes functioned with white clover pollen to produce seeds. Derived plants were confirmed using FISH and were successfully backcrossed to white clover to produce partially fertile breeding populations. Conclusions Although T. occidentale and 6x T. ambiguum are widely separated by geography and ecological adaptation they have maintained enough genomic affinity to produce partially fertile hybrids. Inter-fertility of the hybrids with allotetraploid T. repens showed that T. occidentale can provide a genetic bridge between 6x T. ambiguum and white clover to produce plants with new phenotypes combining the traits of all three species. Use of this information should enable potentially valuable stress tolerance traits from 6x T. ambiguum to be used in white clover breeding for the first time.