Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species

Red clover (Trifolium pratense L.) is used as a forage crop due to a variety of favorable traits relative to other crops. Improved varieties have been developed through conventional breeding approaches, but progress could be accelerated and gene discovery facilitated using modern genomic methods. Existing short-read based genome assemblies of the ~420 Megabase (Mb) genome are fragmented into >135,000 contigs with numerous errors in order and orientation within scaffolds, likely due to the biology of the plant which displays gametophytic self-incompatibility resulting in inherent high heterozygosity. A high-quality long-read based assembly of red clover is presented that reduces the number of contigs by more than 500-fold, improves the per-base quality, and increases the contig N50 statistic by three orders of magnitude. The 413.5 Mb assembly is nearly 20% longer than the 350 Mb short read assembly, closer to the predicted genome size. Quality measures are presented and full-length isoform sequence of RNA transcripts reported for use in assessing accuracy and for future annotation of the genome. The assembly accurately represents the seven main linkage groups present in the genome of an allogamous (outcrossing), highly heterozygous plant species.

Análisis de la sustentabilidad ecológica de distintas estrategias de manejo de malezas en el cultivo de lino oleaginoso (Linum usitatissimum L.) en Argentina

El lino se cultiva generalmente bajo un enfoque productivista, altamente dependiente de insumos externos, modelo que ha causado importantes problemas ecológicos. Entre ellos están los asociados con las malezas cuyo control se realiza principalmente con herbicidas. Ante esta realidad es necesario incorporar estrategias de manejo de malezas que mejoren la sustentabilidad de los agroecosistemas. Se comparó la sustentabilidad ecológica del control de malezas con herbicida respecto al manejo con alta densidad de lino y/o su intercultivo con Trifolium pratense L. En este trabajo se ajustaron y aplicaron algunos indicadores como: diversidad vegetal, % de cobertura de suelo, residuo remanente, materia seca restituida al suelo, % de residuo remanente, uso de herbicida, balance de nitrógeno, eficiencia energética y biomasa vegetal. La metodología usada fue adecuada y fácil de aplicar al objetivo del trabajo. Se observó que el control de malezas en el lino con herbicida fue la práctica de menor aporte a la sustentabilidad, por lo cual si se continúa en el largo plazo con dicha práctica se verá comprometida la misma. Mientras que el intercultivo fue ecológicamente más sustentable que el monocultivo, con puntos más favorables para sustentabilidad sin uso de herbicida, independientemente de la densidad de lino. Esto ocurrió tanto por una mejor conservación de la capacidad productiva del agroecosistema como por ejercer un menor impacto sobre el ambiente externo al mismo. Del presente trabajo surge que existen alternativas al control con herbicidas para el manejo de malezas en lino como es el caso del intercultivo con trébol rojo.

Seed Production of Red Clover (Trifolium pratense L.) under Danish Field Conditions

High and stable seed yield is critical for red clover (Trifolium pratense L.) seed production and the commercial exploitation of the crop. A three-year experiment was conducted from 2013 to 2015 under Danish field conditions to explore the influence of precipitation during peak flowering on the seed yield of three red clover cultivars. We investigated the flowering duration and intensity based on a visual scale assessment, seed yield, and thousand seed weight in all three experimental years. In 2014 and 2015 we measured the seed yield components of floret number per flower head, seed number per flower head, and seed set. During the experimental period, high seed yields of more than 1000 kg ha−1 were obtained for the diploid cultivars ‘Rajah’ and ‘Suez’. Although a relatively high seed yield of 500 kg ha−1 was obtained in the tetraploid cultivar ‘Amos’, this was only around half of the seed yield and seed set of the diploid cultivars. Precipitation during peak flowering positively influenced the seed yield and thousand seed weight for the three cultivars. We conclude that observations of flowering phenology are required to determine the impact of environmental conditions on seed yield in red clover cultivars. Further, adequate water supply during peak flowering is important to obtain the high seed yield of red clover.

Unveiling the Complexity of Red Clover (Trifolium pratense L.) Transcriptome and Transcriptional Regulation of Isoflavonoid Biosynthesis Using Integrated Long- and Short-Read RNAseq

Red clover (Trifolium pratense L.) is used as forage and contains a high level of isoflavonoids. Although isoflavonoids in red clover were discovered a long time ago, the transcriptional regulation of isoflavonoid biosynthesis is virtually unknown because of the lack of accurate and comprehensive characterization of the transcriptome. Here, we used a combination of long-read (PacBio Iso-Seq) and short-read (Illumina) RNAseq sequencing to develop a more comprehensive full-length transcriptome in four tissues (root, stem, leaf, and flower) and to identify transcription factors possibly involved in isoflavonoid biosynthesis in red clover. Overall, we obtained 50,922 isoforms, including 19,860 known genes and 2817 novel isoforms based on the annotation of RefGen Tp_v2.0. We also found 1843 long non-coding RNAs, 1625 fusion genes, and 34,612 alternatively spliced events, with some transcript isoforms validated experimentally. A total of 16,734 differentially expressed genes were identified in the four tissues, including 43 isoflavonoid-biosynthesis-related genes, such as stem-specific expressed TpPAL, TpC4H, and Tp4CL and root-specific expressed TpCHS, TpCHI1, and TpIFS. Further, weighted gene co-expression network analysis and a targeted compound assay were combined to investigate the association between the isoflavonoid content and the transcription factors expression in the four tissues. Twelve transcription factors were identified as key genes for isoflavonoid biosynthesis. Among these transcription factors, the overexpression of TpMYB30 or TpRSM1-2 significantly increased the isoflavonoid content in tobacco. In particular, the glycitin was increased by 50–100 times in the plants overexpressing TpRSM1-2, in comparison to that in the WT plants. Our study provides a comprehensive and accurate annotation of the red clover transcriptome and candidate genes to improve isoflavonoid biosynthesis and accelerate research into molecular breeding in red clover or other crops.

Use of factors of photoperiod duration and growth regulators to increase the yield of mixoploid plants in C0 generation in meadow clover (Trifolium pratense L.)

It has been established that the studied mode of cultivation of meadow clover under controlled environmental conditions, with continuous illumination with an intensity of 18-20 thousand lux and a constant temperature of + 25 °C, causes significant morphological changes in meadow clover. Compared with the optimal (control), the proposed growing regime halves the development time of plants and by the same amount reduces the severity of morphological traits, while simultaneously causing apical dominance in 54% of plants. Spraying plants with phytohormone in the form of naphthylacetic acid in all studied concentrations reduced the growing season by 4-8 days and increased pollen fertility by 8-10%. The optimal concentration of naphthylacetic acid (NLA) was revealed to suppress the growth of secondary meristems in meadow clover under the conditions of an experimental plant growing regime. The most significant effect on the manifestation of apical dominance was shown by the use of naphthylacetic acid at a concentration of 0.001%. The combined use of the proposed growing regime and treatment with naphthylacetic acid at a concentration of 0.001% increased the yield of plants with apical dominance from 54.5% to 76%. The developed method has shown high efficiency for the creation of tetraploid forms of meadow clover. When growing clover C0 generation and selection of mixoploid plants, the yield of mixoploids increased 5.4 times, the survival rate of seedlings increased by 21%, the time to obtain one generation and the selection of mixoploids decreased by 58%.

Influence of inbreeding on the morphobiological characteristics of meadow clover (Trifolium pratense L.)

The influence of inbreeding on the morphobiological characteristics of meadow clover was revealed. Self-pollination, repeated in the number of successive generations, leads to an increase in homozygosity and to inbred depression, which increases from I1 to I3 generation and stabilizes in the I4 generation. It was found that in the I1 generation, according to the main morphobiological characteristics, there is no inward depression, but the maximum release of recessive lethal mutations is manifested, which amounted to 6.2%, and the survival rate of seedlings decreases (91.7%). By the I4 generation, the number of chlorophyll-free seedlings decreases to 1.4%. All the main morpho-biological indicators that determine the productivity of plants decrease from generation I1 to generation I3 by 1.5-2 times and stabilize in generation I4. Obtaining hybrid F1 offspring by crossing a linear material with an I4 induction level leads to the restoration of plant productivity indicators. When creating a linear material, an increase in the number of highly self-compatible genotypes from I1 to I4 generation by 60% is clearly traced. The data obtained make it possible to take into account the survival rate of seedlings, the cleavage of lethal and semi-lethal mutations, the degree of inbred depression in the formation of sample volumes when creating a linear material of meadow clover.