The Identification and Characterization of the <i>KNOX</i> Gene Family as an Active Regulator of Leaf Development in <i>Trifolium repens</i>.

Leaves are the primary and critical feed for herbivores. They directly determine the yield and quality of legume forage. <i>Trifolium repens</i> (<i>T. repens</i>) is an indispensable legume species, widely cultivated in temperate pastures due to its nutritional value and nitrogen fixation. Although the leaves of <i>T. repens</i> are typical trifoliate, they have unusual patterns to adapt to herbivore feeding. The number of leaflets in <i>T. repens</i> affects its production and utilization. The <i>KNOX</i> gene family encodes transcriptional regulators that are vital in regulating and developing leaves. Identification and characterization of <i>TrKNOX</i> gene family as an active regulator of leaf development in <i>T. repens</i> were studied. A total of 21 <i>TrKNOX</i> genes were identified from the T. repens genome database and classified into three subgroups (Class I, Class II, and Class M) based on phylogenetic analysis. Nineteen of the genes identified had four conserved domains, except for <i>KNOX5</i> and <i>KNOX9</i>, which belong to Class M. Varying expression levels of TrKNOX genes were observed at different developmental stages and complexities of leaves. <i>KNOX9</i> was observed to upregulate the leaf complexity of T. repens. Research on <i>TrKNOX</i> genes could be novel and further assist in exploring their functions and cultivating high-quality <i>T. repens</i> varieties.

Genome-Wide Identification, Characterization, and Expression Profiling Analysis of SPL Gene Family during the Inflorescence Development in Trifolium repens.

Trifolium repens is the most widely cultivated perennial legume forage in temperate region around the world. It has rich nutritional value and good palatability, seasonal complementarity with grasses, and can improve the feed intake and digestibility of livestock. However, flowering time and inflorescence development directly affects the quality and yield of T. repens, as well as seed production. The Squa promoter binding protein-like (SPL) gene family is a plant specific transcription factor family, which has been proved to play a critical role in regulating plant formation time and development of flowers. In this study, a total of 37 TrSPL genes were identified from the whole genome of T. repens and were divided into nine clades based on phylogenetic tree. Seventeen TrSPL genes have potential target sites for miR156. The conserved motif of squamosa promoter binding protein (SBP) contains two zinc finger structures and one NLS structure. Gene structure analysis showed that all TrSPL genes contained SBP domain, while ankyrin repeat region was just distributed in part of genes. 37 TrSPL genes were relatively dispersedly distributed on 16 chromosomes, and 5 pairs of segmental repeat genes were found, which indicated that segmental duplication was the main way of gene expansion. Furthermore, the gene expression profiling showed that TrSPL11, TrSPL13, TrSPL22, and TrSPL26 were highly expressed only in the early stage of inflorescence development, while TrSPL1 and TrSPL6 are highly expressed only in the mature inflorescence. Significantly, the expression of TrSPL4 and TrSPL12 increased gradually with the development of inflorescences. The results of this study will provide valuable clues for candidate gene selection and elucidating the molecular mechanism of T. repens flowering regulation.

Unraveling Cadmium Toxicity in Trifolium repens L. Seedling: Insight into Regulatory Mechanisms Using Comparative Transcriptomics Combined with Physiological Analyses.

Trifolium repens (T. repens) can accumulate significant amounts of heavy metal ions, and has strong adaptability to wide environmental conditions, and relatively large biomass, which is considered a potential plant for phytoremediation. However, the molecular mechanisms of T. repens involved in Cd tolerance have not yet been studied in detail. This study was conducted to examine the integrative responses of T. repens exposed to a high-level CdCl2 by investigating the physiological and transcriptomic analyses. The results suggested that T. repens seedlings had a high degree of tolerance to Cd treatment. The roots accumulated higher Cd concentration than leaves and were mainly distributed in the cell wall. The content of MDA, soluble protein, the relative electrolyte leakage, and three antioxidant enzymes (POD, SOD, and APX) was increased with the Cd treatment time increasing, but the CAT enzymes contents were decreased in roots. Furthermore, the transcriptome analysis demonstrated that the differentially expressed genes (DEGs) mainly enriched in the glutathione (GSH) metabolism pathway and the phenylpropanoid biosynthesis in the roots. Overexpressed genes in the lignin biosynthesis in the roots might improve Cd accumulation in cell walls. Moreover, the DEGs were also enriched in photosynthesis in the leaves, transferase activity, oxidoreductase activity, and ABA signal transduction, which might also play roles in reducing Cd toxicity in the plants. All the above, clearly suggest that T. repens employ several different mechanisms to protect itself against Cd stress, while the cell wall biosynthesis and GSH metabolism could be considered the most important specific mechanisms for Cd retention in the roots of T. repens.

R2R3-MYB gene family: Genome-wide identification provides insight to improve the content of proanthocyanidins in Trifolium repens.

The R2R3-MYB family is one of largest transcription factor families in plants playing significant roles in regulating anthocyanin and proanthocyanidin biosynthesis. Proanthocyanidins are one of major objectives to improve the quality of white clover (Trifolium repens L.), which have a beneficial effect on ruminant to prevent the lethal pasture bloat. A total of 133 TrR2R3-MYB genes were identified and distributed on all 16 chromosomes based on the whole genome information of white clover. Also, by exploring the gene structure, motifs and duplication events of TrR2R3-MYBs, as well as the evolutionary relationship with TrR2R3-MYB genes of other species, 10 TrR2R3-MYB genes with the potential to regulate the anthocyanins and proanthocyanidins biosynthesis were screened. These TrR2R3-MYB genes responded significantly to low temperature in white clover. In addition, they have different expression patterns in leaves, petioles and inflorescences of white clover. Importantly, TrMYB116 and TrMYB118 may positively regulate anthocyanin accumulation and low temperature response in white clover. TrMYB118 may also be associated with anthocyanin pigmentation pattern in Purple leaves. This study provides a basis for verifying the function of TrR2R3-MYB and breeding white clover cultivars with high proanthocyanidins.

Genetic diversity and population structure analysis in a large collection of white clover (Trifolium repens L.) germplasm worldwide.

White clover is an important temperate legume forage with high nutrition. In the present study, 448 worldwide accessions were evaluated for the genetic variation and polymorphisms using 22 simple sequence repeat (SSR) markers. All the markers were highly informative, a total of 341 scored bands were amplified, out of which 337 (98.83%) were polymorphic. The PIC values ranged from 0.89 to 0.97 with an average of 0.95. For the AMOVA analysis, 98% of the variance was due to differences within the population and the remaining 2% was due to differences among populations. The white clover accessions were divided into different groups or subgroups based on PCoA, UPGMA, and STRUCTURE analyses. The existence of genetic differentiation between the originally natural and introduced areas according to the PCoA analysis of the global white clover accessions. There was a weak correlation between genetic relationships and geographic distribution according to UPGMA and STRUCTURE analyses. The results of the present study will provide the foundation for future breeding programs, genetic improvement, core germplasm collection establishment for white clover.

Fingerprint identification of white clover cultivars based on SSR molecular markers.

White clover (Trifolium repens L.) is an important perennial legume forage with high productivity and quality. To strengthen the basic research on the genetic characteristics, fingerprint identification and adaptability of white clover germplasm resources, Simple sequence repeat (SSR) molecular markers were applied to 10 white clover cultivars to assess the genetic diversity and related lines of white clover at the molecular level in order to lay a theoretical foundation for the selection of high-quality seeds and cultivars of white clover. A total of 120 different bands were amplified by 29 pairs of SSR primers with good polymorphism, of which 103 (89.5%) were polymorphic. Meanwhile, the PIC of each primer was 0.181-0.588, with an average of 0.329. Analysis of molecular variance revealed that 57% of the genetic variation occurred within cultivars and 43% occurred among cultivars. The results of cluster analysis and the principal coordinate analysis revealed that the parental relationships of the 10 cultivars, with the 'Purple' cultivar very distantly related to the other 9 cultivars and the closest parental relationship between 'Ladino' and 'Sulky'. The fingerprints constructed by three representative primers (gtrs679, gtrs319, and gtrs678) have a strong identification ability. In summary, the SSR markers had good polymorphism and could be used for DNA fingerprint analysis of white clover cultivars.