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BACKGROUND AND AIMS: Leaf variegation is common in plants and confers diverse adaptive functions. However, its genetic underpinnings remain largely unresolved; this is particularly true for variegation that arises through modified leaf tissue structure that affects light reflection. White clover is naturally polymorphic for structure-based white leaf mark variegation. It therefore provides a useful system to examine the genetic basis of this phenotype, and to assess potential costs to photosynthetic efficiency resulting from modified leaf structures. This study sought to map the loci controlling the white leaf mark in white clover and evaluate the relationship between white leaf mark, leaf thickness, and photosynthetic efficiency. METHODS: We generated a high-density genetic linkage map from an F3 mapping population, employing reference genome-based SNP markers. White leaf mark was quantified through detailed phenotypic evaluations alongside leaf thickness to test how tissue thickness may affect the variegation phenotype. Quantitative trait locus (QTL) mapping was performed to characterize their genetic bases. Photosynthetic efficiency measurements were used to test for physiological trade-offs between variegation and photosynthetic output. KEY RESULTS: The V locus, a major gene responsible for the white leaf mark polymorphism, was mapped to the distal end of chromosome 5, and several modifier loci were also mapped that contribute additively to variegation intensity. The presence and intensity of white leaf mark was associated with greater leaf thickness; however, increased variegation did not detectably affect photosynthetic efficiency. CONCLUSIONS: We have successfully mapped the major locus governing the white leaf mark in white clover, along with several modifier loci, revealing a complex basis for this structure-based variegation. The apparent absence of compromised photosynthesis in variegated leaves challenges the notion that variegation creates fitness trade-offs between photosynthetic efficiency and other adaptive functions. This finding suggests that other factors may maintain the white leaf mark polymorphism in white clover.
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White clover (Trifolium repens L.) belongs to the Fabaceae family legume and is cultivated in China for its medicinal properties and ornamental value. White clover is grown around the world for forage, turf , green manure and soil conservation purposes (Zhang el al. 2016). In October 2021, an investigation of a 1,000 m2 plant nursery in Lanzhou, China (36°06'N, 103°83'E) found that 80% of White clover plants were infected, and powdery mildew covered 95% of the leaf area. The disease had seriously destroyed the forage quality and reduced the ornamental value. Initially, thin, radial, irregular white colonies appeared on leaves and gradually spread to stems. The white colonies then expanded and thickened to cover upper surface of the leaf, and microscopic hyphae appeared on the bottom of the leaf. In severe cases, the infection resulted in dieback of the leaf. A small area of sporulating fungus was stripped off from the leaf surface with tape and mounted in sterile water for microscopic examination (Mukhtar et al. 2017). Conidiophores were cylindrical, consisting of a foot cell followed by three to four short cells, measuring 75 to 160 × 7 to 10 µm. Conidiophores had straight, cylindric foot cells ranging from 25 to 40 µm long. Singly produced conidia were hyaline and ranged in shape from oblong to cylindrical. Conidia lacked distinct fibrotic bodies and measured 30 to 45 × 15 to 25 µm in length. Long, unbranched germ tubes formed from the ends of the conidia and nipple-shaped appressoria developed on epiphytic mycelia. Based on these morphological characteristics, the pathogen was initially identified morphologically as Erysiphe polygoni (Braun and Cook 2012). To validate the identity, the internal transcribed spacer (ITS) region of the pathogen (SY77) rDNA was amplified by PCR and sequenced using the ITS1/ITS4 primers (White et al. 1990). The resulting sequences were registered to GenBank (GenBank Accession No.OM280998). The ITS sequence of the SY77 was 100% (640/640) identical to E. polygoni (LC009892) on Polygonum aviculare in the United Kingdom and 99% (638/640) identical to E. polygoni (MK685172) on Antigonon leptopus in Taiwan. MEGA 7.0 was used to conduct the neighbor-joining phylogenetic analysis using the ITS sequences from GenBank. The data indicated that the strain SY77 and E. polygoni clustered together on the same branch. Pathogenicity tests were conducted by gently pressing the infected leaves onto five healthy potted White clover plants, while five non-inoculated plants were used as controls (Michael et al. 2021). The plants were maintained in a growth chamber (25 â, 14 h light, and 10 h dark period, RH > 80%). After 10 days, the inoculated plants developed powdery mildew symptoms, whereas the control plants remained symptom-free. The fungus on the inoculated plants was re-isolated, re-identified, and confirmed as E. polygoni based on morphological observations and molecular identification. There is no previous report on E. polygoni causing powdery mildew on White clover in China. The powdery mildew caused by E. polygoni on Red clover has been reported in China and Bulgaria, respectively (Yuan el al.1991; Galina el al. 2017). To our knowledge, this is the first report of powdery mildew caused by E. polygoni on White clover in China. References: 1. Zheng, L., et al. 2018. Plant Dis. 102:628. 2. Mukhtar, G., et al. 2017. Plant Dis.101:1, 246. 3. Braun, U., and Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No.11. CBS, Utrecht. 4. Michael, R. F., et al. 2021.Plant Dis. First look.( doi.org/ 10.1094/PDIS-09-21-2060-PDN). 5. Yuan, Q. H., el al.1991. Pratacult Sci.05:59 (in Chinese). 6. Galina, N., et al, 2017. BIOTECHNOL Anim Husb.33.127.
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Increased transcriptional levels of genes encoding antioxidant enzymes play important protective roles in coping with excessive accumulation of reactive oxygen species (ROS) in plants exposed to various abiotic stresses. To fully elucidate different evolutions and functions of ROS-scavenging enzymatic genes, we isolated iron superoxide dismutase (FeSOD), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) from white clover for the first time and subsequently tested dynamic expression profiles of these genes together with previously identified other antioxidant enzyme genes including copper zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), glutathione reductase (GR), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in response to cold, drought, salinity, cadmium stress and exogenous abscisic acid (ABA) or spermidine (Spd) treatment. The cloned fragments of FeSOD, DHAR and MDHAR genes were 630, 471 and 669 bp nucleotide sequences encoding 210, 157 and 223 amino acids, respectively. Phylogenetic analysis indicated that both amino acid and nucleotide sequences of these three genes are highly conservative. In addition, the analysis of genes expression showed the transcription of GR, POD, MDHAR, DHAR and Cu/ZnSOD were rapidly activated with relatively high abundance during cold stress. Differently, CAT, APX, FeSOD, Cu/ZnSOD and MnSOD exhibited more abundant transcripts compared to others under drought stress. Under salt stress, CAT was induced preferentially (3-12 h) compared to GR which was induced later (12-72 h). Cadmium stress mainly up-regulated Cu/ZnSOD, DHAR and MDHAR. Interestingly, most of genes expression induced by ABA or Spd happened prior to various abiotic stresses. The particular expression patterns and different response time of these genes indicated that white clover differentially activates genes encoding antioxidant enzymes to mitigate the damage of ROS during various environmental stresses.