Effect of gibberellin on <Emphasis Type="Italic">Arabidopsis</Emphasis> growth,development, and hormonal balance under green and blue light

The role of gibberellins in plant morphology under selective light was studied. A comparison of the effects of green and blue light on growth, development, and hormonal balance was performed for two Arabidopsis thaliana (L.) Heynh. (ecotype Landsberg erecta lines: wild type Ler and its ga4-1 mutant with suppressed GA_4/1 synthesis. The absence of active GA_4/1 from ga4-1 mutant determined its retarded growth, slowed passing through developmental phases, suppressed apical dominance, and reduced seed productivity. The retarded growth and development of the mutant was related to changed hormonal balance in them as compared to wild-type line: IAA content and the IAA/ABA ratio were declined, zeatin riboside and ABA accumulated. Green light retarded stem elongation and branching, reduced leaf specific surface density and plant seed productivity, and retarded plant transition to reproduction to a greater degree at GA_4/1 deficit (ga4-1) than at its normal content (Ler).     

Interaction of jasmonic acid and blue light in the regulation of arabidopsis morphogenesis

The effects of blue light (BL) and jasmonic acid (JA) on morphogenesis of Arabidopsis thaliana (L.) Heynh seedlings of genotypes Col and Ler and their mutants, namely, axr1-3 and jar1-1 mutants resistant to IAA and JA, respectively, and a CRY1 photoreceptor-deficient mutant hy4 were studied. Both 1 μM JA and BL exposure retarded hypocotyl growth of Ler, Col, and jar1-1 seedlings, whereas JA had no effect on hypocotyl growth of axr1-3, but the suppression of hypocotyl growth of this mutant by BL was even more noticeable than that of Ler, Col, and jar1-1. JA and BL applied simultaneously inhibited hypocotyl growth of axr1-3 and especially of Ler, Col, and jar1-1 more than either of factors applied separately. The hy4 mutant did not respond to BL, whereas JA stimulated its hypocotyl growth. JA did not change the cotyledon size of Col, axr1-3, and jar1-1 and reduced the cotyledon size of Ler and hy4. BL enhanced the cotyledon growth of all wild-type and mutant plants used in the study. The cotyledon sizes of all plants except Ler were also increased when JA and BL were applied together. Some of the growth responses correlated with the endogenous IAA and ABA contents. Thus, for example, the hypocotyl and cotyledon growth retardation of Ler seedlings in the presence of JA correlated with a reduced level of free IAA and a considerable increase in the free ABA level in plants grown both in darkness and in BL. Under other growth conditions, no correlation between the endogenous IAA and ABA levels and A. thaliana seedling growth was noted. The interaction between the signal transduction pathways triggered by BL and JA at the early stages of arabidopsis morphogenesis is discussed on the basis of Col, Ler, axr1-3, and jar1-1 hypocotyl growth responses.     

A mutation of casein kinase 2 α4 subunit affects multiple developmental processes in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Key message

Arabidopsis CK2 α4 subunit regulates the primary root and hypocotyl elongation, lateral root formation, cotyledon expansion, rosette leaf initiation and growth, flowering, and anthocyanin biosynthesis.

Abstract

Casein kinase 2 (CK2) is a conserved tetrameric kinase composed of two α and two β subunits. The inhibition of CK2 activity usually results in severe developmental deficiency. Four genes (CKA1–CKA4) encode CK2 α subunit in Arabidopsis. Single mutations of CKA1, CKA2, and CKA3 do not affect the normal growth of Arabidopsis, while the cka1 cka2 cka3 triple mutants are defective in cotyledon and hypocotyl growth, lateral root development, and flowering. The inhibition of CKA4 expression in cka1 cka2 cka3 background further reduces the number of lateral roots and delays the flowering time. Here, we report the characterization of a novel knockout mutant of CKA4, which exhibits various developmental defects including reduced primary root and hypocotyl elongation, increased lateral root density, delayed cotyledon expansion, retarded rosette leaf initiation and growth, and late flowering. The examination of the cellular basis for abnormal root development of this mutant revealed reduced root meristem cells with enhanced RETINOBLASTOMA-RELATED (RBR) expression that promotes cell differentiation in root meristem. Moreover, this cka4-2 mutant accumulates higher anthocyanin in the aerial part and shows an increased expression of anthocyanin biosynthetic genes, suggesting a novel role of CK2 in modulating anthocyanin biosynthesis. In addition, the complementation test using primary root elongation assay as a sample confirms that the changed phenotypes of this cka4-2 mutant are due to the lack of CKA4. Taken together, this study reveals an essential role of CK2 α4 subunit in multiple developmental processes in Arabidopsis.

     

Overexpression of <Emphasis Type="Italic">RcFUSCA3</Emphasis>, a B3 transcription factor from the PLB in <Emphasis Type="Italic">Rosa canina</Emphasis>, activates starch accumulation and induces male sterility in Arabidopsis

A homologue of the Arabidopsis gene FUSCA3 (FUS3), isolated from the protocorm-like body (PLB) of Rosa canina and designated RcFUS3, encodes 331 amino acid residues. It was shown that RcFUS3 is specifically expressed in the PLB of R. canina and that its subcellular localization is in the nucleus. The Arabidopsis fus3-3 mutant phenotype could be rescued by over-expression of RcFUS3, suggesting that RcFUS3 has a function similar to that of Arabidopsis FUS3. Over-expression of RcFUS3 in wild type Arabidopsis resulted in a decrease in endogenous GA and CTK levels, an increase in ABA and IAA contents, starch grain accumulation in the cotyledon and hypocotyl, failure of cotyledon extension and abnormal elongation of the hypocotyl, abnormal stomatal and pavement cells, an increase in branch numbers, prolongation of the growth cycle, and morphological changes in floral organs. Interestingly, over-expression of RcFUS3 in homozygous form resulted in premature degradation of the tapetum, indehiscent anthers and hypogenetic stamens, causing complete male sterility in Arabidopsis; this is the first observation that over-expression of a gene (RcFUS3) homologous to FUS3 can lead to male sterility and starch grain accumulation in Arabidopsis.     

Gibberellin and auxin signals control scape cell elongation and proliferation in <Emphasis Type="Italic">Agapanthus praecox</Emphasis> ssp. <Emphasis Type="Italic">orientalis</Emphasis>

Plant height is determined by the processes of cell proliferation and elongation. Plant hormones play key roles in a species-dependent manner in these processes. We used paclobutrazol (PAC) at 400 mg·L^-1 in this study to spray Agapanthus praecox ssp. orientalis plants in order to induce dwarf scape (inflorescence stem). Morphological examination showed that PAC reduced scape height by inhibiting the cell elongation by 54.56% and reducing cell proliferation by 10.45% compared to the control. Quantification and immunolocalization of endogenous gibberellins (GAs) and indole-3-acetic acid (IAA) showed that the GA₁, GA₃, and GA₄ levels and the IAA gradient were reduced. Among these hormones, GA4 was the key component of GAs, which decreased 59.51-92.01% compared to the control in scape. The expression of cell wall synthesis related genes cellulose synthase (CESA) and UDP-glucuronic acid decarboxylase (UXS) were upregulated significantly, whereas cell wall loosening gene xyloglucan endotransglucosylase 2 (XET2) was downregulated by 99.99% surprisingly. Correlation analysis suggested GA regulated cell elongation and auxin modulated cell proliferation in Agapanthus scape. Additionally, the accumulation of sugars played roles in cell wall synthesis and cell expansion. These results indicated GA and IAA signals triggered a downstream signaling cascade, controlled cell expansion and proliferation during scape elongation.     

Structure–Function Relationships of Four Stereoisomers of a Brassinolide Mimetic on Hypocotyl and Root Elongation of the Brassinosteroid-Deficient <Emphasis Type="Italic">det2-1</Emphasis> Mutant of <Emphasis Type="Italic">Arabidopsis</Emphasis>

Brassinosteroids (BRs) are a group of plant hormones and the bioactive BR, brassinolide (BL), is causally implicated in promoting cell elongation and cell proliferation. In Arabidopsis, the biosynthesis of BL is essential for hypocotyl etiolation in the dark, and application of bioactive BRs can promote both hypocotyl and root elongation, although high concentrations of applied BRs result in inhibition of root elongation. A non-steroidal structure consisting of four stereoisomers was designed to contain subunits bearing key functional groups mimicking those of BL. The bioactivity of each of these individual stereoisomers was tested using the Arabidopsis thaliana det2-1 mutant line, which is deficient in BL, and thus does not etiolate in the dark. Application of BL at each of 0.1, 1.0, and 10.0 µM promotes hypocotyl elongation in dark-grown det2-1 plants while simultaneously inhibiting elongation of their primary root. In contrast, the mimetic structures, when applied to dark-grown det2-1 plants, promote hypocotyl elongation without negatively affecting primary root elongation. In fact, two of the mimetic structures, applied at a 10 µM concentration, significantly promoted both hypocotyl and root elongation. Correlation of this contrasting behavior with the configurations of the hydroxylated stereocenters of the mimetics is described. This is the first example of a non-steroidal BL mimetic where the biological activities of individual stereoisomers were tested and compared.     

Enhancement of hypocotyl elongation by LOV KELCH PROTEIN2 production is mediated by auxin and phytochrome-interacting factors in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

Auxin and two phytochrome-interacting factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, play crucial roles in the enhancement of hypocotyl elongation in transgenic Arabidopsis thaliana plants that overproduce LOV KELCH PROTEIN2 (LKP2).

Abstract

LOV KELCH PROTEIN2 (LKP2) is a positive regulator of hypocotyl elongation under white light in Arabidopsis thaliana. In this study, using microarray analysis, we compared the gene expression profiles of hypocotyls of wild-type Arabidopsis (Columbia accession), a transgenic line that produces green fluorescent protein (GFP), and two lines that produce GFP-tagged LKP2 (GFP-LKP2). We found that, in GFP-LKP2 hypocotyls, 775 genes were up-regulated, including 36 auxin-responsive genes, such as 27 SMALL AUXIN UP RNA (SAUR) and 6 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, and 21 genes involved in responses to red or far-red light, including PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5; and 725 genes were down-regulated, including 15 flavonoid biosynthesis genes. Hypocotyls of GFP-LKP2 seedlings, but not cotyledons or roots, contained a higher level of indole-3-acetic acid (IAA) than those of control seedlings. Auxin inhibitors reduced the enhancement of hypocotyl elongation in GFP-LKP2 seedlings by inhibiting the increase in cortical cell number and elongation of the epidermal and cortical cells. The enhancement of hypocotyl elongation was completely suppressed in progeny of the crosses between GFP-LKP2 lines and dominant gain-of-function auxin-resistant mutants (axr2-1 and axr3-1) or loss-of-function mutants pif4, pif5, and pif4 pif5. Our results suggest that the enhancement of hypocotyl elongation in GFP-LKP2 seedlings is due to the elevated level of IAA and to the up-regulated expression of PIF4 and PIF5 in hypocotyls.

     

Activation of gibberellin 20-oxidase 2 undermines auxin-dependent root and root hair growth in NaCl-stressed <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Although salt stress mainly disturbs plant root growth by affecting the biosynthesis and signaling of phytohormones, such as gibberellin (GA) and auxin, the exact mechanisms of the crosstalk between these two hormones remain to be clarified. Indole-3-acetic acid (IAA) is a biologically active auxin molecule. In this study, we investigated the role of Arabidopsis GA20-oxidase 2 (GA20ox2), a final rate-limiting enzyme of active GA biosynthesis, in IAA-directed root growth under NaCl stress. Under the NaCl treatment, seedlings of a loss-of-function ga20ox2-1 mutant exhibited primary root and root hair elongation, altered GA₄ accumulation, and decreased root Na⁺ contents compared with the wild-type, transgenic GA20ox2-complementing, and GA20ox2-overexpression plant lines. Concurrently, ga20ox2-1 alleviated the tissue-specific inhibition of NaCl on IAA generation by YUCCAs, IAA transport by PIN1 and PIN2, and IAA accumulation in roots, thereby explaining how NaCl increased GA20ox2 expression in shoots but disrupted primary root and root hair growth in wild-type seedlings. In addition, a loss-of-function pin2 mutant impeded GA20ox2 expression, indicating that GA20ox2 function requires PIN2 activity. Thus, the activation of GA20ox2 retards IAA-directed primary root and root hair growth in response to NaCl stress.     

The effects of gibberellins and mepiquat chloride on nitrogenase activity in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA₃) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA₃ or PIX. GA₃ treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA₃-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA₃ and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA₃ treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA₃, respectively, in B. japonicum.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Revealing physiological and genetic properties of a dominant maize dwarf <Emphasis Type="Italic">Dwarf11</Emphasis> (<Emphasis Type="Italic">D11</Emphasis>) by integrative analysis

Plant height is an important agronomic trait involved in lodging resistance and harvest index. The identification and characterization of mutants that are defective in plant height have implications for trait improvement in breeding programs. Two dominant maize dwarf mutants D8 and D9 have been well-characterized. Here, we report the characterization of a dominant maize dwarf mutant Dwarf11 (D11). Dwarf stature of D11 was mainly attributed to the inhibition of longitudinal cell elongation. The levels of bioactive GA₃ were significantly lower in D11. Contrarily, D8 mutant accumulates markedly higher levels of GA₃. The expression of GA biosynthetic and catabolic genes was dramatically decreased in D11. Expression variations of d8 and d9 genes were not observed in D11 mutant. Moreover, genetic suppressors of D11 were identified in inbred line Chang 7-2. Integrated omics data indicated that D11 is a novel dominant maize dwarf. The ultimate D11 gene cloning and its regulatory network elucidation may strengthen our understanding of the genetic basis of plant architecture and provide cues for breeding of crops with plant height ideotypes.     

Identification of the dwarf gene <Emphasis Type="Italic">GmDW1</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) by combining mapping-by-sequencing and linkage analysis

Key message

GmDW1 encodes an ent-kaurene synthase (KS) acting at the early step of the biosynthesis pathway for gibberellins (GAs) and regulates the development of plant height in soybean.

Abstract

Plant height is an important component of plant architecture, and significantly affects crop breeding practices and yield. Here, we report the characterization of an EMS-induced dwarf mutant (dw) of the soybean cultivar Zhongpin 661 (ZDD23893). The dw mutant displayed reduced plant height and shortened internodes, both of which were mainly attributed to the longitudinally decreased cell length. The bioactive GA₁ (gibberellin A₁) and GA₄ (gibberellin A₄) were not detectable in the stem of dw, and the dwarf phenotype could be rescued by treatment with exogenous GA₃. Genetic analysis showed that the dwarf trait of dw was controlled by a recessive nuclear gene. By combining linkage analysis and mapping-by-sequencing, we mapped the GmDW1 gene to an approximately 460-kb region on chromosome (Chr.) 8, containing 36 annotated genes in the reference Willliams 82 genome. Of these genes, we identified two nonsynonymous single nucleotide polymorphisms (SNPs) that are present in the encoding regions of Gmdw1 and Glyma.08G165100 in dw, respectively. However, only the SNP mutation (T>A) at nucleotide 1224 in Gmdw1 cosegregated with the dwarf phenotype. GmDW1 encodes an ent-kaurene synthase, and was expressed in various tissues including root, stem, and leaf. Further phenotypic analysis of the allelic variations in soybean accessions strongly indicated that GmDW1 is responsible for the dwarf phenotype in dw. Our results provide important information for improving our understanding of the genetics of soybean plant height and crop breeding.

     

Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) JAZ3 and SLR1 function in jasmonate and gibberellin mediated epidermal cell differentiation and elongation

Jasmonate ZIM-domain (JAZ) proteins and DELLA proteins are key negative regulators of jasmonates (JAs) and gibberellin (GA) signaling, respectively. In this study, we found JA and GA synergistically promote fiber cell initiation. We characterized the cellular function of a JAZ protein (GhJAZ3), and a DELLA protein (GhSLR1) of cotton (Gossypium hirsutum). GhJAZ3 is specifically expressed in elongating fibers, while GhSLR1 is expressed in different tissues and at a relatively higher level in 3 DPA ovules. GhSLR1 and GhJAZ3 proteins are localized in the cell nucleus. Yeast two-hybrid analysis indicated that GhSLR1, GhJAZ3 and GhDEL65 could interact with each other, and GhSLR1 could also interact with GhBZR1. Overexpression of GhJAZ3 in Arabidopsis increased hypocotyl and root length, leaf trichome length, and plant height, but decreased the number of leaf trichome, while overexpression of GhSLR1 in Arabidopsis decreased hypocotyl length, leaf trichome length and density. Expression of several leaf trichome initiation determinators (GL3, GL2, TTG2 and MYB23) was down-regulated in GhJAZ3 or GhSLR1 transgenic Arabidopsis, while expression of the cell elongation related genes (EXP1, EXP8, EXPL2 and XTH4) was altered in the GhJAZ3 and GhSLR1 transgenic Arabidopsis. Taken together, these results demonstrate that GhJAZ3 and GhSLR1 function in jasmonate and gibberellin mediated epidermal cell differentiation and elongation.     

Engineering gibberellin metabolism in <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. by ectopic expression of gibberellin oxidase genes

Gibberellins (GAs) control many aspects of plant development, including seed germination, shoot growth, flower induction and growth and fruit expansion. Leaf explants of Solanum nigrum (Black Nightshade; Solanaceae) were used for Agrobacterium-mediated delivery of GA-biosynthetic genes to determine the influence of their encoded enzymes on the production of bioactive GAs and plant stature in this species. Constructs were prepared containing the neomycin phosphotransferase (nptII) gene for kanamycin resistance as a selectable marker, and the GA-biosynthetic genes, their expression under the control of the CaMV 35S promoter. The GA-biosynthetic genes comprised AtGA20ox1, isolated from Arabidopsis thaliana, the product from which catalyses the formation of C₁₉-GAs, and MmGA3ox1 and MmGA3ox2, isolated from Marah macrocarpus, which encode functionally different GA 3-oxidases that convert C₁₉-GAs to biologically active forms. Increase in stature was observed in plants transformed with AtGA20ox1, MmGA3ox2 and MmGA3ox1 + MmGA3ox2, their presence and expression being confirmed by PCR and RT-PCR, respectively, accompanied by an increase in GA₁ content. Interestingly, MmGA3ox1 alone did not induce a sustained increase in plant height, probably because of only a marginal increase in bioactive GA₁ content in the transformed plants. The results are discussed in the context of regulating plant stature, since this strategy would decrease the use of chemicals to promote plant growth.     

The <Emphasis Type="Italic">APX4</Emphasis> locus regulates seed vigor and seedling growth in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The amino acid sequence of APX4 is similar to other ascorbate peroxidases (APXs), a group of proteins that protect plants from oxidative damage by transferring electrons from ascorbate to detoxify peroxides. In this study, we characterized two apx4 mutant alleles. Translational fusions with GFP indicated APX4 localizes to chloroplasts. Both apx4 mutant alleles formed chlorotic cotyledons with significantly reduced chlorophyll a, chlorophyll b and lutein. Given the homology of APX to ROS-scavenging proteins, this result is consistent with APX4 protecting seedling photosystems from oxidation. The growth of apx4 seedlings was stunted early in seedling development. In addition, APX4 altered seed quality by affecting seed coat formation. While apx4 seed development appeared normal, the seed coat was darker and more permeable than the wild type. In addition, accelerated aging tests showed that apx4 seeds were more sensitive to environmental stress than the wild-type seeds. If APX4 affects seed pigment biosynthesis or reduction, the seed coat color and permeability phenotypes are explained. apx4 mutants had cotyledon chlorosis, increased H₂O₂ accumulation, and reduced soluble APX activity in seedlings. These results indicate that APX4 is involved in the ROS-scavenging process in chloroplasts.     

Molecular mapping of a rust resistance gene <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">14</Emphasis></Subscript> in cultivated sunflower line PH 3

Sunflower, the fifth largest oilseed crop in the world, plays an important role in human diets. Recently, sunflower production in North America has suffered serious yield losses from newly evolved races of sunflower rust (Puccinia helianthi Schwein.). The rust resistance gene, designated R ₁₄ , in a germplasm line PH 3 originated from a wild Helianthus annuus L. population resistant to 11 rust races. PH 3 has seedling with an extraordinary purple hypocotyl color. The objectives of this study were to map both the R ₁₄ rust resistance gene and the purple hypocotyl gene-designated PHC in PH 3, and to identify molecular markers for marker-assisted breeding for sunflower rust resistance. A set of 517 mapped SSR/InDel and four SNP markers was used to detect polymorphisms between the parents. Fourteen markers covering a genetic distance of 17.0 cM on linkage group (LG) 11 were linked to R ₁₄ . R ₁₄ was mapped to the middle of the LG, with a dominant SNP marker NSA_000064 as the closest marker at a distance of 0.7 cM, and another codominant marker ORS542 linked at 3.5 cM proximally. One dominant marker ZVG53 was linked on the distal side at 6.9 cM. The PHC gene was also linked to R ₁₄ with a distance of 6.2 cM. Chi-squared analysis of the segregation ratios of R ₁₄ , PHC, and ten linked markers indicated a deviation from an expected 1:2:1 or 3:1 ratio. The closely linked molecular or morphological markers could facilitate sunflower rust-resistant breeding and accelerate the development of rust-resistant hybrids.