Repetitive DNA sequences accelerate molecular cytogenetic research in plants with small chromosomes

Repetitive DNA sequences are highly abundant in plant genomes and are favorable probes for chromosome identification in plants. However, it is difficult to conduct studies on the details of metaphase chromosome structures in plants with small chromosomes due to their highly condensed status. Therefore, identification of homologous chromosomes for karyotyping and analyzing chromosome structures is a challenging issue for cytogeneticists without specific probes and precise chromosome stages. In this study, five repetitive DNA probes, i.e., 5S and 45S ribosomal DNAs (rDNAs), melon centromeric sequence (Cmcent), cucumber subtelomeric sequence (Type I), and microsatellite (CT)10 repeats, were used to identify primary constrictions and homologous chromosomes for karyotyping. Four and two loci of 45S rDNA were respectively observed on metaphase and pachytene chromosomes of Abelia × grandiflora. Cmcent was detected on both primary constrictions of melon pachytene and metaphase chromosomes. Furthermore, one pair of 5S rDNA signals were hybridized on melon metaphase chromosomes. Eight and two loci of 45S and 5S rDNA were respectively detected on cucumber chromosomes. Type I and (CT)10 probes were specifically hybridized on subtelomeric and interstitial regions on the chromosomes, respectively. These results suggest that repetitive DNA sequences are versatile probes for chromosome identification in plants with small chromosomes, particularly for karyotyping analyses

Duplication of a well-conserved homeodomain-leucine zipper transcription factor gene in barley generates a copy with more specific functions

Three spikelets are formed at each rachis node of the cultivated barley (Hordeum vulgare ssp. vulgare) spike. In two-rowed barley, the central one is fertile and the two lateral ones are sterile, whereas in the six-rowed type, all three are fertile. This characteristic is determined by the allelic constitution at the six-rowed spike 1 (vrs1) locus on the long arm of chromosome 2H, with the recessive allele (vrs1) being responsible for the six-rowed phenotype. The Vrs1 (HvHox1) gene encodes a homeodomain-leucine zipper (HD-Zip) transcription factor. Here, we show that the Vrs1 gene evolved in the Poaceae via a duplication, with a second copy of the gene, HvHox2, present on the short arm of chromosome 2H. Micro-collinearity and polypeptide sequences were both well conserved between HvHox2 and its Poaceae orthologs, but Vrs1 is unique to the barley tribe. The Vrs1 gene product lacks a motif which is conserved among the HvHox2 orthologs. A phylogenetic analysis demonstrated that Vrs1 and HvHox2 must have diverged after the separation of Brachypodium distachyon from the Pooideae and suggests that Vrs1 arose following the duplication of HvHox2, and acquired its new function during the evolution of the barley tribe. HvHox2 was expressed in all organs examined but Vrs1 was predominantly expressed in immature inflorescence

An improved method for inducing prometaphase chromosomes in plants

Background: Detailed karyotyping using metaphase chromosomes in melon (Cucumis melo L.) remains a challenge because of their small chromosome sizes and poor stainability. Prometaphase chromosomes, which are two times longer and loosely condensed, provide a significantly better resolution for fluorescence in situ hybridization (FISH) than metaphase chromosomes. However, suitable method for acquiring prometaphase chromosomes in melon have been poorly investigated. Results: In this study, a modified Carnoy's solution II (MC II) [6:3:1 (v/v) ethanol: acetic acid: chloroform] was used as a pretreatment solution to obtain prometaphase chromosomes. We demonstrated that the prometaphase chromosomes obtained using the MC II method are excellent for karyotyping and FISH analysis. We also observed that a combination of MC II and the modified air dry (ADI) method provides a satisfactory meiotic pachytene chromosome preparation with reduced cytoplasmic background and clear chromatin spreads. Moreover, we demonstrated that pachytene and prometaphase chromosomes of melon and Abelia × grandiflora generate significantly better FISH images when prepared using the method described. We confirmed, for the first time, that Abelia × grandiflora has pairs of both strong and weak 45S ribosomal DNA signals on the short arms of their metaphase chromosomes. Conclusion: The MC II and ADI method are simple and effective for acquiring prometaphase and pachytene chromosomes with reduced cytoplasm background in plants. Our methods provide high-resolution FISH images that can help accelerate molecular cytogenetic research in plants

Centromeres of Cucumis melo L. comprise Cmcent and two novel repeats, CmSat162 and CmSat189.

Centromeres are prerequisite for accurate segregation and are landmarks of primary constrictions of metaphase chromosomes in eukaryotes. In melon, high-copy-number satellite DNAs (SatDNAs) were found at various chromosomal locations such as centromeric, pericentromeric, and subtelomeric regions. In the present study, utilizing the published draft genome sequence of melon, two new SatDNAs (CmSat162 and CmSat189) of melon were identified and their chromosomal distributions were confirmed using fluorescence in situ hybridization. DNA probes prepared from these SatDNAs were successfully hybridized to melon somatic and meiotic chromosomes. CmSat162 was located on 12 pairs of melon chromosomes and co-localized with the centromeric repeat, Cmcent, at the centromeric regions. In contrast, CmSat189 was found to be located not only on centromeric regions but also on specific regions of the chromosomes, allowing the characterization of individual chromosomes of melon. It was also shown that these SatDNAs were transcribed in melon. These results suggest that CmSat162 and CmSat189 might have some functions at the centromeric regions

Identification of SFBB-Containing Canonical and Noncanonical SCF Complexes in Pollen of Apple (<i>Malus</i> × <i>domestica</i>)

<div><p>Gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a single polymorphic <i>S</i> locus. The <i>S</i> locus contains at least two genes, <i>S-RNase</i> and F-box protein encoding gene <i>SLF/SFB/SFBB</i> that control pistil and pollen specificity, respectively. Generally, the F-box protein forms an E3 ligase complex, SCF complex with Skp1, Cullin1 (CUL1) and Rbx1, however, in <i>Petunia inflata</i>, SBP1 (S-RNase binding protein1) was reported to play the role of Skp1 and Rbx1, and form an SCF^SLF-like complex for ubiquitination of non-self S-RNases. On the other hand, in <i>Petunia hybrida</i> and <i>Petunia inflata</i> of Solanaceae, <i>Prunus avium</i> and <i>Pyrus bretschneideri</i> of Rosaceae, SSK1 (SLF-interacting Skp1-like protein1) is considered to form the SCF^SLF/SFB complex. Here, we isolated pollen-expressed apple homologs of <i>SSK1</i> and <i>CUL1</i>, and named <i>MdSSK1, MdCUL1A</i> and <i>MdCUL1B</i>. <i>MdSSK1</i> was preferentially expressed in pollen, but weakly in other organs analyzed, while, <i>MdCUL1A</i> and <i>MdCUL1B</i> were almost equally expressed in all the organs analyzed. <i>MdSSK1</i> transcript abundance was significantly (>100 times) higher than that of <i>MdSBP1</i>. <i>In vitro</i> binding assays showed that MdSSK1 and MdSBP1 interacted with MdSFBB1-<i>S</i>⁹ and MdCUL1, and MdSFBB1-<i>S</i>⁹ interacted more strongly with MdSSK1 than with MdSBP1. The results suggest that both MdSSK1-containing SCF^SFBB1 and MdSBP1-containing SCF^SFBB1-like complexes function in pollen of apple, and the former plays a major role.</p></div

The Divergence of Chromosome Structures and 45S Ribosomal DNA Organization in Cucumis debilis Inferred by Comparative Molecular Cytogenetic Mapping

Cucumis debilis W.J.de Wilde &amp; Duyfjes is an annual and monoecious plant. This species is endemic to Southeast Asia, particularly Vietnam. However, C. debilis is rarely studied, and no detailed information is available regarding its basic chromosome number, 45S ribosomal DNA (rDNA) status, and divergence among other Cucumis species. In this study, we characterized the morphological characters and determined and investigated the basic chromosome number and chromosomal distribution of 45S rDNA of C. debilis using the fluorescent in situ hybridization (FISH) technique. A maximum likelihood tree was constructed by combining the chloroplast and internal transcribed spacer of 45S rDNAs to infer its relationship within Cucumis. C. debilis had an oval fruit shape, green fruit peel, and protrusion-like white spots during the immature fruit stage. FISH analysis using 45S rDNA probe showed three pairs of 45S rDNA loci located at the terminal region in C. debilis, similar to C. hystrix. Meanwhile, two, two, and five pairs of 45S rDNA loci were observed for C. melo, C. metuliferus, and C. sativus, respectively. One melon (P90) and cucumber accessions exhibited different chromosomal localizations compared with other members of Cucumis. The majority of Cucumis species showed the terminal location of 45S rDNA, but melon P90 and cucumber exhibited terminal&ndash;interstitial and all interstitial orientations of 45S rDNA loci. Based on molecular cytogenetics and phylogenetic evidence, C. debilis is more closely related to cucumber than melon. Therefore, C. debilis may serve as a potential parental accession for genetic improvement of cucumber through interspecific hybridization