Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2 mmol/L hydroxyurea for 18 h, a 4.5-h recovery in hydroxyurea-free medium, 2 h incubation with 10 micromol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20 min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4 x 10(5) morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The parity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.

• MeSH
• buněčný cyklus MeSH
• chromozomy rostlin genetika   MeSH
• Cicer genetika   MeSH
• cytogenetika MeSH
• DNA rostlinná genetika   metabolismus   MeSH
• fyzikální mapování chromozomů metody   MeSH
• genetická vazba MeSH
• genom rostlinný * MeSH
• hybridizace in situ fluorescenční MeSH
• indoly MeSH
• karyotypizace MeSH
• kořeny rostlin genetika   MeSH
• metafáze MeSH
• mikrosatelitní repetice MeSH
• místa se sekvenční adresou MeSH
• mitóza MeSH
• polymerázová řetězová reakce MeSH
• průtoková cytometrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DAPI MeSH Prohlížeč
• DNA rostlinná MeSH
• indoly MeSH

The dioecious white campion Silene latifolia (syn. Melandrium album) has heteromorphic sex chromosomes, XX in females and XY in males, that are larger than the autosomes and enable their separation by flow sorting. The group of MROS genes, the first male-specifically expressed genes in dioecious plants, was recently identified in S. latifolia. To localize the MROS genes, we used the flow-sorted X chromosomes and autosomes as a template for PCR with internal primers. Our results indicate that the MROS3 gene is located in at least two copies tandemly arranged on the X chromosome with additional copy(ies) on the autosome(s), while MROS1, MROS2, and MROS4 are exclusively autosomal. The specificity of PCR products was checked by digestion with a restriction enzyme or reamplification using nested primers. Homology search of databases has shown the presence of five MROS3 homologues in A. thaliana, four of them arranged in two tandems, each consisting of two copies. We conclude that MROS3 is a low-copy gene family, connected with the proper pollen development, which is present not only in dioecious but also in other dicot plant species.

• MeSH
• DNA primery MeSH
• hybridizace in situ fluorescenční MeSH
• Magnoliopsida genetika   MeSH
• molekulární sekvence - údaje MeSH
• pohlavní chromozomy * MeSH
• polymerázová řetězová reakce MeSH
• rostlinné geny * MeSH
• rostlinné proteiny chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA primery MeSH
• rostlinné proteiny MeSH