<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Genome-wide comparative analysis of <Emphasis Type="Italic">LEAFY</Emphasis> promoter sequence in angiosperms

Regulation of the flowering mechanism is influenced by many environmental factors. Dissecting the regulatory processes upstream of the LFY (LEAFY) gene will help us to understand the molecular mechanisms of floral induction. In total, 53 LFY sequences were identified in 37 species. Among the 53 selected LFY promoters and after eliminating the short sequences, 47 LFY promoters were analyzed. Comparative genome studies for LFY promoters among plants showed that TATA-box existed in all herbaceous plants. The 1345-bp promoter sequence upstream to hickory LFY gene was cloned and analyzed, together with functional studies. The result of sequence alignment showed that the region of the hickory LFY promoter has only two conserved auxin response elements (AuxRE), whereas other plants had four. The positions of AuxRE in hickory and walnut were the same, but they were different from the positions from other plants. Furthermore the sequence analysis showed that the promoter have TATA-box and CAAT-box motifs. Deletion analysis of these motifs did not block β-glucuronidase (GUS) activity during the transient expression assay, suggesting that it may be a TATA-less promoter. Low temperature and light significantly induced the full-length promoter to increase about two folds of the GUS enzymatic activity, suggesting these environmental factors induced flowering in hickory.     

Characterisation of <Emphasis Type="Italic">Thinopyrum bessarabicum</Emphasis> chromosomes through genome-wide introgressions into wheat

Key message

Genome-wide introgressions of Thinopyrum bessarabicum into wheat resulted in 12 recombinant lines. Cytological and molecular techniques allowed mapping of 1150 SNP markers across all seven chromosomes of the J genome.

Abstract

Thinopyrum bessarabicum (2n = 2x = 14, JJ) is an important source for new genetic variation for wheat improvement due to its salinity tolerance and disease resistance. Its practical utilisation in wheat improvement can be facilitated through development of genome-wide introgressions leading to a variety of different wheat–Th . bessarabicum translocation lines. In this study, we report the generation of 12 such wheat–Th . bessarabicum recombinant lines, through two different crossing strategies, which were characterized using sequential single colour and multi-colour genomic in situ hybridization (sc-GISH and mc-GISH), multi-colour fluorescent in situ hybridization (mc-FISH) and single nucleotide polymorphic (SNP) DNA markers. We also detected 13 lines containing different Th. bessarabicum chromosome aberrations through sc-GISH. Through a combination of molecular and cytological analysis of all the 25 lines containing Th. bessarabicum recombinants and chromosome aberrations we were able to physically map 1150 SNP markers onto seven Th. bessarabicum J chromosomes which were divided into 36 segmental blocks. Comparative analysis of the physical map of Th. bessarabicum and the wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed that Th. bessarabicum contains the 4/5 translocation also present in the A genome of wheat. These wheat–Th . bessarabicum recombinant lines and SNP markers provide a useful genetic resource for wheat improvement with the latter having a wider impact as a tool for detection of introgressions from other Thinopyrum species containing the J or a closely-related genome such as Thinopyrum intermedium (J^rJ^rJ^vsJ^vsStSt) and Thinopyrum elongatum (E^eE^e), respectively.