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Identification and analysis of DYAD: a gene required for meiotic chromosome organisation and female meiotic progression in Arabidopsis

Bhavna Agashe, Chellapilla Krishna Prasad^* and Imran Siddiqi

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007, India
^* Present address: University of Arkansas for Medical Sciences, 4301 West Markham, Slot No. 518, Little Rock, Arkansas-72205, USA

View larger version (76K): [in a new window]   Fig. 1. (A,B) Mature ovule of wild type (A) and dyad (B) plants. Bar: 10 µm. (C) Localisation of dyad on chromosome 5. The region between the markers nga129 and KMR gives the positions of DNA markers and number of crossovers in the respective intervals that were used to localise dyad. The portion of P1 clone MFG13 in which dyad was mapped to a 25 kb segment between the markers MDL and Mf3.2 is indicated below. The 25 kb region was subcloned as 2 SalI fragments (S1,S2) and 2 XbaI fragments (X1,X2) each of which was tested for complementation.

View larger version (31K): [in a new window]   Fig. 2. (A) Sequence analysis of the DYAD gene. The top panel is a box diagram representation of DYAD showing the positions of the exons. Arrowheads indicate positions of primers used for examination of expression by RT-PCR. The predicted sequence of the DYAD protein is given below, and the region showing similarity to MS1 is underlined. The putative nuclear localisation signal is in bold. The dyad mutation causes a frameshift at aa position 505. (B) Analysis of DYAD expression by RT-PCR. The left panel compares DYAD expression in leaves (Lea) and inflorescence (Inf) of wild type. The right panel examines expression of DYAD in the inflorescence of wild type (wt) and mutant (dy) plants.

View larger version (147K): [in a new window]   Fig. 3. Expression of DYAD in male and female meiocytes. RNA in situ hybridisation of DYAD antisense RNA to sections of flower buds. Expression is seen (A) in pollen mother cells and (B) in megaspore mother cells at an early stage of ovule development at the time of integument initiation. (C) Magnification of the distal portion of the upper two ovules shown in B. The signal appears localised towards the apical end of the MMC in both ovules. (D) Whole- mount optical section of a cleared ovule at the same developmental stage as in B,C. Bar: 100 µm (A,B) and 20 µm (C,D).

View larger version (78K): [in a new window]   Fig. 4. Chromosome analysis in various stages of female meiosis in wild type and dyad plants. (A-F) Wild type, (G-M) dyad. (A,G) Chromosomes first become visible as elongated strands during leptotene. (B) Synapsis takes place during zygotene and synapsed regions can be detected as thicker segments. (C) Synapsis is complete at pachytene and chromosomes have a shorter and thicker appearance. (D) Late diplotene stage, when bivalents have undergone partial decondensation of the arms but not centromeric regions and the two halves of bivalents can be seen. (E) Metaphase stage showing five condensed bivalents. (F) Dyad stage after completion of meiosis 1. Each of the two cells contains 5 univalents at prometaphase 2. (H) In dyad, 10 univalents are observed instead of 5 bivalents indicating failure to undergo synapsis. (I) The univalents undergo partial decondensation of the arms. (J) Metaphase plate containing 10 chromosomes. (K) Anaphase 1 resembles an equational division. (L) Dyad stage at prometaphase 2 after completion of meiosis 1. (M) Magnification of the upper cell of the dyad in L. The cell is estimated to contain 10 chromosomes (white arrows). Black arrowhead indicates a chromosome with a pair of sister chromatids suggesting an additional round of replication has occurred. Bar: 5 µm (J,M) and 10 µm (A-I,K,L).

View larger version (159K): [in a new window]   Fig. 5. Expression of pAtDMC1::GUS in wild-type and dyad plants. (A,B) Wild type. (C-E) dyad. (A,C) GUS expression is specific to the MMC in stage 2-4 ovules. (B) After meiosis expression is seen in degenerating spores but not in the developing embryo sac at ovule stage 3-2. (D) Pistil containing ovules from stages 2-5 onwards. Ovules towards the basal end of the pistil (right) are slightly more advanced in development than those at the stigmatic end (left). Several of the basal ovules show intense GUS expression in the division products of the MMC consistent with defective meiotic progression. (E) Four examples of division products of the MMC where persistent expression is seen in one or both of the cells. Bar: 20 µm (A-C); 50 µm (D); 10 µm (E).