Cloning and Characterization of Meiotic Genes Rec8 and Cdc5 and Subcellular Analysis of Kinetochore Orientation in Wheat

Abstract

Meiosis is a specialized cell division that halves chromosomes and generates haploid gametes in eukaryotes. It is a dynamic cellular process governed by a complex genetic network. Two key players of this network, Rec8 and Cdc5, were cloned and analyzed using comparative genomics and subcellular immunolocalization methodologies in wheat (Triticum turgidum L., genome AABB). TtRec8 and TtCdc5 were localized to group 1 and 5 chromosomes, with two homoeoalleles in sub-genome A and B, respectively. One of the two TtRec8 homoeoalleles, TtRec8-A1, contains 20 exons in a 6.5 kb-genomic DNA fragment, and the coding region encodes 608 amino acids. Two homoeoalleles of TtCdc5 separately encode 1,081 and 1,084 amino acids. The expression profilings of TtRec8 and TtCdc5 were meiotic tissue dominant in LDN, and the highest levels of TtRec8 and TtCdc5 were at interphase through early prophase I and at pachytene stage of meiosis, separately, and then decreased as meiosis proceeded. TtRec8 protein was detected along the entire chromosomes through the early stages of prophase I. Thereafter, TtRec8 protein was mostly removed from the chromosomes. The DNA sequences and conserved domains of TtRec8 and TtCdc5 as well as their kinetics through the meiotic process in LDN were very similar as the cohesion protein Rec8 and polo-like kinase Cdc5 in models, suggesting their specific roles in meiosis. Chromosome pairing (or synapsis) may play a role in kinetochore orientation during meiosis. Special genotypes that contained both paired (bivalents) and unpaired (univalent) chromosomes in the LDN background were constructed to determine the orientation of sister kinetochores in the univalent and bivalent chromosomes in meiosis I. Among the special genotypes included the hybrids from the crosses of the disomic LDN D-genome substitution lines LDN 1D(1B), LDN 2D(2A), LDN 2D(2B), LDN 3D(3A), LDN 4D(4B), LDN 6D(6A), LDN 6D(6B), LDN 7D(7A), and LDN 7D(7B) with LDN, LDN 1D(1A) with rye (Secale cereale L., genome RR) `Gazelle', LDN with Aegilops tauschii (genome DD) RL5286, and LDN 1D(1B) with Ae. tauschii RL5286. All univalents were found amphitelically orientated and all bivalents syntelically orientated at metaphase I, suggesting meiotic pairing mediates kinetochore orientation and subsequently chromosome segregation in LDN.