The Fine Physical Mapping Of Rice Chromosome 4 And The Structural Analysis Of The Rice Centromere 4 Centromere

As part of an international effort to completely sequence the rice genome, we have produced a fine bacterial artificial chromosome (BAC)-based physical map of the Oryza sativa japonica Nipponbare chromosome 4 through an integration of 114 sequenced BAC clones from a taxonomically related subspecies O. sativa indica Guangluai 4 and 182 RFLP and 407 expressed sequence tag (EST) markers with the fingerprinted data of the Nipponbare genome and other physical map construction approaches. The map consisted of 4 contigs with a total length of 35.78 Mb and covered 99% of the whole chromosome size, including the entire centromere region. BAC clones corresponding to telomeres, as well as to the centromere position and the gap sizes between contigs, were determined by BAC-pachytene chromosome fluorescence in situ hybridization (FISH). The genetic-to-physical distance ratio and FISH analysis indicated that the short arm and the pericentromeric region of the long arm were recombinationally inactive regions and rich in heterochromatin, which occupied 45% of the chromosome, indicating that this chromosome is likely very difficult to sequence. To our knowledge, this map provided the first example of a rapid and reliable physical mapping on the basis of the integration of the data from two taxonomically related subspecies. With this strategy, we also rapidly constructed another chromosome 4 physical map of indica Guangluai 4 and have covered ~65% chromosome region.We performed some genome-wide comparative analysis of the DNA sequences of these two major cultivated rice subspecies, indica and japonica. The sequence alignment between 2.3 Mb collinear sequences revealed their extensive microcolinearity in gene order and content. The deviations from colinearity are frequent owing to insertions/deletions and single nucleotide polymorphisms (SNP). These variations may have some effect on gene structures and gene expression, and then contribute to intraspecific phenotypic diversities.A complete sequence of a chromosome centromere is very necessary for fully understanding centromere function. We reported the sequence structures of the first complete rice chromosome centromere through identifying and sequencing a large BAC clone-based contig, which covered the rice chromosome 4 centromere. Complete sequencing of the 124-kb rice chromosome 4 centromere revealed that it consisted of eighteen tracts of 379 tandemly arrayed repeats known as CentO and a total of nineteen centromeric retroelements (CRs) but no unique sequences were detected. Eighteen CentO tracts were flanked by nineteen retroelements and four tracts, composed of 65 CentO repeats, were located in the opposite orientation. The CRs were classified into four types, and the type I retroelements appeared to be more specific to rice centromeres. The preferential insert of the CRs among CentO repeats indicated that the centromere-specific retroelements may contribute to centromere expansion during evolution. The presence of three intact retrotransposons in the centromere suggests that they may be responsible for functional centromere initiation through a transcription-mediated mechanism. The complete sequence of rice chromosome 4 is the first centromere sequence in higher eukaryotic species. The structural information may shed some light on the functional dissection of a centromere and it is also useful for constructing rice or plant artificial chromosomes.