Formation Characteristics Analysis Of The Endogenous Neocentromere On Chromosome 8 In Indica Rice

Centromere is essential for the faithful segregation of chromosomes in eukaryotes.Although centromere typically contains tandem repetitive DNA sequences,its function is the epigenetic regulation process which related to but not entirely rely on these DNA modifs.If canonical centromeric DNA sequences are lost,a new region without the typical centromeric DNA repeats will constitute a de novo-formed centromere,termed neocentromere,which was found in human,wheat-barley,oat-maize addition lines and maize.However,the forming feature of neocentromere has not been very clear yet.Especially,neocentromere has been rarely reported in normal diploid rice.In the previous study from our laboratory,it was identified that an indica rice line,derived from rice variety Zhongxian 3037,without centromeric reperitive sequences in the centromere of chromosome 8(Cen8)region.In addition,the mitosis and meiosis of chromosome 8 were totally normal suggesting the endogenous de novo centromere was formed.Based on this material,the present study continued to explore the formation characteristics of endogenous neocentromere on chromosome 8.The main results are as follows:1.By Oligo-FISH,the rice chromosome without centromere specific DNA sequences(CentO)was further identified as chromosome 8.The material was temporarily named Del 8,and the normal control was named Nor 8.2.Because of lacking applicable reference sequencing genome of Zhongxian 3037,a BAC library was constructed and the clones containing Cen8 sequences were selected by PCR.Then the cytological method was used to verify that the two BAC clones spanning the Cen8 region contained CentO sequences.3.The selected BAC clones were sequenced on the PacBio platform.The sequencing results showed that 12,412,6670 and 7585 reads were respectively obtained from the 3 BAC clones and the each length was successively 133,443bp,128,178bp and 103,389bp after assembly.Subsequently,these sequences were supposed to be assembled as reference.4.To make clear the location of the neocentromeric location and its structure characteristics,we performed CENH3-ChIP-seq(Centromere special histone H3-Chromatin Immunoprecipitation-sequencing)analysis.The result showed that neocentromere expanded to a 43kb region containing three expressed genes,locating at the long arm of chromosome 8 in Del 8,which indicate canonical histone H3 was replaced by CENH3 in this region.5.Combined with RNA-seq,the three genes showed low or no transcription in neocentromeric region,and another two high transcriptional genes without binding CENH3 were location between the deletion region and the neocentromere.These results revealed that the neocentromere expansion tend to choose the region which contains no genes or inactive genes and skip the region which contains active genes.6.Furture to explore epigenetic characteristics of genes in the de novo neocentromere,the levels of H3K36me3,H3K27ac,H4K5ac,H4K8ac,H4K12ac and H4K16ac on the three genes were analyzed by ChIP-qPCR.The ChIP-qPCR results showed that the levels of six histone modification on the three genes were almost decreased in Del 8.It was deduced that the binding of CENH3 altered histone modification.In short,our findings reveal the genomic and epigenetic characteristics of neocentromeric formation,and provide a powerful support for the study of eukaryotic centromere evolution.