Comparative Cytology Reserch Of Humulus Based On Repetitive Sequences

Repetitive sequences,including tandem repetitive sequences and transposons,are important driving forces for the evolution of genomes and the origin of sex chromosomes.Humulus scandens and Humulus lupulus of Humulus belong to dioecious plants with X and Y sex chromosomes,but their exact karyotype composition,sex chromosome composition of male and female plants and the origin mechanism of sex chromosomes are still unclear.Therefore,in this study,firstly,the repetitive sequences of Humulus and its related species Cannabis sativa were annotated and identified by Repeat Explorer,and different types of repeat sequences were located by metaphase chromosome fluorescence in situ hybridization(FISH).Thus,it provides important technical support and theoretical data for the study of karyotype and sex chromosome evolution of Humulus.The main results are as follows:1.The analysis of the composition and content of repetitive sequences in the genomes of Humulus by Repeat Explorer showed that there were a large number of repetitive sequences in the genomes of the three plants,of which the content in the genome of Humulus was the highest,but the repetitive sequence content of the two species in the genus Humulus varied greatly.For example,the repetitive sequence content of Humulus lupulus(59.9%)is closer to that of Cannabis sativa(56.7%)of the same family.Secondly,there was a small difference in repetitive sequence content between male and female plants,which was 71.4%in female and 70.4%in male plants.According to the types of repetitive sequences,the most abundant repeat sequences in the three plants were LTR retrotransposons.Among them,the contents of LTR/Gypsy retrotransposons in male and female Humulus scandens and Humulus lupulus were the highest,accounting for 77.5%of the total repetitive sequences,77.0%and 65.8%of the total repetitive sequences,respectively,and the subfamilies with the most content were Tekay and Retand retrotransposons.The most abundant LTR retrotransposon in the same family is LTR/Copia,accounting for 38.0%of its repetitive sequences,indicating that the composition of interspecific genomic repeat sequences in Humulus is more conservative.2.In order to analyze the specific position information of transposons in different subfamilies,we randomly selected 14 sequences from different subfamilies of LTR transposons from Humulus scandens,Humulus lupulus and reference species of Cannabis sativa for metaphase chromosome fluorescence in situ hybridization.The results showed that a total of four probes could obtain stable hybridization signals,in which the CRM retrotransposon hybridization signals were located in the centromere and its flanking region in two species of Humulus and their reference species Cannabis sativa.The Ikeros and Athila retrotransposon subfamilies were also mainly located in the centromere and its surrounding regions in Humulus,and the other retrotransposon subfamilies were mostly dispersed on the whole chromosome in the three species.It is worth noting that the distribution of the Athila subfamily of Gypsy retrotransposons is obviously different on the X and Y chromosomes of Humulus.It is only located in the centromere position on the X chromosome,but diffusely distributed on the whole chromosome in the two Y chromosomes,indicating that LTR retrotransposons may be involved in the evolution of Humulus sex chromosomes.3.Satellite DNA is one of the high-content repetitive sequences in the genome of Humulus.In order to analyze whether satellite DNA is also involved in the evolution of its sex chromosomes,we used the TARAEN function of Repeat Explorer software to analyze the satellite DNA in the genomes of Humulus scandens,Humulus lupulus and reference species of Cannabis sativa.The results showed that the content of satellite DNA in these three species was low,accounting for 1.87-2.02%of the genome,respectively.0.71%and 1.18%.We amplified the sequences of 8 highly reliable satellite DNA from three species of plants and performed fluorescence in situ hybridization(FISH)in metaphase chromosomes and meiosis.The results showed that there were significant differences in the distribution of satellite DNA on X and Y sex chromosomes,in which satellite DNA Hs CL33 was located at the centromere of all chromosomes in female plants,but not on Y₁and Y₂ sex chromosomes in male plants.The positions of satellite DNA Hs CL37 and Hs CL111 on the chromosomes of male and female plants are similar.They are located at the ends of other chromosomes except chromosome 7 in female plants,but only in one of the two Y chromosomes in male plants,while the other Y chromosome signal is also distributed at the end of one side like the X chromosome.The obvious signal of Humulus satellite DNA Hs CL111 was also hybridized at the end of Humulus lupulus,indicating that there is an interspecific conserved satellite DNA sequence in the genome of Humulus.The metaphase chromosome mapping of three satellite DNA in hops showed that except Hl CL239 only distributed on chromosome 3,the other two satellite DNA were located at the end of chromosome on autosomal.Secondly,satellite DNA Hl CL10 only had a hybridization signal on the short arm of X chromosome,and other signals of autosomal and Y chromosomes were only distributed in the end,so XY sex chromosomes could be identified in cytology.The hybridization signal of satellite DNA Cs CL2 of Cannabis sativa was distributed on both sides of most chromosomes of female plants,but only on the end of short arm of chromosome 9,and on the end of Y chromosome and centromere of male plants,while the obvious signal of X chromosome could only be observed at the end of chromosome 9.Thus it can be seen that the satellite DNA is mainly distributed in the terminal position on the chromosomes in the genomes of Humulus and its related species of Cannabis sativa.