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Cloning and physical localization of male-biased repetitive DNA sequences in Spinacia oleracea (Amaranthaceae).

ABSTRACT: Spinach (Spinacia oleracea Linnaeus, 1753) is an ideal material for studying molecular mechanisms of early-stage sex chromosome evolution in dioecious plants. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) technique facilitates the retrotransposon-relevant studies by enriching specific repetitive DNA sequences from a micro-dissected single chromosome. We conducted genomic subtractive hybridization to screen sex-biased DNA sequences by using the DOP-PCR amplification products of micro-dissected spinach Y chromosome. The screening yielded 55 male-biased DNA sequences with 30 576 bp in length, of which, 32 DNA sequences (12 049 bp) contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats, and DNA/CMC-EnSpm. Among these repetitive DNA sequences, four DNA sequences that contained a fragment of Ty3-gypsy retrotransposons (SP73, SP75, SP76, and SP77) were selected as fluorescence probes to hybridization on male and female spinach karyotypes. Fluorescence in situ hybridization (FISH) signals of SP73 and SP75 were captured mostly on the centromeres and their surrounding area for each homolog. Hybridization signals primarily appeared near the putative centromeres for each homologous chromosome pair by using SP76 and SP77 probes for FISH, and sporadic signals existed on the long arms. Results can be served as a basis to study the function of repetitive DNA sequences in sex chromosome evolution in spinach.

SUBMITTER: Zhou J

PROVIDER: S-EPMC8087614 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDioecious spinach (Spinacia oleracea L.), a commercial and nutritional vegetable crop, serves as a model for studying the mechanisms of sex determination and differentiation in plants. However, this mechanism is still unclear. Herein, based on PacBio Iso-seq and Illumina RNA-seq data, comparative transcriptome analysis of male and female flowers were performed to explore the sex differentiation mechanism in spinach.ResultsCompared with published genome of spinach, 10,800 transcripts were newly annotated; alternative splicing, alternative polyadenylation and lncRNA were analyzed for the first time, increasing the diversity of spinach transcriptome. A total of 2965 differentially expressed genes were identified between female and male flowers at three early development stages. The differential expression of RNA splicing-related genes, polyadenylation-related genes and lncRNAs suggested the involvement of alternative splicing, alternative polyadenylation and lncRNA in sex differentiation. Moreover, 1946 male-biased genes and 961 female-biased genes were found and several candidate genes related to gender development were identified, providing new clues to reveal the mechanism of sex differentiation. In addition, weighted gene co-expression network analysis showed that auxin and gibberellin were the common crucial factors in regulating female or male flower development; however, the closely co-expressed genes of these two factors were different between male and female flower, which may result in spinach sex differentiation.ConclusionsIn this study, 10,800 transcripts were newly annotated, and the alternative splicing, alternative polyadenylation and long-noncoding RNA were comprehensively analyzed for the first time in spinach, providing valuable information for functional genome study. Moreover, candidate genes related to gender development were identified, shedding new insight on studying the mechanism of sex determination and differentiation in plant.

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Cloning and physical localization of male-biased repetitive DNA sequences in Spinacia oleracea (Amaranthaceae).

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