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.

中文翻译：

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菠菜（A菜）中男性偏向的重复DNA序列的克隆和物理定位

菠菜（Spinacia oleracea Linnaeus，1753年）是研究雌雄异株植物早期性染色体进化的分子机制的理想材料。简并寡核苷酸引发的聚合酶链反应（DOP-PCR）技术通过从显微切割的单个染色体中富集特定的重复DNA序列，促进了与反转座子相关的研究。我们进行了基因组消减杂交，通过使用微解剖的菠菜Y染色体的DOP-PCR扩增产物来筛选性别偏爱的DNA序列。筛选产生了55个男性偏爱的DNA序列，长度为30576 bp，其中32个DNA序列（12049 bp）包含重复的DNA序列，包括LTR / Copia，LTR / Gypsy，简单重复和DNA / CMC-EnSpm 。在这些重复的DNA序列中，选择包含Ty3-gypsy逆转座子片段的四个DNA序列（SP73，SP75，SP76和SP77）作为荧光探针，与男性和女性菠菜核型杂交。SP73和SP75的荧光原位杂交（FISH）信号主要捕获在着丝粒及其周围区域的每个同源物上。通过使用FISH的SP76和SP77探针，杂交信号主要出现在每个同源染色体对的假定着丝粒附近，而长臂上则存在零星信号。研究结果可作为研究菠菜性染色体进化中重复DNA序列功能的基础。SP73和SP75的荧光原位杂交（FISH）信号主要捕获在着丝粒及其周围区域的每个同源物上。通过使用FISH的SP76和SP77探针，杂交信号主要出现在每个同源染色体对的假定着丝粒附近，而长臂上则存在零星信号。研究结果可作为研究菠菜性染色体进化中重复DNA序列功能的基础。SP73和SP75的荧光原位杂交（FISH）信号主要捕获在着丝粒及其周围区域的每个同源物上。通过使用FISH的SP76和SP77探针，杂交信号主要出现在每个同源染色体对的假定着丝粒附近，而长臂上则存在零星信号。研究结果可作为研究菠菜性染色体进化中重复DNA序列功能的基础。