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Background <p><i>Ginkgo biloba</i> is a dioecious gymnosperm that has remained morphologically stable for over 200&#xa0;million years, offering a unique system for studying early plant reproductive evolution. However, the molecular mechanisms underlying its sex determination remain largely unknown due to its large genome and the lack of genetic transformation tools. The purpose of this study was to precisely map the sex-determining region and decipher the gene regulatory network governing sexual differentiation in this ancient species.</p> Results <p>Through bulked segregant analysis sequencing (BSA-seq) and transcriptomic profiling of male and female pools, we mapped a sex-determining region (SDR) to a nine-megabase interval on chromosome two. Within this region, a B-class MADS-box transcription factor, <i>GbAP3a</i>, was identified as a core candidate promoting male development. It exhibits strict male-specific expression in microstrobili, and its sequence and function are highly conserved due to tandem duplication and strong purifying selection. We also characterized a male-specific long noncoding RNA, <i>GbLINC-CNR1</i> (Cellular Negative Regulator 1), located within this region. This long noncoding RNA regulates sex differentiation via two parallel pathways: it represses the MADS transcription factor <i>GbAGL32a</i> to abort ovule development, and it acts as a competing endogenous RNA for microRNA 159 to prevent the degradation of <i>GbMYB33</i> transcripts, thereby sustaining pollen development.</p> Conclusions <p>Sex determination in <i>Ginkgo</i> is orchestrated by the functional divergence of two MADS-box genes, tightly integrated with dual regulatory inputs from a sex-linked long noncoding RNA. These findings provide mechanistic insights into the evolution of sex chromosomes and reproductive strategies in gymnosperms, offering potential molecular targets for early sex identification and marker-assisted breeding in dioecious trees.</p>

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Functional divergence of MADS-box genes and the dual regulatory role of a long noncoding RNA underlie sex determination in Ginkgo biloba

  • 期刊论文

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  • 发表日期：

    2026年7月10日

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  • Huaitong Wu,
  • Baoyu Zheng,
  • Kaiyuan Liu,
  • Tongming Yin,
  • Suyun Wei

摘要

Background

Ginkgo biloba is a dioecious gymnosperm that has remained morphologically stable for over 200 million years, offering a unique system for studying early plant reproductive evolution. However, the molecular mechanisms underlying its sex determination remain largely unknown due to its large genome and the lack of genetic transformation tools. The purpose of this study was to precisely map the sex-determining region and decipher the gene regulatory network governing sexual differentiation in this ancient species.

Results

Through bulked segregant analysis sequencing (BSA-seq) and transcriptomic profiling of male and female pools, we mapped a sex-determining region (SDR) to a nine-megabase interval on chromosome two. Within this region, a B-class MADS-box transcription factor, GbAP3a, was identified as a core candidate promoting male development. It exhibits strict male-specific expression in microstrobili, and its sequence and function are highly conserved due to tandem duplication and strong purifying selection. We also characterized a male-specific long noncoding RNA, GbLINC-CNR1 (Cellular Negative Regulator 1), located within this region. This long noncoding RNA regulates sex differentiation via two parallel pathways: it represses the MADS transcription factor GbAGL32a to abort ovule development, and it acts as a competing endogenous RNA for microRNA 159 to prevent the degradation of GbMYB33 transcripts, thereby sustaining pollen development.

Conclusions

Sex determination in Ginkgo is orchestrated by the functional divergence of two MADS-box genes, tightly integrated with dual regulatory inputs from a sex-linked long noncoding RNA. These findings provide mechanistic insights into the evolution of sex chromosomes and reproductive strategies in gymnosperms, offering potential molecular targets for early sex identification and marker-assisted breeding in dioecious trees.