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Article

Sex Identification and Male–Female Differences in Ginkgo Biloba Hybrid F1 Generation Seedlings

by
Xiaoge Gao
1,
Yaping Hu
1,2,
Fangdi Li
1,
Fuliang Cao
1 and
Qirong Guo
1,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
2
Key Laboratory of Plant Innovation and Utilization, Institute of Subtropical Crops of Zhejiang Province, Wenzhou 325005, China
*
Author to whom correspondence should be addressed.
Forests 2024, 15(9), 1636; https://doi.org/10.3390/f15091636
Submission received: 7 August 2024 / Revised: 1 September 2024 / Accepted: 13 September 2024 / Published: 16 September 2024
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
In exploring the male–female differentiation and differences in the seedling stage of the F1 generation of ginkgo hybrids, an early selection test for the cultivation and research of leafy or medicinal ginkgo industry was conducted, which may provide a basis for boosting the precision of the ginkgo industry. The hybrid F1 generation obtained from the cross-mating was used as material for the determination of growth and development, as well as of the physiology and biochemistry of the monocots, and the male and female differential genes were obtained based on the data of SNPs obtained from the GBS sequencing of the hybrid progeny. In the seedling stage of ginkgo hybrid offspring, male plants had a significantly higher nutrient growth capacity than female plants, while the total flavonoid and terpene lactone contents in female plants were higher than those of the male plants. This result can provide a corresponding theoretical basis for the use of ginkgo germplasm resources, which can make full use of the male and female differences in the seedling stage and maximize the benefits of early sex identification.

1. Introduction

Whether angiosperms or gymnosperms, dioecious plants exhibit sex differentiation and distinct differences in growth, development, physiology, biochemistry, and gene expression due to reproductive differences. In recent years, research has delved deeper into early sex identification in plants [1,2], the mechanisms of sex regulation [3,4], sexual dimorphism [5,6], and differences in stress resistance between males and females under various adversities [7].
In the study of sexual dimorphism in plants, the sources of male and female specimens generally fall into two categories. One involves randomly selecting pairs from natural populations, while the other involves using male and female specimens from the same family line. The first method can eliminate the positional effects and spatial heterogeneity of male and female plants, but since dioecious plants in natural environments are outcrossing species with frequent genetic exchanges, there is considerable genetic variation between different individuals. The second method, compared to the first, can reduce genetic differences between male and female individuals. However, since the male and female parents in the same family line may not be consistent due to hybridization, there can still be some genetic inconsistencies. Therefore, in the study of sexual dimorphism in plants, the best approach is to fix the hybrid parents and conduct the research within a full-sibling family. This ensures a consistent genetic background for both male and female specimens.
Ginkgo biloba L. is a dioecious, wind-pollinated plant, and research on the differences between male and female ginkgo trees has consistently attracted the attention of scientists. Studies on the sexual dimorphism of ginkgo mainly focus on transcriptional differences between male and female trees [8], differences in antioxidant capacities [9], and the identification of male and female trees [10,11]. However, past studies have faced the issue of inconsistent genetic backgrounds in the male and female specimens used. These studies primarily focused on adult ginkgo trees that had already begun flower bud differentiation or used grafted plants obtained from branches of adult ginkgo trees. Since research on sex identification in ginkgo seedlings has only recently made breakthroughs, there is no precedent for using full-sibling families of ginkgo to identify sex at the seedling stage and subsequently study the differences between male and female ginkgo trees.
In this study, PCR primers designed from specific regions on the Y chromosome of male ginkgo trees were used to perform sex identification in seedlings from controlled hybridization experiments. This study also analyzed the differences between male and female offspring in terms of growth and development, physiological and biochemical characteristics, and whole-genome SNPs. This comprehensive analysis aimed to further elucidate the characteristics and potential mechanisms underlying the differences between male and female ginkgo trees.

2. Materials and Methods

2.1. Hybrid F1 Leaf Sample Collection

Starting around 20 March 2018, observations of male plant flower bud development were conducted every morning and evening. When the male flowers began to turn yellow, pollen collection started. Before 9:00 in the morning on sunny days, the inflorescences were cut, placed on sulfuric acid paper for shade drying, and pollen was collected by sieving and stored in a refrigerator at 4 °C for later use. The female parents used for hybridization were all located in the Chinese ginkgo seed orchard, with each female parent having three replicates. Before pollen shedding from the male plants, the branches of the female parents were bagged with anhydrous sulfuric acid paper. After collecting pollen from the male plants, artificial pollination was performed uniformly. The bags were resealed immediately after pollination and opened five days later. Hybrid combinations are listed in Table 1. Starting in mid-October of the same year, seeds from the controlled hybridization were collected and subjected to cold stratification to promote germination. Seeds collected from a naturally pollinated ginkgo tree (CK) were also subjected to the same treatment for germination.
The F1 and CK were sown at the China Ginkgo Seed Base (118.03° E, 34.39° N), and a free-pollinated ginkgo seed plant was collected at the same time and subjected to the same treatments (CK). Three years later, the young leaves of all the individual plants in the CK and F1 populations were collected and transferred to the laboratory in dry-ice sampling cassettes. They were stored in an ultra-low temperature refrigerator at −80 °C for subsequent DNA extraction and sequencing. In addition, nine male and female Ginkgo biloba leaves were collected from the campus of Nanjing Forestry University to verify the validity of the primers.

2.2. DNA Extraction, PCR Amplification, and Electrophoresis of the Hybrid F1

The DNA of all hybrid offspring was extracted using the HiPure SF Plant DNA Maxi Kit® (Magen, Guangzhou, China). The specific PCR primer sequences for the key ginkgo sex-regulating gene GbMADS18 were provided by Yunpeng Zhao’s research team at Zhejiang University [12]. The primer sequences were GbSD-F: 5′-TCAAGACACATAAAAACGCAATG-3′, GbSD-R: 5′-GGTGCATTCAACATTGTACACC-3′(DynaScience Biotechnology (Shanghai) Co., Shanghai, China). PCR was amplified using Applied Biosystem’s PCR instrument. The total PCR reaction system was 25 μL, to which 1 μL of primer, 22 μL of T3 Super PCR Mix (Beijing Tsingke Biotech Co., Ltd.), and 1 μL of DNA Template were added. The reaction conditions were 98 °C for 3 min (98 °C, 10 s; 58 °C, 10 s; 72 °C, 20 s) × 35 cycles and finally, 72 °C for 2 min to end the reaction.

2.3. Determination of F1 Growth and Physiological and Biochemical Indices

2.3.1. Determination of Growth in the F1 Population of Hybrids

During the seedling growth period, each individual plant in the F1 population was measured monthly using a steel tape measure and a vernier caliper. In October of the same year, after the seedlings had completed their growth period, the steel tape measure was used to assess the growth of new shoots for each individual plant.

2.3.2. Determination of Physiological and Biochemical Indices in F1 Hybrid Populations

A CIRAS-3 portable photosynthesis system (PP Systems, Amesbury, MA, USA) was used to measure the photosynthetic characteristics and light response curves of two-year-old seedlings from both the hybrid parents and the F1 population.
The soluble sugar content in ginkgo leaves was measured using the anthrone colorimetric method [13]. Chlorophyll was extracted from ginkgo leaves using a 95% ethanol extraction method, and chlorophyll content was calculated using a spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan) [14]. The soluble protein content in ginkgo leaves was determined using the Coomassie Brilliant Blue G-250 method. Flavonoid and ginkgolide contents were measured using HPLC [15].

2.4. Genotyping-by-Sequencing and Specific Gene Enrichment Analysis

GBS libraries were sequenced on the Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd., (Guangzhou, China). The filtered reads were aligned to the latest Ginkgo reference genome [16] using the mem algorithm of the alignment software BWA [17] (v 0.7.12) with the alignment parameter of -k 32 -M. The results of the alignment were tagged using the software PICARD (v1.129). Population SNPs were detected using the variant detection software GATK [18] (v.3.4-46), and the functional annotation of the detected variant was performed using ANNOVAR [19] (v. 2).
The gene IDs of the target gene set were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database terms. The number of genes for each term was calculated, and hypergeometric testing was used to define KEGG terms that are significantly enriched in the target gene set compared to the background genes. The enrichment results were visualized using TBtools-II (v2.119) [20].
All of the raw sequencing data were submitted to the National Genomics Data Center (NGDC) with accession number PRJCA008950, ensuring that the data are publicly accessible and reproducible.

2.5. Data Processing

All raw data were organized using Microsoft Excel 2021, statistical analyses were performed using SPSS 24.0, and plots were generated with Origin 2021 and R (v4.2.0). The significance of the differences between males and females was assessed using an Independent Sample t-Test, with a significance level of p < 0.05.

3. Results

3.1. F1 Male-to-Female Segregation Ratio

Specific primers were designed for a unique 2.7 Mb region on the Y chromosome of ginkgo No. 2, which effectively differentiates the sex of the plant. PCR validation was performed on ginkgo with known sex using these primers. The amplification product from male samples consistently showed a 617 bp band, while no such band was present in female samples (Figure 1a). This indicates that the primers can accurately be used for sex identification in ginkgo, meeting the requirement for early sex determination in F1 progeny.
PCR amplification using GbSD primers was conducted on 798 F1 ginkgo plants to identify their sex (Figure 1b). Statistics showed that the sex ratio of males and females in the total offspring population was 56:44. This means that females accounted for 44% of the total, while males made up 56% (Figure 1). The proportion of female plants in each progeny group ranged from 38% to 44%. Among them, the A3 group had the lowest proportion of female plants (38%), while the B3 group had the highest proportion of female plants (44%), with both groups having a majority of males.

3.2. Male and Female Differences in the Growth of Ginkgo F1

In the entire F1 population, the average seedling height of male plants was 78.69 ± 3.74 cm, which was significantly higher than the average seedling height of female plants (70.36 ± 4.13 cm, Figure 2b). The average seedling height of male plants was 1.84% that of female plants. In all hybrid progeny groups, male seedlings were taller than female seedlings. Except for the C2 and CK groups, the differences in seedling height between male and female plants in other groups were statistically significant (p = 0.042 < 0.05).
The average stem diameter of male ginkgo seedlings was also greater than that of female seedlings (Figure 2c). The average stem diameter of male plants was 11.27 ± 1.16 mm, significantly higher than the average stem diameter of female plants (9.89 ± 0.76 mm). The average stem diameter of male plants was 113.95% of that of female plants. Unlike the seedling height, the average stem diameter of male plants was slightly lower than that of female plants in the B1 and C2 groups. In contrast, the average stem diameter of male plants was significantly larger than that of female plants in the A1, A2, A3, and C3 groups.
The average new-shoot growth of male ginkgo seedlings was greater than that of female seedlings (Figure 2d). The average new-shoot growth for male plants was 23.09 ± 1.97 cm, significantly higher than the average new-shoot growth of female plants (21.49 ± 1.56 cm). The average new-shoot growth of male plants was 107.46% of that of female plants. In all hybrid progeny groups, male plants exhibited greater new-shoot growth than female plants. Except for the A2, B1, C2, and CK groups, the differences in the average new-shoot growth between males and females in other groups were statistically significant (p = 0.038 < 0.05).
Overall, male ginkgo seedlings exhibit significantly higher vegetative growth capacity compared to female seedlings during the nursery stage.

3.3. Male and Female Differences in Photosynthesis in Ginkgo F1

In the F1 population, the average net photosynthetic rate of male ginkgo plants was significantly higher than that of female plants (Figure 3). The average net photosynthetic rate for male plants was 3.41 ± 0.22 μmol CO2·m−2s−1, which is 117.65% of the average net photosynthetic rate of female plants (2.89 ± 0.24 μmol CO2·m−2s−1). Among the groups, males in the A1, A2, B2, and B3 groups showed significantly higher net photosynthetic rates compared to females, while the differences in other groups were not significant.
In the F1 population, there were no significant differences in the average transpiration rate, stomatal conductance, and intercellular CO2 concentration between male and female ginkgo (p = 0.041 < 0.05). The average transpiration rate and stomatal conductance of male plants were slightly lower than those of female plants. Specifically, the average transpiration rate of male plants was 22.98 ± 1.61 μmol H2O·m−2s−1, which is 90.99% of the female plants’ average rate (25.26 ± 2.27 μmol H2O·m−2s−1). The average stomatal conductance of male plants was 0.70 ± 0.05 mmol·m−2s−1, which is 97.15% of the female plants’ average (0.72 ± 0.06 mmol·m−2s−1). Conversely, the average intercellular CO2 concentration of male plants was slightly higher than that of female plants, with male plants at 223.61 ± 15.62 μmol·m−2s−1, which is 101.39% of the female plants’ average (220.54 ± 15.62 μmol·m−2s−1).
Overall, during the nursery stage, male ginkgo seedlings have higher net photosynthetic rates compared to female seedlings, while other photosynthetic parameters do not show significant differences.

3.4. Male and Female Differences in Chlorophyll Content of Ginkgo F1

In the F1 population, the average total chlorophyll, chlorophyll a, and chlorophyll b content in male ginkgo were significantly higher than in female plants (p = 0.039 < 0.05, Figure 4). The average total chlorophyll content in male plants was 2.18 ± 0.19 mg·g−1 FW, which is 108.79% of the content in female plants (2.01 ± 0.12 mg·g−1 FW). Significant differences in total chlorophyll content were observed between males and females in the A1, A2, B1, B2, and CK groups. The patterns of differences in chlorophyll a and chlorophyll b content followed the same trend as total chlorophyll. Specifically, the average chlorophyll content in male plants (1.11 ± 0.10 mg·g−1 FW) was 120.91% of that in female plants (0.92 ± 0.06 mg·g−1 FW), while the average chlorophyll b content in male plants (0.58 ± 0.05 mg·g−1 FW) was 144.60% of that in female plants (0.40 ± 0.02 mg·g−1 FW).
In the progeny, there were no significant differences in carotenoid content between male and female ginkgo plants. Except for the A1 and A2 groups, where male plants had significantly higher carotenoid content than female plants, no significant differences were observed across the other F1 groups. In the A2, B1, B3, and C3 groups, the average carotenoid content in female plants was higher than in male plants.
Overall, during the nursery stage, male ginkgo seedlings had higher total chlorophyll, chlorophyll a, and chlorophyll b content compared to female seedlings, while carotenoid content did not show significant differences.

3.5. Male and Female Differences in Soluble Sugar Content of Ginkgo F1

In all hybrid progeny of ginkgo, the average soluble sugar content in male plants was 8.77 ± 0.62 mg·g−1, which was significantly higher than that in female plants (7.81 ± 0.69 mg·g−1, p = 0.021 < 0.05, Figure 5a). The average soluble sugar content in male plants was 112.35% of that in female plants. In progeny groups A1, A3, B1, and B2, male plants had a higher average soluble sugar content than female plants. In the remaining groups, the differences in the average soluble sugar content between males and females were not statistically significant. The largest difference between sexes was observed in the A1 group, where the average soluble sugar content in male plants was 123.57% of that in female plants.
Soluble sugars play a crucial role in plant growth and development by providing the necessary energy and metabolic intermediates [21]. The faster growth and development of male ginkgo seedlings compared to females is consistent with their higher soluble sugar content. High soluble sugar content is one of the key factors supporting the faster growth and development rate of male ginkgo.
Overall, during the nursery stage, male ginkgo seedlings have significantly higher soluble sugar content compared to female seedlings.

3.6. Male and Female Differences in Soluble Protein Content of Ginkgo F1

In all hybrid progeny of ginkgo, the pattern of differences in soluble protein between male and female plants is consistent with the differences observed in soluble sugar content. The average soluble protein content in male plants across all progenies was 0.92 ± 0.07 mg·g−1, significantly higher than that in female plants (0.76 ± 0.05 mg·g−1, p = 0.030 < 0.05, Figure 5b). Male plants had 121.15% of the soluble protein content found in female plants. In progeny groups, except for B2 and B3, where the average soluble protein content in female plants was not significantly lower than that in males, all other groups showed significantly lower average soluble protein content in female plants. The greatest difference was observed in the CK group, where the average soluble protein content in male plants was 139.72% of that in female plants.
Soluble proteins are crucial for enhancing plant resistance and are an important source of nutrients. The higher average soluble protein content in male ginkgo seedlings indicates that they have stronger stress resistance. The elevated levels of both soluble protein and soluble sugar in male plants may collectively contribute to their faster growth and development. Overall, male ginkgo seedlings have significantly higher soluble protein content compared to female seedlings during the nursery stage.

3.7. Male and Female Differences in Total Flavonoid Content of Ginkgo F1

Identifying the differences in total flavonoid content between male and female ginkgo plants has significant implications for the development and advancement of the flavonoid industry. The comparison shows that in hybrid progeny, the average total flavonoid content in male plants was 16.93 ± 1.04 mg·g−1, which is significantly lower than the average content in female plants (18.89 ± 1.47 mg·g−1, p = 0.044 < 0.05, Figure 5c). The average total flavonoid content in male plants was 89.60% of that in female plants. In progeny groups A2, B2, B3, and CK, the average total flavonoid content in female plants was significantly higher than in male plants, while differences were not significant in the other groups. The greatest difference was observed in the B1 group, where the average total flavonoid content in female plants was 113.74% of that in male plants.
Identifying the sex of ginkgo seedlings during the nursery stage can enable targeted cultivation of male plants for applications such as medicinal leaf orchards, which can effectively enhance economic benefits. Overall, female ginkgo seedlings have significantly higher total flavonoid content compared to male seedlings during the nursery stage.

3.8. Male and Female Differences in the Terpene Lactone Content of Ginkgo F1

The pattern of differences in terpenoid lactone content between male and female ginkgo seedlings does not align with the pattern observed for total flavonoid content. In hybrid progeny, the average terpenoid lactone content in male plants was 11.04 ± 0.98 mg·g−1, which was lower than the average content in female plants (11.56 ± 0.89 mg·g−1, Figure 5d), but the difference was not statistically significant. The average terpenoid lactone content in male plants was 95.52% of that in female plants. Significant differences in terpenoid lactone content between sexes were observed in the B2, B3, C2, and C3 groups, where female plants had higher average content compared to males, but no significant differences were noted in other groups. Notably, in the A3 group, the terpenoid lactone content in female plants (12.56 ± 1.11 mg·g−1) was slightly lower than that in male plants (12.65 ± 1.18 mg·g−1).
Overall, during the nursery stage, female ginkgo seedlings have higher terpenoid lactone content than male seedlings, but the difference does not reach statistical significance.

3.9. Male and Female Specific Genes and Functional Annotation of Ginkgo F1

Based on the SNP data obtained from GBS sequencing of the hybrid progeny, the genes annotated to the female and male populations in the progeny were compared, in which there were a total of 48 endemic genes in the female population and a total of 64 endemic genes in the male population (Figure 6a, Table S1). To further analyze the differences between male and female ginkgo, KEGG enrichment analysis was carried out for male- and female-specific genes (Figure 6b,c). The results showed that female ginkgo-specific genes were mainly enriched in caffeine metabolism, aminoacyl-tRNA biosynthesis selenocompound metabolism, and selenocompound metabolism; male ginkgo-specific genes were mainly enriched in thiamine metabolism, Glycosylphosphatidylinositol, and KEGG pathways such as pantothenate and CoA biosynthesis.

4. Discussion

In this study, we further analyzed the differences between males and females after identifying the sex of F1 progeny at the seedling stage (Figure 7). Using PCR primers designed based on unique regions of the ginkgo Y chromosome, we performed the sex identification of ginkgo hybrid progeny at the seedling stage. The results show that 44% of the progeny were female and 56% were male. In nature, the sex ratio of plants is usually 1:1, but there are also instances of imbalance [22]. In nature, the sex ratio of Salix polaris and Premna herbacea is approximately 6:4 [23].
Based on the determination of soluble sugars and soluble proteins in both male and female ginkgo plants, it was found that both soluble sugars and soluble proteins were higher in male plants than in female plants. As important osmoregulatory substances and nutrient sources in plants, the higher soluble sugars and soluble proteins in male plants increased the resilience of male ginkgo plants. It is therefore possible that male and female plants will react and cope differently under different environmental conditions. Secondly, the sample size of 798 plants used for male and female identification and difference analysis in this study is large, but a large sample size is needed to more accurately reflect the separation of males and females and their differences.
Male ginkgo trees have a higher advantage in vegetative growth compared to female ginkgo trees, a pattern commonly observed in dioecious plants. In nature, especially among perennial woody plants, male plants often surpass female plants in terms of biomass accumulation, leaf area, and wood volume [24,25]. It is generally believed that male plants invest less in reproductive growth and grow faster in terms of vegetative growth. However, in species with shorter growth cycles, the opposite is true. For example, female plants of S. sachalinensis have a significant growth advantage over male plants, with greater biomass and more vegetative branches [26].
The faster growth ability of male ginkgo trees is evidenced by their higher net photosynthetic rate. The average net photosynthetic rate of male ginkgo trees is 117.65% of that of female trees. The higher net photosynthetic rate in male ginkgo trees is attributed to their higher chlorophyll content. Overall, in the seedling stage of ginkgo progeny, male plants have higher total chlorophyll, chlorophyll a, and chlorophyll b content compared to female plants, but there is no significant difference in carotenoid content. In natural populations of Populus yunnanensis [27] and Hippophae rhamnoides [28], male plants exhibit higher net photosynthetic rates, and greater efficiency in the use of light energy and water compared to female plants, mirroring the trends observed in ginkgo. This physiological advantage makes male ginkgo seedlings more competitive in the early growth stage, especially under natural conditions of limited resources or environmental stress. Through optimizing light conditions, the photosynthetic efficiency of male ginkgo seedlings may be further enhanced, leading to faster growth. The faster growth rate of male ginkgo trees gives them a more pronounced advantage over female trees in terms of timber production and other uses, making them more targeted for selective cultivation.
The faster growth of male ginkgo is supported by its higher net photosynthetic rate, which is 117.65% of the rate observed in female trees. This increased rate in males is due to their higher chlorophyll content. In the seedling stage, male ginkgo progeny exhibit higher levels of total chlorophyll, chlorophyll a, and chlorophyll b compared to female progeny, although carotenoid content shows no significant difference between the sexes. Similarly, in the natural community of P. yunnanensis [27], male trees demonstrate superior net photosynthetic rates and more efficient use of light and water than female trees, mirroring the trends observed in ginkgo. The faster growth rate of male ginkgo gives them a more significant advantage over female trees for timber use and other applications, making them more suitable for targeted cultivation.
The flavonoid and terpene lactone content in ginkgo are important economic components and primary aspects of its value. During the seedling stage, female ginkgo plants have higher total flavonoid and terpene lactone content than male plants, offering greater utilization value. Research indicates that in 10-year-old and 20-year-old ginkgo trees, the total flavonoid content is higher in male trees than in female trees. However, the effect of sex differences on flavonoid content is minimal. The flavonoid content in ginkgo is dynamic and varies with different seasons and growth stages [29]. Throughout the annual growth cycle, the flavonoid and terpene lactone contents gradually increase from those in spring, reaching a peak in April, and showing a second peak in October, displaying a bimodal variation trend [30]. Additionally, as the trees age, the total flavonoid content in ginkgo gradually decreases [31,32]. Based on the understanding of the dynamic changes in the flavonoid and terpene lactone content in ginkgo, it is still uncertain whether the differences between male and female plants during the seedling stage persist into adulthood. Female plants may require more flavonoids to protect reproductive structures (e.g., seeds and fruits), thus ensuring their development and maturation. This protective mechanism may make female plants more active in flavonoid synthesis and accumulation [33,34]. However, the significant feature that female ginkgo seedlings have higher total flavonoid and terpene lactone content than male seedlings can be effectively utilized in medicinal leaf harvesting and other applications. This characteristic can maximize the benefits of early sex identification.
Male and female ginkgo trees display significant differences in growth, development, and physiological and biochemical traits, largely due to variations in gene expression. After annotating the whole-genome SNPs of male and female ginkgo, 48 unique genes were identified in males and 64 in females. The unique genes in female ginkgo are predominantly enriched in pathways related to caffeine metabolism, highlighting caffeine’s crucial role in plant reproductive growth [35]. The unique genes in male ginkgo are primarily enriched in pathways related to thiamine metabolism. Thiamine, in the form of thiamine pyrophosphate (TPP), serves as an essential coenzyme in crucial metabolic pathways such as glycolysis and the citric acid cycle. A thiamine deficiency can lead to reduced metabolic rates, decreased photosynthesis, and impaired chlorophyll synthesis in plants [36]. The triggering of wheat seeds by thiamine can be effective in increasing osmotic pressure regulation and antioxidant capacity, thus effectively resisting salt stress [37]. This is consistent with the physiological characteristics of male ginkgo and the result that unique genes are mainly enriched in the thiamine metabolic pathway. Thus, the unique gene-enriched pathways of male ginkgo help to explain some of the observed differences, but a more comprehensive understanding of these differences requires an integrated analysis of gene transcription, translation, and expression across the genome.
During plant evolution, sex-specific physiological and biochemical functions have evolved as a result of reproductive and survival needs. Male plants have evolved a higher photosynthetic capacity and faster growth rates to compete for more pollination opportunities to ensure that they can rapidly enter the reproductive phase and attract pollinators. In contrast, female plants have evolved stronger resource reserves and protective mechanisms to ensure successful seed and fruit development [38]. In a resource-limited environment, the two sexes have gradually developed different resource allocation strategies [39]. Male plants may devote more resources to rapid growth and the development of reproductive structures, whereas female plants are more inclined to use resources to support the development of offspring and seed survival [40]. This is in line with the physiological, biochemical, and genetic-level differences observed in the different sexes of ginkgo in this study.
Sex differences in ginkgo provide new research directions for breeding and genetic improvement and are particularly important in breeding strategies for selecting sex ratios or optimizing physiological traits. By rationally utilizing male and female sex differences, cultivators can manage cropping systems more efficiently, improve productivity and product quality, and reduce resource wastage at the same time. We will follow up with an in-depth investigation of the long-term effects of environmental stressors on ginkgo populations, explore the genetic mechanisms of sex determination, further explore specific genes, and cultivate varieties with obvious advantages in target traits (e.g., growth rate, stress tolerance).

5. Conclusions

This study used male-specific primers to identify the sex of hybrid ginkgo seedlings during the seedling stage and further analyzed the differences in growth, development, and physiological, biochemical, and genomic aspects between male and female plants. The results showed that the sex ratio of the hybrid progeny was 56:44. During the seedling stage, male plants demonstrated significantly higher nutritional growth capacity compared to female plants. The soluble sugar and soluble protein content, as well as the total flavonoid content, were significantly higher in male seedlings. The female ginkgo population had 48 specific genes annotated, while the male population had 64 specific genes. Specific genes in female ginkgo were predominantly enriched in pathways related to caffeine metabolism, whereas specific genes in male ginkgo were primarily enriched in pathways related to thiamine metabolism.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f15091636/s1; Table S1: Genes specific to male and female populations.

Author Contributions

Conceptualization, X.G. and Q.G.; methodology, Y.H.; software, X.G.; validation, F.L.; formal analysis, X.G.; resources, Y.H.; writing—original draft preparation, X.G.; writing—review and editing, Q.G.; project administration, F.C.; funding acquisition, Q.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31971648), the Talent Introduction Project Study of Nanjing Forestry University (GXL2018001) on Ginkgo biloba and other important tree germplasm resources, and the Postgraduate Research &Practice Innovation Program of Jiangsu Province (KYCX23_1250).

Data Availability Statement

The article contains all the information required to support its conclusions.

Acknowledgments

Authors gratefully acknowledge all lab members for their help in collecting ginkgo seed and data organization.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. (a) PCR electropherograms of ginkgo male and female controls. (b) PCR electrophoresis of part of hybrid progeny. The number is the serial number of the progeny.
Figure 1. (a) PCR electropherograms of ginkgo male and female controls. (b) PCR electrophoresis of part of hybrid progeny. The number is the serial number of the progeny.
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Figure 2. (a) Sex ratio of males and females in F1 population. The numbers in the figure are the proportions of male plants in each population. (b–d) Differences in (b) height growth, (c) ground diameter growth, and (d) shoot growth of ginkgo seedlings by sex. * indicates significant difference among females in the same group (p < 0.05).
Figure 2. (a) Sex ratio of males and females in F1 population. The numbers in the figure are the proportions of male plants in each population. (b–d) Differences in (b) height growth, (c) ground diameter growth, and (d) shoot growth of ginkgo seedlings by sex. * indicates significant difference among females in the same group (p < 0.05).
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Figure 3. Differences in (a) net photosynthetic rate (Pn), (b) transpiration rate (Tr), (c) stomatal conductance (Gs), (d) intercellular CO2 concentration (Ci) of ginkgo by sex. * Indicates significant difference among females in the same group (p < 0.05).
Figure 3. Differences in (a) net photosynthetic rate (Pn), (b) transpiration rate (Tr), (c) stomatal conductance (Gs), (d) intercellular CO2 concentration (Ci) of ginkgo by sex. * Indicates significant difference among females in the same group (p < 0.05).
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Figure 4. Differences in (a) total chlorophyll, (b) chlorophyll a, (c) chlorophyll b content, and (d) carotenoid content of ginkgo by sex. * Indicates significant difference among females in the same group (p < 0.05).
Figure 4. Differences in (a) total chlorophyll, (b) chlorophyll a, (c) chlorophyll b content, and (d) carotenoid content of ginkgo by sex. * Indicates significant difference among females in the same group (p < 0.05).
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Figure 5. Differences in (a) soluble sugar content, (b) soluble protein content, (c) total flavonoid content, and (d) terpene lactone content of ginkgo sex. * Indicates significant difference among females in the same group (p < 0.05).
Figure 5. Differences in (a) soluble sugar content, (b) soluble protein content, (c) total flavonoid content, and (d) terpene lactone content of ginkgo sex. * Indicates significant difference among females in the same group (p < 0.05).
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Figure 6. (a) Veen diagram of annotated genes in male and female ginkgo populations; ginkgo-specific gene KEGG enrichment in female (b) and male plants (c) in the top 20 pathways.
Figure 6. (a) Veen diagram of annotated genes in male and female ginkgo populations; ginkgo-specific gene KEGG enrichment in female (b) and male plants (c) in the top 20 pathways.
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Figure 7. Comparison of ginkgo differences by gender (baseline with female plant index as 100). * indicates significant difference between male and female (p < 0.05).
Figure 7. Comparison of ginkgo differences by gender (baseline with female plant index as 100). * indicates significant difference between male and female (p < 0.05).
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Table 1. Hybrid combination.
Table 1. Hybrid combination.
♂1
(Nanlinxiongyi)		2
(Guangfosixiong)		3
(Dahuasui)
♀
A
(Fozhi)		A1A2A3
B
(Zhongzi)		B1B2B3
C
(Changzi)		C2 *C2C3
* During the collection process, C1 and C2 were accidentally mixed, so the C1 and C2 populations were collectively referred to as C2.
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Gao, X.; Hu, Y.; Li, F.; Cao, F.; Guo, Q. Sex Identification and Male–Female Differences in Ginkgo Biloba Hybrid F1 Generation Seedlings. Forests 2024, 15, 1636. https://doi.org/10.3390/f15091636

AMA Style

Gao X, Hu Y, Li F, Cao F, Guo Q. Sex Identification and Male–Female Differences in Ginkgo Biloba Hybrid F1 Generation Seedlings. Forests. 2024; 15(9):1636. https://doi.org/10.3390/f15091636

Chicago/Turabian Style

Gao, Xiaoge, Yaping Hu, Fangdi Li, Fuliang Cao, and Qirong Guo. 2024. "Sex Identification and Male–Female Differences in Ginkgo Biloba Hybrid F1 Generation Seedlings" Forests 15, no. 9: 1636. https://doi.org/10.3390/f15091636

APA Style

Gao, X., Hu, Y., Li, F., Cao, F., & Guo, Q. (2024). Sex Identification and Male–Female Differences in Ginkgo Biloba Hybrid F1 Generation Seedlings. Forests, 15(9), 1636. https://doi.org/10.3390/f15091636

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