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Communication

Male-Specific Sequence in Populus simonii Provides Insights into Gender Determination of Poplar

by
Ziyue Wang
1,
Yijing Lei
1,
Guanqing Liu
2,3,
Yihang Ning
1,
Runxin Ni
1,
Tao Zhang
2,3,* and
Mengli Xi
1,*
1
State Key Laboratory of Tree Genetics and Breeding, Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
2
Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding/Zhongshan Biological Breeding Laboratory/Key Laboratory of Plant Functional Genomics of the Ministry of Education, Agricultural College of Yangzhou University, Yangzhou 225009, China
3
Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops/Jiangsu Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou 225009, China
*
Authors to whom correspondence should be addressed.
Forests 2023, 14(12), 2385; https://doi.org/10.3390/f14122385
Submission received: 3 November 2023 / Revised: 3 December 2023 / Accepted: 5 December 2023 / Published: 6 December 2023
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
The genus Populus is composed of dioecious woody plants and adult females produce large numbers of seed hairs that can affect public health and pose a potential fire risk. However, it is difficult to distinguish between males and females based on their morphology at the seedling stage. Therefore, developing a technology that identifies the gender of poplar seedlings is crucial for controlling seed hairs. In this study, we developed an approach for the early gender identification of Tacamahaca and Aigeiros species based on the male-specific sequence in Populus simonii. The gender of Tacamahaca and Aigeiros species can be accurately identified by PCR. The sequencing results showed that the male-specific sequence was conserved in P. simonii and its F1 progenies. Interestingly, there were three nucleobase differences between Tacamahaca and Aigeiros species. Sequence alignment showed that the male-specific sequence had not been assembled on the pseudochromosome. Subsequently, fluorescence in situ hybridization (FISH) was used to locate this specific sequence at the short arm end of chromosome 19 in male P. simonii. This study provides an efficient and convenient method for early gender determination of Tacamahaca and Aigeiros species and lays the groundwork for exploring key sex-determination genes.

1. Introduction

Poplar is an important woody plant for shelter forests, artificial timber forests, bioremediation, and landscaping due to its rapid growth, straight trunk, versatile timber, and strong stress resistance [1]. It is a dioecious plant and its sex is controlled by genetics [2,3]. Adult female trees produce a large number of seed hairs that can cause allergies, fires, and other problems [4,5,6]. Therefore, female trees should not be used for landscaping. However, poplar has a long juvenile period; it takes 6 years for the first blossoms to appear, and it is difficult to accurately identify gender from its morphology at the seedling stage. Therefore, feasible methods for the early identification of gender during the poplar seedling stage need to be developed. Previous studies analyzed isozymes of peroxidase and esterase in leaves and buds from adult Populus davidiana, P. pseudo-simonii, and P. alba × (P. davidiana × P. simonii) [7]. The male and female plants showed differences in most of the zymograms, but the differences varied depending on enzymes, organs, and species. Thus, this method could not accurately identify the gender of the three poplar species at the seedling stage. However, the rapid development and application of molecular markers has allowed simple sequence repeats (SSRs) molecular markers that are only suitable for P. davidiana sex identification to be developed [8].
In recent years, the iterative upgrading of sequencing technology and bioinformatics analysis has improved the sex identification of poplar. Next-generation sequencing of male and female aspens has identified male-specific TOZ19 as a sex candidate gene in P. tremuloides and P. tremula [9]. Based on this gene sequence, a PCR-based molecular marker has been developed to identify the gender of P. tremuloides and P. tremula. However, the sex-linked marker TOZ19 could not identify the gender of P. grandidentata which belongs to the same section (Leuce) as P. tremuloides and P. tremula. Therefore, the ARR17 inverted repeat was used as a male-specific marker to distinguish P. grandidentata males and females [10]. Furthermore, a male-specific sex-linked single nucleotide polymorphism (SNP) marker has been identified in P. trichocarpa and P. balsamifera by combining whole genome resequencing, the genome-wide association approach (GWAS), and polymerase chain reaction, followed by restriction fragment length polymorphism (PCR-RFLP) [11]. Whereas, using the female P. trichocarpa genome as the reference [12] can lead to false positive markers, and the PCR-RFLP assay is a time-consuming procedure. Moreover, three sex discrimination markers based on the Y-specific hemizygous sequence have been developed using P. deltoides, and the PCR products were only generated in the males [13]. However, these markers can only be used for P. deltoids sex identification. Furthermore, the study by Kim et al. [10] also demonstrated that ARR17 could be used as a female-specific marker in P. alba, P. adenopoda, and their hybrids. In summary, these sex identification molecular markers developed from poplars are not very transferable among species.
Populus deltoids (section Aigeiros) and P. simonii (section Tacamahaca) are important parents for poplar hybrid breeding. It has been suggested that the F1 hybrid population of P. deltoids and P. simonii has obvious heterosis, showing fast growth, straight trunks, disease resistance, and stress tolerance [14,15,16]. The development of a molecular marker for the sex identification of P. deltoids, P. simonii, and their hybrid population at the seedling stage could significantly improve breeding efficiency. A male-specific DNA sequence was previously identified using high-throughput sequencing and bioinformatics analysis of the genomic DNA from female and male P. simonii [17]. To test the reliability of this sequence in the sex identification of poplar, this study selected 32 adult poplars of known sex from sections Tacamahaca and Aigeiros and analyzed them using the PCR method. The results demonstrated that this male-specific DNA sequence can be used to identify the gender of Tacamahaca and Aigeiros seedlings and that the sequence was mapped to the distal region of the short arm of chromosome 19 in male P. simonii. These findings provide a rapid, simple, and accurate method for the early gender identification of Tacamahaca and Aigeiros and lay a foundation for further exploring the sexual determination mechanism in poplar.

2. Materials and Methods

2.1. Plant Materials

Thirty-two adult poplar individuals were selected as materials, which were one P. simonii, one P. deltoides × P. euramericana ‘Nanlin895′, twenty-one P. deltoides, and nine P. deltoides ‘I-69′ × P. simonii hybrid progeny (Table 1). Fresh leaf samples from the selected plants were collected, frozen immediately in liquid nitrogen, and stored at −80 °C until needed.

2.2. DNA Extraction, PCR Amplification, and Gel Electrophoresis

DNA from the frozen leaf tissue was extracted using a Plant Genomic DNA kit according to the manufacturer’s instructions (Tiangen, Beijing, China). Elongation factor 1-alpha (EF1α) was used as an internal control fragment [18,19]. PCR amplification was performed using a Veriti™ 96-Well Thermal Cycler (Thermo Fisher, Waltham, MA, USA) and the PCR mix (25 μL) contained 12.5 μL Premix Taq™ (Takara, Beijing, China), 1 μL (10 μM) forward primer (5′-GGCAAGGAGAAGGTACACAT-3′), 1 μL (10 μM) reverse primer (5′-CAATCACACGCTTGTCAATA-3′), 3 μL (10 ng·μL−1) template DNA, and 7.5 μL ddH2O. The PCR amplification procedure was as follows: 94 °C for 3 min, 30 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 14 s, and a final extension at 72 °C for 10 min.
Primers for the P. simonii male-specific sequence were designed using Primer Premier 5.0 software (http://www.premierbiosoft.com, accessed on 5 June 2023). The PCR mix (25 μL) contained 12.5 μL Premix Taq™ (Takara, Beijing, China), 1 μL (10 μM) forward primer MS-F (5′-CACAACCTAAGCAATAGTTGGCA-3′), 1 μL (10 μM) reverse primer MS-R (5′-ATGTCTTTGAGCTTTGGTGCTG-3′), 3 μL (10 ng·μL−1) template DNA, and 7.5 μL ddH2O. The PCR amplification procedure was as follows: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min.
The PCR products of EF1α and the male-specific sequence were mixed in equal volume and detected by 1.5% gel electrophoresis. The fragments of EF1α were expected to be about 100 bp, while male-specific fragments were about 400 bp.

2.3. Sanger Sequencing and SNP Analysis

Fragments of the male-specific sequence were PCR amplified from genomic DNA of seven male poplars (3-130, 3-16, 3-167, 3420, 3415, T20, and P. simonii) using the primers MS-F and MS-R. The PCR amplification was completed with TaKaRa LA Taq (Takara, Beijing, China) and the procedure was as follows: 94 °C for 3 min, 32 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min. The PCR products were sent to Sangon Biotech (Shanghai, China) for Sanger sequencing and the results were analyzed using Mega 7 software (version 7.0) [20].

2.4. Chromosome Preparation

Root tips about 0.5~1 cm long were cut from P. simonii (2n = 2x = 38) and pretreated with 0.7 mM cycloheximide for 3 h at room temperature. Mitotic metaphase chromosome slides were performed as described in published protocols [3].

2.5. Fluorescence In Situ Hybridization (FISH) Analysis

The P. simonii genome database (https://www.ncbi.nlm.nih.gov/data-hub/genome/GCA_007827005.2/, accessed on 20 June 2023) and related annotation files were used to search for sequences based on previously published procedures [3,21]. The selected sequence in P. simonii was used to design forward (5′-CCATCTGCAGAACCAGCTGA-3′) and reverse (5′-TTTCTCGCCATTTCCCCTCC-3′) primers to amplify the sequence. The amplified and purified DNA were labeled with digoxigenin-dUTP (digoxigenin-11-dUTP, 11093088910, Roche, Basel, Switzerland) using nick translation. The biotin-labeled single-stranded oligos prepared from the libraries were used as FISH probes. The FISH experiment was performed following published protocols [22] and the images were digitally captured using a CCD camera (C11440-42U, Hamamatsu, Shizuoka, Japan) attached to an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan). Final image contrasting was performed using Adobe Photoshop 2020 software (version 21.0.1).

3. Results and Discussion

3.1. Poplar Gender Verification and Other Sex Markers in Populus

A total of 32 poplar genotypes of known sex were subjected to PCR detection using male-specific sequence and primers derived from P. simonii. Each individual represented a distinct genotype. The electrophoresis results revealed a clear band of approximately 400 bp in all 19 male individuals, whereas none of the females showed this band (Figure 1). These findings confirm the sex-linked inheritance of the male-specific sequence. Previous studies on Populus species, including Tacamahaca and Aigeiros, have consistently suggested an XY system, where males are the heterogametic sex [11,23,24]. These observations suggest that the male-specific sequence is located in the sex determination region (SDR) of the Y chromosome in P. simonii. Consequently, the presence or absence of the male-specific DNA sequence can be used to determine the gender of Tacamahaca, Aigeiros, and their offspring during the seeding stage.
To date, the majority of sex molecular markers have only been developed for specific poplar species, such as the sex-specific molecular markers for P. deltoides, and cannot be universally applied to other Populus species [13]. Furthermore, distinguishing the sex of different poplar species within the Leuce section requires the use of different sex molecular markers, none of which are universal [9,10]. Geraldes et al. [11] used a PCR-RFLP assay to demonstrate that a sex-linked SNP marker developed for P. trichocarpa can identify P. deltoides males and females. However, this marker did not prove effective for certain P. trichocarpa and P. balsamifera individuals, both of which belong to the Tacamahaca section. This suggests that there may be issues with the assembly of the reference genome because there were SNPs associated with sex across multiple chromosomes [11]. The PCR-RFLP assay requires the digestion of amplified products by restriction endonucleases to produce fragments of varying sizes. Identifying males and females is then based on the number and size of the gel bands, which means that this method can be time-consuming and complex [11,25]. In contrast, sex identification based on male-specific sequence PCR is simpler, faster, and more reliable compared to the PCR-RFLP method, which relies on sex-linked SNP markers. Consequently, the sex identification method developed here can be used to effectively identify the gender of Tacamahaca and Aigeiros populations on a large scale.

3.2. SNP Detection of the Male-Specific Sequence

A total of 7 of the 19 male individuals were selected to further elucidate the characteristics of the male-specific sequence in P. simonii. A PCR amplification of the genomic DNA from these seven male individuals was performed using primers specific to the male-specific sequence in P. simonii and the amplified products were subjected to Sanger sequencing. The sequencing results revealed that the P. simonii male-specific sequence was consistent in P. simonii and offspring generated with P. simonii as the male parent and P. deltoids as the female parent, which suggested that the region where this sequence is located has undergone recombination inhibition. Related studies have shown that X and Y chromosomes will gradually undergo recombination inhibition during evolution and cannot exchange genetic materials [26,27]. These inhibition regions are called the sex-determining regions of sex chromosomes. There were only three nucleotide differences in this male-specific sequence between the Aigeiros and Tacamahaca sections. For example, in P. simonii and the F1 generation obtained when P. simonii was the male parent, the nucleotide at position 34 is A, at position 54 is C, and at position 98 is A, while in P. deltoides, the nucleotide at position 34 changes to C, position 54 is missing, and it changes to T at position 98 (Figure 2). This suggests that the male-specific sequence can be faithfully transmitted to all male offspring without any recombination during meiosis. Although the sequence was different between P. deltoides and P. simonii, it was conserved among the three sequenced P. deltoides. This suggests that the three P. deltoides were from the same male parent.
The male-specific sequence was aligned to the P. simonii reference genome sequence (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_007827005.2/, version 2.0, accessed on 2 June 2023) to determine whether the male-specific sequence in P. simonii was located on the sex chromosome, but it could not be detected [28]. However, the unassembled scaffold 000293F did contain this sequence. This may be due to significant recombination suppression in this region, resulting in a lack of segregating DNA markers. As a result, this scaffold could not be anchored to the chromosome based on the genetic linkage map [3]. In recent years, the telomere-to-telomere (T2T) sequencing technology applied to some plants has achieved gap-free assembly of T2T sequences for all chromosomes and has become a powerful tool in plant-related research [29,30]. In the future, T2T sequencing and assembly could be performed on P. simonii. The T2T genome has fewer gaps or no gaps at all compared with the existing genome. Therefore, the assembly and annotation of high-quality reference genomes can fill the gaps that are not assembled at present and reveal the sex determination mechanism in poplar [31,32].

3.3. Cytogenetic Location of the Male-Specific Sequence in P. simonii

Fluorescence in situ hybridization can physically map unassembled genomic sequences onto chromosomes [33,34]. Chromosome painting probes for all 19 chromosomes were developed based on the P. trichocarpa reference genome. These probes can accurately identify each P. trichocarpa chromosome and can be universally applied to other poplar species [22]. After using oligonucleotide fluorescence in situ hybridization (oligo-FISH), the unassembled scaffold22 and scaffold25 in the P. trichocarpa genome were successfully mapped to the centromere region of chromosome 3 and the distal end of the short arm of chromosome 19, respectively [22]. Previous studies have shown that chromosome 19 is the sex chromosome in poplar [2,24,35]. To further determine the location of male-specific sequences on chromosomes in P. simonii, a chromosome 19 painting probe for poplar and the male-specific DNA sequence in P. simonii were selected as probes. These probes were then hybridized to the metaphase chromosomes prepared from P. simonii. The oligo-FISH results demonstrated that the oligo probe for 19 produced a bright green signal on a pair of chromosomes, but there was an unstained area at one end of the chromosome (Figure 3). Interestingly, bright red fluorescent signals produced by the male-specific sequence probe were present at the end of chromosome 19, which coincided with the area not labeled by the oligo probe. Previous experiments indicated that this region, not stained by the oligo probes, was located at the end of the short arm of chromosome 19 [3]. Therefore, the male-specific DNA sequence is located at the end of the short arm of chromosome 19.
Identifying sex-determining regions and genes is key to understanding the formation and evolution of sex chromosomes. However, sex-determining regions usually have a high repeat density, which means that the assembly process is difficult [36,37]. Previous genetic mapping studies have mapped the sex-determining region of poplar to the proximal telomeric end or centromere region of chromosome 19 [24,35]. A cytological analysis showed that the short arm ends of chromosome 19 in P. tomentosa and P. deltoides do not pair at the pachytene stage in 22%–24% of meiotic cells, which further suggests that the short arm ends of chromosome 19 are the sex-determining regions [3]. A case study on P. deltoides revealed that the male trees harbored two Y-specific genes, FERR-R and MSL, which inhibit female and promote male development, respectively [2]. In P. simonii, most of the FERR-R sequences were located on the unassembled scaffold 000293F. The P. simonii male-specific sequence was also located on this scaffold at the end of the short arm of chromosome 19. The results from this study suggest that this scaffold is related to sex determination in P. simonii. Therefore, a comprehensive sequence analysis of this scaffold should be conducted to identify sex-related genes and verify their functions, thus laying a foundation for studying the sex determination mechanism in poplar.

4. Conclusions

This study utilized the male-specific sequence in P. simonii to accurately identify the gender of Tacamahaca and Aigeiros species with known gender. In addition, Sanger sequencing was performed on PCR amplification products from selected male samples. The results revealed that the specific sequence in P. simonii showed sex-linked inheritance characteristics. Therefore, this sequence may be located within the sex-determining region of P. simonii. However, the sequence alignment results showed that the scaffold containing the sequence had not been assembled on the chromosome. Therefore, FISH was used to precisely locate the sequence at the end of the short arm of chromosome 19 in male P. simonii. This study introduces a highly efficient and simple method for early sex identification of Tacamahaca and Aigeiros species and establishes the groundwork for further investigations into the molecular mechanism controlling sex determination and sex chromosome evolution in the family Salicaceae.

Author Contributions

M.X. and T.Z. conceived and designed the research; Z.W., Y.L., G.L., Y.N. and R.N. performed experiments. M.X. and Z.W. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the STI 2030-Major Projects (2023ZD04057).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. PCR amplification of P. simonii male-specific DNA sequence as a molecular marker to determine sex in poplars. Samples were run on 1.5% agarose gel. The upper bands represent the male-specific fragment and the lower bands the control fragment. Lane M: DL2,000 DNA Marker. Lane PSX: male P. simonii. Lane T20, 74-4, 48-1, 3420, 3415, 88-4, 11-1, 101-4, 100-4, 60-2, 62-3, and 95-44: male P. deltoides. Lane 72-1, 3016, 80-4, 35-2, 74-1, 88-7, 131-1, 24-3, and NL15: female P. deltoides. Lane NL895: female P.deltoides × P. euramericana ‘Nanlin895′. Lane 3-35, 3-104, 3-160, 3-167, 3-16, and 3-130: male P. deltoides ‘I-69′ × P. simonii hybrids. Lane 3-46, 3-89, and 3-53: female P. deltoides ‘I-69′ × P. simonii hybrids. Lane W: water control.
Figure 1. PCR amplification of P. simonii male-specific DNA sequence as a molecular marker to determine sex in poplars. Samples were run on 1.5% agarose gel. The upper bands represent the male-specific fragment and the lower bands the control fragment. Lane M: DL2,000 DNA Marker. Lane PSX: male P. simonii. Lane T20, 74-4, 48-1, 3420, 3415, 88-4, 11-1, 101-4, 100-4, 60-2, 62-3, and 95-44: male P. deltoides. Lane 72-1, 3016, 80-4, 35-2, 74-1, 88-7, 131-1, 24-3, and NL15: female P. deltoides. Lane NL895: female P.deltoides × P. euramericana ‘Nanlin895′. Lane 3-35, 3-104, 3-160, 3-167, 3-16, and 3-130: male P. deltoides ‘I-69′ × P. simonii hybrids. Lane 3-46, 3-89, and 3-53: female P. deltoides ‘I-69′ × P. simonii hybrids. Lane W: water control.
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Forests 14 02385 g002
Figure 2. Sequencing results of SNP. P. deltoides: 3420, 3415, and T20; P. deltoides ‘I-69′ × P. simonii: 3-130, 3-16, and 3-167; P. simonii: PSX.
Figure 2. Sequencing results of SNP. P. deltoides: 3420, 3415, and T20; P. deltoides ‘I-69′ × P. simonii: 3-130, 3-16, and 3-167; P. simonii: PSX.
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Figure 3. The FISH mapping of a male-specific sequence associated with P. simonii (red) and the oligo-based painting of chromosome 19 probe (green) on a metaphase cell of P. simonii. Bars = 10 μm. (a) 4′-6-Diamidino-2-phenylindole-stained chromosomes of a metaphase cell. (b) Same metaphase cell in (a) was hybridized to male-specific DNA (red) probes. (c) Same metaphase cell in (a) was hybridized to chromosome 19 (green) painting probes. (d) Merged picture from (a–c).
Figure 3. The FISH mapping of a male-specific sequence associated with P. simonii (red) and the oligo-based painting of chromosome 19 probe (green) on a metaphase cell of P. simonii. Bars = 10 μm. (a) 4′-6-Diamidino-2-phenylindole-stained chromosomes of a metaphase cell. (b) Same metaphase cell in (a) was hybridized to male-specific DNA (red) probes. (c) Same metaphase cell in (a) was hybridized to chromosome 19 (green) painting probes. (d) Merged picture from (a–c).
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Table 1. Plant materials used in this study.
Table 1. Plant materials used in this study.
NumberSpeciesGenotypeSex
1P. deltoidesT20male
2P. deltoides ‘I-69′ × P. simonii F13-35male
3P. deltoides72-1female
4P. deltoides74-4male
5P. deltoides3016female
6P. deltoides ‘I-69′ × P. simonii F13-104male
7P. deltoides48-1male
8P. deltoides ‘I-69′ × P. simonii F13-46female
9P. deltoides ‘I-69′ × P. simonii F13-160male
10P. deltoides ‘I-69′ × P. simonii F13-89female
11P. deltoides ‘I-69′ × P. simonii F13-53female
12P. deltoides ‘I-69′ × P. simonii F13-167male
13P. deltoides × P. euramericanaNanlin895 (NL895)female
14P. deltoides3420male
15P. deltoides3415male
16P. deltoides80-4female
17P. simoniiP. simoniimale
18P. deltoides35-2female
19P. deltoides ‘I-69′ × P. simonii F13-16male
20P. deltoides88-4male
21P. deltoides74-1female
22P. deltoides ‘I-69′ × P. simonii F13-130male
23P. deltoides11-1male
24P. deltoides88-7female
25P. deltoides131-1female
26P. deltoides101-4male
27P. deltoides24-3female
28P. deltoides100-4male
29P. deltoides60-2male
30P. deltoides62-3male
31P. deltoidesNanLin15 (NL15)female
32P. deltoides95-44male
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MDPI and ACS Style

Wang, Z.; Lei, Y.; Liu, G.; Ning, Y.; Ni, R.; Zhang, T.; Xi, M. Male-Specific Sequence in Populus simonii Provides Insights into Gender Determination of Poplar. Forests 2023, 14, 2385. https://doi.org/10.3390/f14122385

AMA Style

Wang Z, Lei Y, Liu G, Ning Y, Ni R, Zhang T, Xi M. Male-Specific Sequence in Populus simonii Provides Insights into Gender Determination of Poplar. Forests. 2023; 14(12):2385. https://doi.org/10.3390/f14122385

Chicago/Turabian Style

Wang, Ziyue, Yijing Lei, Guanqing Liu, Yihang Ning, Runxin Ni, Tao Zhang, and Mengli Xi. 2023. "Male-Specific Sequence in Populus simonii Provides Insights into Gender Determination of Poplar" Forests 14, no. 12: 2385. https://doi.org/10.3390/f14122385

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