Next Article in Journal
Net Photosynthesis and Biomass Production in Stevia, Eggplant, and Cowpea Can Be Improved by Fertilization with Cyanobacteria (Limnospira maxima)
Previous Article in Journal
Triploid Hybrids of 2x Lingonberry (Vaccinium vitis-idaea) by 2x Black Highbush Blueberry (V. fuscatum) and 2x Elliott’s Blueberry (V. elliottii) as Evidence of a Genome Balance Requirement for Hybridization Success
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 9
Issue 12
10.3390/horticulturae9121310
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

A MADS-Box Gene-Based InDel Marker Discriminating Sex in Actinidia arguta

by
Sewon Oh
1,2,
Jung Kim
1,
Yumi Kim
1,
Mockhee Lee
3 and
Daeil Kim
1,*
1
Department of Horticulture, Chungbuk National University, Cheongju 28644, Republic of Korea
2
Fruit Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 55365, Republic of Korea
3
Research Institute of Climate Change and Agriculture, National Institute of Horticultural and Herbal Science, Rural Development Administration, Jeju 63240, Republic of Korea
*
Author to whom correspondence should be addressed.
Horticulturae 2023, 9(12), 1310; https://doi.org/10.3390/horticulturae9121310
Submission received: 9 November 2023 / Revised: 27 November 2023 / Accepted: 1 December 2023 / Published: 6 December 2023
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
Browse Figures
Review Reports Versions Notes

Abstract

:
Gene-based markers are valuable tools in breeding programs due to their direct linkage to traits of interest. In dioecious plants, such as kiwifruit (Actinidia spp.), sex-discriminating markers can shorten the breeding cycle by enabling the selection of preferred sexes at the juvenile stage. To develop a gene-based sex-discriminating marker, resequencing was conducted on female and male A. arguta accessions, and insertion and deletion (InDel) variations within sex-related genes were explored. A total of 203,116 InDels were detected between female and male A. arguta accessions, and 118,865 InDels were heterozygous between the two accessions. Sequence similarity between thirty-seven sex-related genes from seven dioecious species and the kiwifruit reference genome was investigated, revealing that ten genes exhibited similarities ranging from 59 to 79%. Among the 118,865 InDels, seven InDels were located on four sex-related genes encoding agamous-like MADS-box genes and hypothetical proteins. A 20 bp insertion in male A. arguta located in the agamous-like MADS-box gene was converted into an InDel marker, which clearly discriminates female and male A. arguta accessions and the interspecific hybrid cultivar. The InDel marker was designated CBk25id01 and produced approximately 350 bp amplicon only in the male A. arguta. The CBk25id01 linked to the agamous-like MADS-box gene involved in floral organ development may help understand sex differentiation and accelerate the breeding of kiwifruits.

1. Introduction

In dioecious plants, the sex-determination system is characterized by distinct male and female individuals, with each plant exclusively exhibiting one sex. Male plants exclusively produce male reproductive structures, while female plants exclusively produce female ones. The sex determination in dioecious plants is primarily regulated by genetic factors. In the genetic sex-determination system of plants, sex is determined by specific genes within their genome. These genes, commonly known as sex-determination genes or sex chromosomes, play a crucial role in orchestrating the development of male or female reproductive structures [1,2].
Two sex-determination systems, namely the XY and ZW systems, have been identified in plants. The XY system functions similarly to the sex-determination system observed in animals, where females possess two identical sex chromosomes (XX), while males have two different sex chromosomes (XY). On the other hand, the ZW system has similarities with the XY system, but in this case, females have two different sex chromosomes (ZW), and males possess two identical sex chromosomes (ZZ). Among dioecious plants, the XY system has been frequently observed as the predominant sex-determination system [3].
Previous studies on Y chromosome evolution in plants, particularly silene (Silene latifolia), have revealed non-recombining male-determining regions containing multiple genes [4]. Recently, a genome assembly in garden asparagus (Asparagus officinalis) has led to the identification of two sex-determining genes: a potential male-promoting factor (M) and a potential SuF named SOFF, which is hypothesized to have arisen from a gene-duplication event followed by the acquisition of a new function, distinct from that of the ancestral gene [5]. In diploid persimmon (Diospyros lotus), on the other hand, sex determination is currently hypothesized to be under the control of a pair of paralogous genes called MeGI (Male Growth Inhibitor) and OGI (Oppressor of MeGI) [6,7]. In the case of ginkgo (Ginkgo biloba), it has been discovered that four MADS-box genes (GbMADS1, GbMADS2, GbMADS4, and GbMADS18) are exclusively located on the Y chromosome. Furthermore, these four MADS-box genes displayed elevated expression levels in the staminate strobilus compared to female organs [8]. Additionally, two single-copy genes, CYP703 and GPAT3, have been identified in male date palms, and they are associated with the inhibition of female flower development [9]. Another dioecious plant, spinach (Spinacia oleracea), possesses 12 genes linked to the male-determining locus in the male-specific region of the Y chromosome [10].
Kiwifruit (Actinidia spp.), a dioecious plant, also follows the XY sex-determination system. Two sex-determination genes, namely Shy Girl (SyGl) and Friendly Boy (FrBy), have been identified in kiwifruits [11,12]. SyGl acts as a dominant suppressor of carpel development, while FrBy plays a crucial role in maintaining male fertility. However, the regulatory mechanism underlying the functions of SyGl and FrBy remains yet to be elucidated.
Sex-discriminating markers in dioecious plants can enhance breeding efficiency by enabling the selection of the desired sexes corresponding to the breeding objectives during the seedling stage. In kiwifruits, sex-discriminating markers linked to the sex chromosomes have been developed [13,14,15,16,17,18]. However, these markers have not been employed in the marker-assisted selection (MAS) of kiwifruit breeding.
Gene-based markers linked to genes of interest offer advantages in MAS, primarily by enhancing the selection precision for desired traits. Due to the conserved nature of gene sequences, variations occurring within a specific gene are promising candidates for gene-based markers. In a previous study, we developed a cleaved amplified polymorphic sequence (CAPS) marker that enables the differentiation of sexes in A. arguta. This marker relies on the presence of a single nucleotide polymorphism (SNP) located on the sex chromosome [18]. However, the CAPS marker does not exhibit linkage to any sex-discriminating gene(s), and its application was limited to A. arguta.
Recently, Ye et al. [19] proposed the involvement of MADS-box genes in floral sex differentiation in kiwifruit. This highlights the involvement of multiple sex-determination genes in the complex process of sexual differentiation within the Actinidia species. These genes could be candidates for the development of sex-discriminating markers in Actinidia species. Therefore, we developed a gene-based sex-discriminating marker with relatively wide transferability by examining the sequence similarities of previously identified sex-determination genes in dioecious plants.

2. Materials and Methods

2.1. Plant Materials and DNA Extraction

Fresh young leaves from 37 accessions, including 16 females and 21 males (Table 1), were collected at Namhae Branch, the National Institute of Horticultural and Herbal Science, Rural Development Administration, Korea (34°48′59.8″ N, 127°55′36.6″ E). The 16 females were comprised of 15 A. arguta accessions and 1 hybrid ((A. arguta × A. deliciosa) × A. arguta). All 21 males belonged to the A. arguta. The collected fresh young leaves were washed and stored at −70 °C.
The genomic DNA of 37 accessions was extracted from the leaves using a DNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The quality and quantity of DNA samples were measured using a Denovix DS-11 spectrophotometer (Denovix, Wilmington, DE, USA).

2.2. Resequencing and Polymorphic InDel Detection

Out of the 37 A. arguta accessions, K5-10-5 (female) and K5-3-12 (male), included in the core set of A. arguta native to Korea [20], were selected to perform resequencing.
Sequencing libraries were constructed using DNA samples (≥1 µg) from both the female and male accessions using the TruSeq Nano DNA kit (Illumina, San Diego, CA, USA). The libraries were sequenced on the Illumina Hiseq X platform with paired-end sequencing. The trimming of 151 bp short reads was performed using the Cutadapt [21] and the SolexaQA v1.13 package [22]. After passing the trimming process, cleaned reads were mapped to the reference genome (A. chinensis Red5) [23] using Burrows–Wheeler alignment with default options [24]. Raw InDels were detected using SAMtools v0.1.16 [25] with BAM files, and consensus sequences were extracted. To detect polymorphic InDels between female and male accessions, an integrated InDel matrix was created, and the InDels were classified as homozygous (reads rate ≥ 90%), heterozygous (40% ≤ read rate ≤ 60%) InDels, or other types. Polymorphic InDel structures were also confirmed in the Integrative Genomics Viewer (IGV) by comparing read alignments in BAM files.

2.3. Detection of Candidate InDels Discriminating Sex

A total of 37 genes related to the XY sex-determination system in kiwifruit [11,12], persimmon [6,7], ginkgo [8], date palm [9], spinach [10], asparagus [5,26], and silene [27,28,29,30,31,32,33,34] (Table S1) were compared with the kiwifruit reference genome (A. chinensis Red5). BLASTx analysis was performed using the coding DNA sequences of the 37 genes, and genes located on chromosome 25, known as the sex chromosome of kiwifruit, were selected as sex-related genes based on the criteria of e-value ≤ 1 × 10−10 and identity ≥ 40%. Polymorphic InDels between female and male A. arguta accessions were investigated, specifically those occurring in the sex-related genes.

2.4. Development of a Sex-Discriminating InDel Marker

Among the polymorphic InDels observed in the sex-related genes, a candidate InDel marker targeting InDel structures over 10 bp was selected to develop an agarose gel-based marker system. Flanking sequences of approximately 600 bp, which include the InDel and its 300 bp up and downstream sequences, were extracted. Primer sequences were designed using Primer 3 with the following criteria: (1) 20 bp length of primer sequences, (2) 50% GC content, and (3) an annealing temperature of 60 °C. Specifically, the forward primer sequences were designed to contain the InDel sequences.
The PCR reaction mixture (10 μL) consisted of 2× HS Prime Taq Plus Premix (GeNetBio, Daejeon, Republic of Korea), 10 ng·μL−1 of genomic DNA, 10 pmole‧μL−1 of InDel primer pair, and distilled water. The PCR conditions consisted of pre-denaturation at 95 °C for 5 min, followed by 34 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, an extension at 72 °C for 1 min, and a final extension step at 72 °C for 5 min. Subsequently, PCR amplicons were checked using 3% agarose gel electrophoresis.

3. Results

3.1. Resequencing and InDel Detection

In female and male A. arguta accessions, a total of 171.9 and 156.6 million read pairs were produced, respectively. After trimming the raw sequencing reads, an average of 66% of read pairs were retained. Among these, approximately 86% of the trimmed reads were mapped to the kiwifruit reference genome (A. chinensis Red5) [23], resulting in 26.9 and 24.5× of genome coverage for the female and male accessions, respectively (Table S2).
After variant calling, a total of 2,045,913 and 2,033,740 InDels were detected in the female and male accessions, respectively, compared to the reference genome. Approximately 17% of these InDels exhibited heterozygosity between A. arguta and the reference genome (Table S3). Subsequently, an investigation into InDels between the female and male accessions revealed a total of 203,116 InDels. Among these, 14,251 InDels were identified as homozygous, while 188,865 InDels were classified as heterozygous between the female and male accessions.

3.2. Selection of Polymorphic InDels Occurring in Sex-Related Genes

After conducting a BLASTx analysis of the 37 sex-related genes (Table S1) against the kiwifruit reference genome, it was confirmed that 10 genes showed sequence similarities ranging from 59 to 79% with 8 sex-related genes found in ginkgo, data palm, asparagus, spinach, and silene (Table 2). Specifically, the gene Acc28871, encoding an agamous-like MADS-box protein, displayed a sequence similarity of 68% (1.08 × 10−41) with GbMADS1, 2, 4, and 18 from ginkgo. Another gene, Acc28333, encoding a cytochrome P450 CYP736A12-like protein, showed 58.8% homology with CYP703 from date palms. Furthermore, several other genes exhibited sequence similarity with genes from asparagus. These included the hypothetical protein Acc28900, the transcription factor-like proteins Acc28821, Acc28639, and Acc28638, and the Myb-related protein Acc28429, which displayed sequence similarities ranging from 68.6 to 78.6 with TDF1. The gene Acc28776, encoding a GDSL esterase/lipase, exhibited similarity to the GDSL esterase/lipase found in spinach. Lastly, the gene AP3Y from silene showed sequence similarities of 62.2 to 69.5% with Acc28719 and Acc28871.
Seven InDels were identified in four out of ten sex-related genes when comparing female and male A. arguta accessions (Table 3). These four genes (Acc28719, Acc28871, Acc28717, and Acc28900) containing the seven detected InDels displayed similarities to sex-related genes found in ginkgo, silene, and asparagus (Table 2). Among these InDels, five were detected in three agamous-like MADS-box genes, which shared homology with AP3Y from silene (Acc28719 and Acc28717) and GbMADS from ginkgo (Acc28871). In the female A. arguta accession, a 2 bp insertion (5′-AT-3′) and a 4 bp deletion (5′-TGAT-3′) were detected in the exon region of Acc28719 and Acc28717, respectively. Furthermore, in the male A. arguta accession, 1 and 20 bp of insertions were found in the intron region of Acc28871. Additionally, two InDels, including a 1 bp deletion in the male A. arguta accession, were identified in the intron region of Acc28990 (hypothetical protein), which shared homology with TDF1 from asparagus.

3.3. Development of a Sex-Discriminating InDel Marker

An InDel primer set was designed to target a 20 bp insertion specific to male A. arguta (Table S4). The targeted insertion resides in the third intron region of Acc28871, which encodes an agamous-like MADS-box protein (Figure 1a). Sequencing-read alignment results showed that the targeted 20 bp insertion was only in male A. arguta (Figure 1b). The forward primer was designed to include the inserted sequences. As a result of PCR amplification, no sex-specific amplicons were observed in female accessions, whereas ~350 bp amplicons were exclusively detected in male accessions (Figure 2). The InDel primer pair was designated as CBk25id01 (Table S4).

4. Discussion

The presence or absence of sex-determination genes located on the X and Y chromosomes is responsible for the development of male or female reproductive organs in dioecious plants. The sex-determination genes can vary among different plant species but play a crucial role in the development of floral structures, including stamens and pistils [35]. Sequence variation in the sex-related gene is a powerful tool for MAS and gene-based sex-discriminating markers that have been developed in dioecious plants [36,37]. Therefore, we explored InDels in sex-related genes to develop a gene-based sex-discriminating marker in A. arguta.
As a result of resequencing, the amount of raw and trimmed reads was lower in male accession than in female accession, while the length of the mapped region and the mapping rate showed similarity in both accessions (Table S2). Although the length of the mapped region was shorter than the genome size of the reference genome (A. chinensis Red5, 637 Mb) [23], this could potentially be influenced by the genetic distance between A. arguta and A. chinensis [20].
For the development of gene-based sex-discriminating markers, polymorphic InDels between female and male accessions were explored (Table S3). The number of InDels between both accessions and the reference genome was similar, but ~93% of the 203,116 InDels between female and male accessions were observed to be heterozygous. Therefore, heterozygosity between female and male accessions was thought to be high.
A total of 8 of the 37 sex-related genes (Table S1) originating from kiwifruit, persimmon, ginkgo, date palm, asparagus, spinach, and silene showed sequence similarity with 10 genes from the kiwifruit reference genome (Table 1). Notably, two sex-determination genes in kiwifruit, SyGl and FrBy, displayed no sequence similarity in the kiwifruit reference genome. Because the SyGl and FrBy are located on the Y chromosome [11,12] and the female reference genome lacks a Y-specific region, there were no genes exhibiting sequence similarity to SyGl and FrBy. In addition, none of the genes showed sequence similarity with MeGI and OGI from persimmon [6,7]. MeGI is located on an autosome, and the methylation levels of MeGI decide the development of male and female flowers [7], while OGI encodes small RNAs and plays a role in the repression of MeGI [6]. The differences in the principle of sex determination between the two species likely account for the absence of sequence similarities.
The 8 sex-related genes (GbMADS1, GbMADS2, GbMADS4, GbMADS18, CYP703, TDF1, GDSL esterase/lipase, and AP3Y) identified in ginkgo (Ginkgo biloba), date palm (Phoenix spp.), asparagus (Asparagus officinalis), spinach (Spinacia oleracea), and silene (Silene latifolia) displayed sequence similarity (>58.8%) to the 10 genes located on the sex chromosome of kiwifruit (Table 2). These 10 genes are known to be male-specific genes. The four MADS-box genes of ginkgo exhibited significant expression levels in staminate strobilus [8]. The CYP703 in date palm also demonstrated high expression in male flowers, while the female date palm does not have CYP703 [9]. The TDF1, located on the Y chromosome of asparagus, serves as a promoter with a function in another development [26]. The GDSL esterase/lipase in spinach and AP3Y in silene are genes linked to the male-determining locus, and they are included within the male-specific region of the Y chromosome [10,27]. This means that the sex chromosome of kiwifruit contains not only sex-determination genes (SyGl and FrBy) but also genes involved in the development of male flowers.
Sequence variations in genes have low polymorphisms, but markers based on them are desirable for marker-assisted selection. Thus, we explored InDels located within sequences of 10 genes showing sequence similarity with sex-related genes (Table 1). A total of seven InDels were detected in four genes, and most of them occurred in the sequences of agamous-like MADS-box genes, except for two InDels located in sequences of hypothetical proteins (Table 2). MADS-box genes are involved in various biological processes, including floral organ development [38]. In particular, the agamous-like MADS box gene is a C-type gene involved in stamen and carpel development in the ABCDE model that regulates floral organ development [39]. In addition, Ye et al. [19] suggested that C-type MADS-box genes were expressed in the flower of female kiwifruit, and Varkonyi-Gasic [40] confirmed that a transgenic Arabidopsis plant over-expressed agamous MADS-box gene from A. chinensis exhibited early flowering compared to the wild-type plant. Therefore, the five InDels located in three agamous-like MADS-box genes may affect floral organ development in male and female A. arguta accessions.
Among the five InDels detected in agamous-like MADS-box genes, three InDels were detected in both female A. arguta, and the reference genome was excluded because InDels in females could not be utilized in sex discrimination. The 20 bp insertion (5′-ATTCGCTGAATCTGACACTT-3′) in male A. arguta was selected as a candidate marker for sex discrimination. We expected a 20 bp size difference between female and male A. arguta accessions, but this differentiation was hindered due to the presence of three regions exhibiting high sequence similarity in the second and third introns of the agamous-like MADS-box (Figure 1a). Therefore, the forward primer sequence was designed, including the inserted sequence, to prevent amplicon production in female A. arguta. In male A. arguta, an amplicon of 93 bp was expected, but a larger amplicon of ~350 bp was generated (Figure 2b). The alignment of sequence reads revealed additional insertions following the 20 bp insertion, which is thought to contribute to the production of the ~350 bp amplicon. In addition, a non-targeted DNA fragment (~500 bp) was generated in the female ‘Ilse’, but no male-specific amplicon was observed. This might be due to the presence of a complementary genomic region in ‘Ilse’. Nevertheless, the InDel marker (CBk25id01) effectively enables sex discrimination not only in A. arguta but also in a hybrid cultivar.
The sex-discriminating marker in kiwifruit breeding is an essential tool that helps shorten breeding cycles and promote the development of improved cultivars [41]. Previously, we developed a CAPS marker discriminating female and male A. arguta accessions [18], but it requires restriction enzyme treatment. In this study, we have developed CBk25id01, a sex-discriminating InDel marker, which offers a relatively simple method for sex identification. In addition, the CBk25id01 is based on a sex-related agamous-like MADS-box gene; therefore, it could be reliably used in kiwifruit breeding programs.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae9121310/s1, Table S1: Sex-related genes identified in dioecious plants with XY sex-determination system; Table S2: Alignment results of sequencing reads from female and male Actinidia arguta accessions to the kiwifruit reference genome; Table S3: The number of insertion and deletions (InDels) detected in female and male Actinidia arguta accessions; Table S4: Information on insertion and deletion (InDel) marker discriminating sex in Actinidia arguta accessions.

Author Contributions

Conceptualization, S.O. and D.K.; methodology, S.O. and D.K.; software, S.O.; validation, S.O. and Y.K.; formal analysis, S.O. and J.K.; investigation, S.O. and J.K.; resources, M.L.; data curation, S.O.; writing—original draft preparation, S.O.; writing—review and editing, D.K.; visualization, S.O.; supervision, D.K.; project administration, D.K.; funding acquisition, D.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. RS-2020-RD009281)” Rural Development Administration, Republic of Korea.

Data Availability Statement

Data are contained within the article and supplementary materials.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Wellmer, F.; Riechmann, J.L.; Alves-Ferreira, M.; Meyerowitz, E.M. Genome-wide analysis of spatial gene expression in Arabidopsis flowers. Plant Cell 2004, 15, 1314–1326. [Google Scholar] [CrossRef]
  2. Zhang, X.; Feng, B.; Zhang, Q.; Altman, N.; Ma, H. Genome-wide expression profiling and identification of gene activities during early flower development in Arabidopsis. Plant Mol. Biol. 2005, 58, 401–419. [Google Scholar] [CrossRef] [PubMed]
  3. Ming, R.; Bendahmane, A.; Renner, S.S. Sex chromosomes in land plants. Annu. Rev. Plant Biol. 2011, 62, 485–514. [Google Scholar] [CrossRef] [PubMed]
  4. Kazama, Y.; Ishii, K.; Aonuma, W.; Ikeda, T.; Kawamoto, H.; Koizumi, A.; Filatov, D.A.; Chibalina, M.; Bergero, R.; Charlesworth, D.; et al. A new physical mapping approach refines the sex-determining gene positions on the Silene latifolia Y-chromosome. Sci. Rep. 2016, 6, 18917. [Google Scholar] [CrossRef] [PubMed]
  5. Harkess, A.; Zhou, J.; Xu, C.; Bowers, J.E.; Ven der Hulst, R.; Ayyampalayam, S.; Mercati, F.; Riccardi, P.; McKain, M.R.; Kakrana, A.; et al. The asparagus genome sheds light on the origin and evolution of a young Y chromosome. Nat. Commun. 2017, 8, 1279. [Google Scholar] [CrossRef]
  6. Akagi, T.; Henry, I.M.; Tao, R.; Comai, L. A Y-chromosome-encoded small RNA acts as a sex determinant in persimmons. Science 2014, 346, 646–650. [Google Scholar] [CrossRef] [PubMed]
  7. Akagi, T.; Henry, I.M.; Kawai, T.; Comai, L.; Tao, R. Epigenetic regulation of the sex-determination gene MeGI in polyploid persimmon. Plant Cell 2016, 28, 2905–2915. [Google Scholar] [CrossRef]
  8. Liao, Q.; Du, R.; Gou, J.; Shen, H.; Liu, H.; Nguyen, J.K.; Ming, R.; Yin, T.; Huang, S.; Yan, J. The genomic architecture of the sex-determining region and sex-related metabolic variation in Ginkgobiloba. Plant J. 2020, 104, 1399–1499. [Google Scholar] [CrossRef] [PubMed]
  9. Torres, M.F.; Mathew, L.S.; Ahmed, I.; Al-Azwani, I.K.; Krueger, R.; Rivera-Nuñez, D.; Mohamoud, Y.A.; Clark, A.G.; Suhre, K.; Malek, J.A. Genus-wide sequencing supports a two-locus model for sex-determination in Phoenix. Nat. Commun. 2018, 9, 3969. [Google Scholar] [CrossRef]
  10. Okazaki, Y.; Takahata, S.; Hirakawa, H.; Suzuki, Y.; Onodera, Y. Molecular evidence for recent divergence of X- and Y-linked gene pairs in Spinacia oleracea L. PLoS ONE 2019, 14, e0214949. [Google Scholar] [CrossRef]
  11. Akagi, T.; Henry, I.M.; Ohtani, H.; Morimoto, T.; Beppu, K.; Kataoka, I.; Tao, R. A Y-encoded suppressor of feminization arose via lineage-specific duplication of a cytokinin response regulator in kiwifruit. Plant Cell 2018, 30, 780–795. [Google Scholar] [CrossRef] [PubMed]
  12. Akagi, T.; Pilkington, S.M.; Varkonyi-Gasic, E.; Henry, I.M.; Sugano, S.S.; Sonoda, M.; Firl, A.; McNeilage, M.A.; Douglas, M.J.; Wang, T.; et al. Two Y-chromosome-encoded genes determine sex in kiwifruit. Nat. Plant 2019, 5, 801–809. [Google Scholar] [CrossRef] [PubMed]
  13. Harvey, C.F.; Fraser, L.G.; Gill, G.P. Sex determination in Actinidia. Acta Hortic. 1997, 444, 85–88. [Google Scholar] [CrossRef]
  14. Gill, G.P.; Harvey, C.F.; Gardner, R.C.; Fraser, L.G. Development of sex-linked PCR markers for gender identification in Actinidia. Theor. Appl. Genet. 1998, 97, 439–445. [Google Scholar] [CrossRef]
  15. Shirkot, P.; Sharma, D.R.; Mohapatra, T. Molecular identification of sex in Actinidia deliciosa var. deliciosa by RAPD markers. Sci. Hortic. 2022, 94, 33–39. [Google Scholar] [CrossRef]
  16. Zhang, Q.; Liu, C.; Liu, Y.; VanBuren, R.; Yao, X.; Zhong, C.; Huang, H. High-density interspecific genetic maps of kiwifruit and the identification of sex-specific markers. DNA Res. 2015, 22, 367–375. [Google Scholar] [CrossRef] [PubMed]
  17. Hale, I.; Melo, A.T.O.; Gustafson, H. Sex-linked molecular markers for two cold-hardy kiwifruit species, Actinidia arguta and A. kolomikta. Eur. J. Hortic. Sci. 2018, 83, 236–246. [Google Scholar] [CrossRef]
  18. Kim, J.; Oh, S.; Kim, Y.; Lee, M.; Kwack, Y.B.; Kim, D. Development of a CAPS marker for discriminating sex in Actinidia arguta. Hortic. Sci. Technol. 2022, 40, 233–241. [Google Scholar] [CrossRef]
  19. Ye, L.X.; Luo, M.M.; Wang, Z.; Bai, F.X.; Luo, X.; Gao, L.; Peng, L.; Chen, Q.H.; Zhang, L. Genome-wide analysis of MADS-box gene family in kiwifruit (Actinidia chinensis var. chinensis) and their potential role in floral sex differentiation. Front. Genet. 2022, 13, 1043178. [Google Scholar] [CrossRef]
  20. Oh, S.; Lee, M.; Kim, K.; Han, H.; Won, K.; Kwack, Y.B.; Shin, H.; Kim, D. Genetic diversity of kiwifruit (Actinidia spp.), including Korean native A. arguta, using single nucleotide polymorphisms derived from genotyping-by-sequencing. Hortic. Environ. Biotehcnol. 2019, 60, 105–114. [Google Scholar] [CrossRef]
  21. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011, 17, 10–12. [Google Scholar] [CrossRef]
  22. Cox, M.P.; Peterson, D.A.; Biggs, P.J. Solexa QA: At-a-glance quality assessment of Illumina second-generation sequencing data. BMC Bioinform. 2010, 11, 485. [Google Scholar] [CrossRef] [PubMed]
  23. Pilkington, S.M.; Crowhurst, R.; Hilario, E.; Nardozza, S.; Fraser, L.; Peng, Y.; Gunaseelan, K.; Simpson, R.; Tahir, J.; Deroles, S.C.; et al. A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants. BMC Genom. 2018, 19, 257. [Google Scholar] [CrossRef] [PubMed]
  24. Li, H.; Durbin, R. Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 2009, 25, 1754–1760. [Google Scholar] [CrossRef]
  25. Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R.; 1000 Genome Project Data Processing Subgroup. The sequence alignment/map format and SAMtools. Bioinformatics 2009, 25, 2078–2079. [Google Scholar] [CrossRef] [PubMed]
  26. Harkess, A.; Huang, K.; ven der Hulst, R.; Tissen, B.; Caplan, J.L.; Kloppula, A.; Batish, M.; Meyers, B.C.; Leebens-Mack, J. Sex determination by two Y-linked genes in garden asparagus. Plant Cell 2020, 32, 1790–1796. [Google Scholar] [CrossRef]
  27. Cegan, R.; Marais, G.A.B.; Kubekova, H.; Blavet, N.; Widmer, A.; Vyskot, B.; Doležel, J.; Šafář, J.; Hobza, R. Structure and evolution of Apetala3, a sex-linked gene in Silene latifolia. BMC Plant Biol. 2010, 10, 180. [Google Scholar] [CrossRef] [PubMed]
  28. Guttman, D.S.; Charlesworth, D. An X-linked gene with a degenerate Y-linked homologue in a dioecious plant. Nature 1998, 393, 263–266. [Google Scholar] [CrossRef]
  29. Delichère, C.; Veuskens, J.; Hernould, M.; Barbacar, N.; Nouras, A.; Negrutiu, I.; Monéger, F. SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD-repeat protein. EMBO J. 1999, 18, 4169–4179. [Google Scholar] [CrossRef]
  30. Atanassov, I.; Delichère, C.; Filatov, D.A.; Charlesworth, D.; Negrutiu, I.; Monéger, F. Analysis and evolution of two functional Y-linked loci in a plant sex chromosome system. Mol. Biol. Evol. 2001, 18, 2162–2168. [Google Scholar] [CrossRef]
  31. Moore, R.C.; Kozyreva, O.; Lebel-Hardenack, S.; Siroky, J.; Hobza, R.; Vyskot, B.; Grant, S.R. Genetic and functional analysis of DD44, a sex-linked gene from the dioecious plant Silene latifolia, provides clues to early events in sex chromosome evolution. Genetics 2003, 163, 321–334. [Google Scholar] [CrossRef] [PubMed]
  32. Qiu, S.; Bergero, R.; Forrest, A.; Kaiser, V.B.; Charlesworth, D. Nucleotide diversity in Silene latifolia autosomal and sex-linked genes. Proc. R. Soc. B 2010, 227, 3283–3290. [Google Scholar] [CrossRef] [PubMed]
  33. Muir, G.; Bergero, R.; Charlesworth, D.; Filatov, D.A. Does local adaptation cause high population differentiation of Silene latifolia Y chromosomes? Evolution 2011, 62, 3368–3380. [Google Scholar] [CrossRef]
  34. Weingartner, L.A.; Delph, L.F. Neo-sex chromosome inheritance across species in Silene hybrids. J. Evol. Biol. 2014, 27, 1491–1499. [Google Scholar] [CrossRef] [PubMed]
  35. Chanderbali, A.S.; Yoo, M.J.; Zahn, L.M.; Brockington, S.F.; Wall, P.K.; Gitzendanner, M.A.; Albert, V.A.; Leebens-Mack, J.; Altman, N.S.; Ma, H.; et al. Conservation and canalization of gene expression during angiosperm diversification accompany the origin and evolution of the flower. Biol. Sci. 2010, 107, 22570–22575. [Google Scholar] [CrossRef] [PubMed]
  36. Men, Y.; Li, J.R.; Shen, H.L.; Yang, Y.M.; Fan, S.T.; Li, K.; Guo, Y.S.; Lin, H.; Liu, Z.D.; Guo, X.W. VaAPRT3 gene is associated with sex determination in Vitis amurensis. Front. Genet. 2022, 12, 727260. [Google Scholar] [CrossRef] [PubMed]
  37. Khanum, P.; Khan, A.A.; Khan, I.A.; Chaffar, A.; Khan, Z. TPD1-like gene as a suitable marker for early sex determination in date palm (Phoenix dactylifera L.). Genes 2023, 14, 907. [Google Scholar] [CrossRef]
  38. Kong, X.; Wang, F.; Geng, S.; Guan, J.; Tao, S.; Jia, M.; Sun, G.; Wang, Z.; Wang, K.; Ye, X.; et al. The wheat AGL6-like MADS-box gene is a master regulator for floral organ identity and a target for spikelet meristem development manipulation. Plant Biotechnol. J. 2021, 20, 75–88. [Google Scholar] [CrossRef]
  39. Shen, G.; Yang, C.H.; Shen, C.Y.; Huang, K.S. Origination and selection of ABCDE and AGL6 subfamily MADS-box genes in gymnosperms and angiosperms. Biol. Res. 2019, 52, 25. [Google Scholar] [CrossRef]
  40. Varkony-Gasic, E.; Moss, M.S.; Voogd, G.; Wu, R.; Lough, R.H.; Wnag, Y.Y.; Hellens, R.P. Identification and characterization of flowering genes in kiwifruit: Sequence conservation and role in kiwifruit flower development. BMC Plant Biol. 2011, 11, 72. [Google Scholar] [CrossRef]
  41. De Mori, G.; Testolin, R.; Cipriani, G. A molecular protocol for early sex discrimination (ESD) in Actinidia spp. J. Berry Res. 2022, 12, 249–266. [Google Scholar] [CrossRef]
Horticulturae 09 01310 g001
Figure 1. Insertion and deletion (InDel) structure located in the agamous-like MADS-box protein (Acc28871) (a). The yellow boxes are coding regions, and the black lines without coding regions are intron. The asterisk regions on the second and third introns have ~99% sequence similarity. Sequences highlighted in blue are insertions in the agamous-like MADS-box protein of male Actinidia arguta. Alignment of sequencing readings indicate insertion into reads from male A. arguta (b). Purple squares with numbers in gray bars are insertions and inserted size (bp). Red bars are insertions that are larger than expected, and other colored bars are the reads from different chromosomes.
Figure 1. Insertion and deletion (InDel) structure located in the agamous-like MADS-box protein (Acc28871) (a). The yellow boxes are coding regions, and the black lines without coding regions are intron. The asterisk regions on the second and third introns have ~99% sequence similarity. Sequences highlighted in blue are insertions in the agamous-like MADS-box protein of male Actinidia arguta. Alignment of sequencing readings indicate insertion into reads from male A. arguta (b). Purple squares with numbers in gray bars are insertions and inserted size (bp). Red bars are insertions that are larger than expected, and other colored bars are the reads from different chromosomes.
Horticulturae 09 01310 g001
Horticulturae 09 01310 g002
Figure 2. Insertion and deletion (InDel) marker based on the 20 bp InDel structure was applied to female (a) and male (b) Actinidia arguta accessions. The black triangle indicates a male-specific amplicon. M, 100 bp ladder marker.
Figure 2. Insertion and deletion (InDel) marker based on the 20 bp InDel structure was applied to female (a) and male (b) Actinidia arguta accessions. The black triangle indicates a male-specific amplicon. M, 100 bp ladder marker.
Horticulturae 09 01310 g002
Table 1. Thirty-seven kiwifruit accessions used for sex discrimination.
Table 1. Thirty-seven kiwifruit accessions used for sex discrimination.
SexSpeciesAccession
FemaleActinidia argutaK5-1-8K5-1-12K5-2-3K5-2-13
K5-2-18K5-4-7K5-7-3K5-7-5
K5-10-1K5-10-5K5-12-6Chiak
Hardy RedIlseAutumn Sense
(A. arguta × A. deliciosa) × A. argutaSkinny Green
MaleA. argutaK5-2-22K5-3-3K5-3-6K5-3-7
K5-3-8K5-3-12K5-3-13K5-3-18
K5-4-8K5-5-1K5-5-12K5-5-14
K5-6-10K5-9-1K5-9-8K5-11-1
K5-11-2K5-11-3K5-11-8K5-13-6
K5-14-1
Table 2. Candidate genes showing homology (sequence identity ≥ 40% and E-value ≤ 1 × 10−10) to previously identified sex-related genes in the kiwifruit reference genome.
Table 2. Candidate genes showing homology (sequence identity ≥ 40% and E-value ≤ 1 × 10−10) to previously identified sex-related genes in the kiwifruit reference genome.
Sex-Related GeneGene ID 1DescriptionIdentity (%)E-Value
GbMADS1Acc28871Agamous-like MADS-box protein68.331.08 × 10−41
GbMADS2
GbMADS4
GbMADS18
CYP703Acc28333Cytochrome P450 CYP736A12 like58.841.85 × 10−75
TDF1Acc28900Hypothetical protein78.637.05 × 10−57
Acc28821Transcription factor like73.851.77 × 10−49
Acc28639Transcription factor like68.642.73 × 10−39
Acc28638Transcription factor like68.642.73 × 10−39
Acc28429Myb-related protein73.261.98 × 10−30
GDSL esterase/lipaseAcc28776GDSL esterase/lipase, partial74.789.52 × 10−145
AP3YAcc28719Agamous-like MADS-box protein69.495.06 × 10−12
Acc28717Agamous-like MADS-box protein62.222.03 × 10−22
1 Gene ID of Red5 from the kiwifruit genome database.
Table 3. Insertion and deletions (InDels) detected in the genes that show similarity to sex-related genes.
Table 3. Insertion and deletions (InDels) detected in the genes that show similarity to sex-related genes.
VariationPosition (bp)InDel (Reference/Alternative)Gene ID 1RegionDescription
FemaleMale
Insertion12,919,896-/AT-/-Acc28719ExonAgamous-like MADS-box protein
14,557,224-/AG-/-Acc28871IntronAgamous-like MADS-box protein
14,559,075-/--/TAcc28871IntronAgamous-like MADS-box protein
14,570,161-/--/ATTCGGTGAATCTGACACTTAcc28871IntronAgamous-like MADS-box protein
Deletion12,896,671TGAT/-TGAT/TGATAcc28717ExonAgamous-like MADS-box protein
14,814,902A/AA/-Acc28900IntronHypothetical protein
14,815,837-/--/TAcc28900IntronHypothetical protein
1 Gene ID of Red5 from the kiwifruit genome database.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Oh, S.; Kim, J.; Kim, Y.; Lee, M.; Kim, D. A MADS-Box Gene-Based InDel Marker Discriminating Sex in Actinidia arguta. Horticulturae 2023, 9, 1310. https://doi.org/10.3390/horticulturae9121310

AMA Style

Oh S, Kim J, Kim Y, Lee M, Kim D. A MADS-Box Gene-Based InDel Marker Discriminating Sex in Actinidia arguta. Horticulturae. 2023; 9(12):1310. https://doi.org/10.3390/horticulturae9121310

Chicago/Turabian Style

Oh, Sewon, Jung Kim, Yumi Kim, Mockhee Lee, and Daeil Kim. 2023. "A MADS-Box Gene-Based InDel Marker Discriminating Sex in Actinidia arguta" Horticulturae 9, no. 12: 1310. https://doi.org/10.3390/horticulturae9121310

APA Style

Oh, S., Kim, J., Kim, Y., Lee, M., & Kim, D. (2023). A MADS-Box Gene-Based InDel Marker Discriminating Sex in Actinidia arguta. Horticulturae, 9(12), 1310. https://doi.org/10.3390/horticulturae9121310

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop