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Article

Authenticity Identification of F1 Hybrid Offspring and Analysis of Genetic Diversity in Pineapple

by
Panpan Jia
1,2,3,†,
Shenghui Liu
1,3,†,
Wenqiu Lin
1,3,4,*,
Honglin Yu
1,3,
Xiumei Zhang
1,3,
Xiou Xiao
1,3,
Weisheng Sun
1,3,4,
Xinhua Lu
1,3 and
Qingsong Wu
1,3,4,*
1
South Subtropical Crop Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524091, China
2
School of Horticulture and Forestry, Huazhong Agricultural University, Wuhan 430070, China
3
Laboratory of Tropical Fruit Biology, Ministry of Agriculture, Zhanjiang 524091, China
4
Hainan Provincial Engineering Research Center for Pineapple Germplasm Innovation and Utilization, Zhanjiang 524091, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(7), 1490; https://doi.org/10.3390/agronomy14071490
Submission received: 21 May 2024 / Revised: 28 June 2024 / Accepted: 5 July 2024 / Published: 9 July 2024
(This article belongs to the Special Issue Advances in Crop Molecular Breeding and Genetics)
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Abstract

:
Breeding is an effective method for the varietal development of pineapple. However, due to open pollination, it is necessary to conduct authentic identification of the hybrid offspring. In this study, we identified the authenticity of offspring and analyzed the genetic diversity within the offspring F1 hybrids resulting from crosses between ‘Josapine’ and ‘MD2’ by single nucleotide polymorphism (SNP) markers. From the resequencing data, 26 homozygous loci that differentiate between the parents have been identified. Then, genotyping was performed on both the parents and 36 offspring to select SNP markers that are suitable for authentic identification. The genotyping results revealed that 2 sets of SNP primers, namely SNP4010 and SNP22550, successfully identified 395 authentic hybrids out of 451 hybrid offspring. We randomly selected two true hybrids and four pseudohybrids for sequencing validation, and the results have shown that two true hybrids had double peaks with A/G, while pseudohybrids had single peaks with base A or G. Further study showed that the identification based on SNP molecular markers remained consistent with the morphological identification results in the field, with a true hybridization rate of 87.58%. K-means clustering and UPGMA tree analysis revealed that the hybrid offspring could be categorized into two groups. Among them, 68.5% of offspring aggregated with MD2, while 31.95% were grouped with Josapine. The successful application of SNP marker to identify pineapple F1 hybrid populations provides a theoretical foundation and practical reference for the future development of rapid SNP marker-based methods for pineapple hybrid authenticity and purity testing.

1. Introduction

Pineapple (Ananas comosus (L.) Merr) is indigenous to southern Brazil and Paraguay, and is one of the most economically significant tropical fruit in the world; it is also the only edible fruit of the Bromeliaceae family. The fruit is the sold fresh, dried, or in fruit juices, and is generally used as a source of flavors and fragrances. It has been cultivated in more than 80 countries or regions all around the world [1]. In recent years, the total world production for pineapples has increased, reaching about 29,361,138 metric tons in 2022 (FAOSTAT). China is one of the ten top pineapple-producing countries all over the world [2]. It is predominantly cultivated in provinces, such as Guangdong, Hainan, Yunnan, Guangxi and Fujian, which plays a pivotal role in the tropics and significantly contributes to rural revitalization in China [3]. ‘Comte de Paris’ is the major cultivar in China, with a history spanning nearly a century, which resulting in concentrated harvesting and diminished quality, thereby impeding the development of the pineapple industry. The selection and promotion of new varieties represent effective measures for adjusting varietal structures and enhancing industrial quality. Crossbreeding can effectively improve the objective trait, such as high yield, high quality, strong disease resistance, and good adaptability, and this is the key way to cultivate new varieties of pineapple. ‘MD-2’ (PRI hybrid 73-114), a hybrid bred by the Hawaiian Pineapple Research Institute in the United States with ‘Smooth Cayenne’ as the parent, is among the most important fresh pineapple varieties in the world and has a comparable yield and a good sugar profile to balance acid during the winter months [4]. Cabral et al. have bred new variety ‘Imperia’ from a crossing between ‘Perolera’ and ‘Smooth Cayenne’ [5]. In China, crossbreeding has made remarkable progress. New varieties of pineapple bred through hybrid selection in mainland China include ‘Yue Cui’, ‘Yue Tong’, ‘Yue tian’, and ‘Renong56’ [6,7].
Given the self-incompatibility of pineapples, pollination between of the same variety does not result in seed production [8]. Therefore, the crossbreeding of pineapple is mainly carried out through artificial pollination directly on the mother plants without the removal of stamens. Moreover, due to prolonged developmental pollination patterns, bees and other insects are involved in cross-pollination, leading to the outcome that the offspring might not come from the intended parents [9]. Pineapple, a perennial monocotyledonous herbaceous fruit tree, typically requires a minimum of 2.5 years from seed sowing to fruiting. Usually, the authenticity identification of hybrid offspring is investigated in field, but this is time-consuming and labor-intensive. Identifying early hybrid seedlings proves effective in preventing the wastage of resources during the subsequent breeding of individuals that do not align with the breeding objectives. Furthermore, seedling identification enables the early detection and elimination of potentially undesirable gene combinations, fostering variety improvement and supporting genetic studies [10].
Morphological characteristics observation, fluorescence in situ hybridization, chromosome counting, and molecular labeling are the common identification methods for the identification of offspring. Currently, hybrid authenticity identification primarily relies on observing the morphological characteristics of hybrid offspring in pineapple. However, the concentrated use of parental material results in diminishing morphological differences among hybrid offspring. Furthermore, morphological identification is vulnerable to the impact of external environmental factors and subjective breeder judgment. It also faces challenges, such as being time-consuming and suffering from difficulties in quantification [11,12]. Fluorescence in situ hybridization (FISH) and chromosome counting are also used to authenticate the plants. However, due to the fact that these techniques are complex, technically demanding, time-consuming, and labor-intensive, requiring strict sample preparation and processing and relying on professional equipment, they are not suitable for large-scale applications [13]. Additionally, the biochemical labeling identification method is constrained by expensive experimental techniques, time-consuming processes, and the need for professional knowledge and high-quality samples. These shortcomings restrict its efficiency in large-scale applications [13]. With the development of sequencing, molecular markers are widely use in hybrid authenticity and purity identification; this method has the advantages of high accuracy, high efficiency, not being affected by environmental conditions, and scientific data quantification, which can fully reveal the genetic information between offspring and parents from the genome level [14].
Molecular markers have been successfully applied to the identification of hybrid authenticity and genetic diversity analysis in maize, gourd, mango, rape, and citrus [15,16,17,18,19,20], including random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restricted fragment length polymorphism (RFLP), simple repeat sequence (SSR), and inter simple sequence repeat (ISSR) [15,16,17,18,19,20]. However, these molecular markers still have shortcomings in terms of stability, polymorphism and automation level of operation and hard to data sharing [21,22,23,24,25]. The single nucleotide polymorphism marker (SNP) is the latest generation of molecular markers, which refers to the DNA sequence polymorphism caused by a single nucleotide variation at the genome level [26]. This technique relies on allele-specific oligonucleotide elongation and fluorescence resonance energy transfer for signal detection [27]. SNP markers have the advantages of high polymorphism, easy detection, good stability, and high throughput, and they are suitable for genome-wide association studies [28,29,30]. They have also been recognized by the International Union for the Protection of New Varieties of Plants (UPOV) BMT Molecular Detection Guidelines as a standard method for determining variety or seed purity.
The advancement of whole-genome sequencing technology has significantly facilitated the application of SNP markers in verifying hybrid authenticity in several crops, such as maize, rice, melon, and cowpea [31,32,33,34]. Josia et al. used 92 SNP markers to assess the genetic purity of 26 inbred lines, 4 doubled haploid lines, and 158 single-cross maize hybrids, revealing that 67% of the inbred lines were pure, while 33% showed heterozygosity levels exceeding 5% [31]. In rice, 41 SNP markers were used for both Inpari Blas (i.e., line number 16, 21 and 22) and Inpari HDB (i.e., line number 10, 15 and 18) identification by inter-varietal genotyping [32]. Kishor et al. employed 96 genome-wide SNP markers and high-throughput Fluidigm genotyping technology to successfully distinguish 85 melon F1 hybrids, their parental lines, and 6 PT melon breeding lines [33]. In cowpea F1 plants, 79% of the putative were true hybrids, 14% were selfed plants, and 7% were undetermined by 17 SNP markers [34]. However, there is currently no research on the application of SNP markers to authenticate the hybrid F1 generation population of pineapples.
In this study, both homozygous and discrepant loci of parental were screened according to the genome resequencing data of ‘Josapine’ and ‘MD2’ to develop SNP markers. SNP markers were used to identify the hybrid offspring of ‘Josapine’ and ‘MD2’, and we verified the results of SNP typing by using DNA sequencing. Subsequently, K-means and UPGMA methods were used to analyze the genetic diversity of the offspring. Fast and accurate identification of target offspring at the seedling stage saves time and money, improves selection efficiency, increases genetic diversity, and ensures excellent traits in the planted varieties. This development not only promotes the breeding process of new varieties, but also makes genetic analysis of key traits possible.

2. Materials and Methods

2.1. Plant Materials and DNA Extraction

The hybrid population was constructed with ‘MD2’ as the male parent and ‘Josapine’ as the female parent (Figure 1A,B). A total of 451 hybrid offspring (named as JM1–JM451) resulting from the crossbreeding through artificial pollination was obtained in March–April 2020. These parents and offspring were cultivated in the South Subtropical Crop Research Institute Zhanjiang, Guangdong, China (21_1002″ N; 110_16034″ E).
The white part of the base of fresh young pineapple leaves were extracted using the improved CTAB method [35] (Figure 1C), and the DNA of two parents and hybrid F1 generation leaves were extracted. The DNA sample concentration was uniformly diluted with ddH2O to 10–100 ng/μL. The uniform concentration DNA was stored in a 96-well plate and placed in a −20 °C refrigerator for subsequent PCR amplification.

2.2. Establishment of System for Authenticity Verification

In this study, SNP markers were obtained from pineapple germplasm resources by resequencing. Homozygous SNP loci with different parental genotypes (‘MD2’ and ‘Josapine’) were screened to authenticate the hybrid offspring. Seventeen SNPs exhibiting strong polymorphisms were selected for genetic diversity analysis, considering four parameters: minimum allele frequency (MAF) > 0.43, deletion rate < 3%, PIC > 0.37, and heterozygosity < 0.4. Primers were designed based on the SNP loci information using the primer design tool (http:/www.snpway.com (accessed on 6 May 2023)) on the webpage of Wuhan Jingpeptide Biotechnology Co., Wuhan, China). For each SNP loci, two allele-specific primers and one universal primer were designed. The primers were synthesized by Wuhan Sangon Biological Co., Ltd. (Wuhan, China) with FAM- or HEX-tails (FAM tail: 5-GAAGGTGACCAAGTTCATGCT-3′; HEX tail: 5′-GAAGGTCGGAGTCAACGGATT-3′). The 2×PARMS mix reagent was procured from Wuhan Jingpeptide Biotechnology Co. (Wuhan, China). The PARMS assay was performed in a 6 μL PCR system/condition that consisted of 3 μL of PARMS master mix (Wuhan Jingpeptide Biotechnology Co., Wuhan, China), approximately equal to 0.45 μL of primer, 1.55 μL of H2O, and 1 μL of DNA at a concentration of 10–100 ng/μL. The sample DNA of ‘Josapine’, ‘MD2’, and 451 offspring, along with the specified system, were dispensed into 384-well plates using an INTEGR pipette. The PCR program was as follows: 15 min at 94 °C, 10 touchdown cycles of 94 °C for 20 s and 65–55 °C for 60 s (decreasing by 0.7 °C percycle), and 26 cycles of 94 °C for 20 s and 57 °C for 60 s. After the PCR, fluorescence data were read and analyzed using the ABI QuantStudio6 QS6 instrument (USA, AppliedBiosystems, Waltham, MA, USA).

2.3. Authenticity Verification of Hybrids

PARMS utilizes FAM and HEX as the reporter fluorescence for the 2 alleles, with ROX fluorescence serving as the internal reference fluorescence. Genotyping is fluorescence-based, and the corresponding signals appear when the allele undergoes amplification. SNP markers are employed for genotyping parents and hybrid progenies through competitive allele-specific PCR, detecting the fluorescence signal value of the measured locus [36,37]. The genomic DNA of ‘Josapine’, ‘MD2’, and 451 hybrid offspring were genotyped using the selected primers. The genotypes of each sample were tallied. If the sample’s genotype was pure, it was categorized as a pseudohybrid, and if the sample had a heterozygous genotype, it was classified as a true hybrid.
Ensembl Plant (https://plants.ensembl.org/index.html (accessed on 30 May 2023)) was utilized to fetch before and after 250 bp sequences of the 5578047 SNP site from contig18, which was from the SNP22550 marker. Specific primers were designed using NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 16 June 2023)) to demonstrate genotyping reliability. The primers’ information was followed: (F22550: 5′-ATCATTCTCGCTTGCCTCCG-3′; R22550: 5′-TCC ATGTAACTCCAGCATTTCAGA-3′). The DNA of two offspring of each genotypes used as a template were randomly selected for PCR amplification, followed by electrophoresis detection. Subsequently, these PCR products were sent to Sangon Biotech (Shanghai, China) for sequencing. If the sequencing peak graph of the SNP locus exhibited a single peak, this indicated a pure genotype. Conversely, if these sequencing peak graphs showed overlapping peaks, this signified a heterozygous genotype.

2.4. Genetic Diversity Analysis

‘MD2’, ‘Josapine’, and their hybrid offspring underwent genotyping using 18 sets of SNP markers. Hybrid offspring with a high deletion rate were excluded. Subsequently, the genotypic data of the parents and 313 hybrid offspring were analyzed. The SNP genotype data were converted into binary coded data using Excel 2016 software, with the wild type indicated as (1, 1), the mutant as (2, 2), heterozygous genotypes as (1, 2), and deletion sites recorded as (0, 0). Genetic diversity parameters, such as minimum allele frequency (MAF), gene diversity (GD), heterozygosity (He), and polymorphism information content (PIC) were calculated using the Powermarker V3.25 software [38]. We used R language (# install. Packages (“factoextra”) and # install. Packages (“cluster”)) calculated the value of the K-means to carry out the cluster analysis. The two-by-two genetic distance matrix of the genotyping data was calculated, and a neighbor-joining tree (NJ) was constructed by Nei (1973) [39] using the standard genetic distance; it was visualized on MEGA 11(version 11.0.13) [40].

3. Results

3.1. Screening of Pure Co-Dominant SNP Markers in Hybrid Parents

Based on the information of pineapple whole-genome resequencing SNP data, we screened for SNP markers that exhibited different pure genotypes between ‘Josapine’ and ‘MD2’. According to this criterion, a total of 26 sets of primers were obtained by screening between the parents of ‘Josapine’ and ‘MD2’. These primers were distributed across LG1, LG2, LG3, LG5, LG6, LG7, LG8, LG11, LG13, LG14, LG15, LG18, LG19, LG21, and LG24, covering a total of 15 chromosomes (Supplementary Materials, Table S1). Primer screenings for the 26 sets of SNP markers involved ‘Josapine’, ‘MD2’, and 36 F1 progenies, and only 6 representative typing results were cited in this paper (Figure 2).
Out of 26 polymorphic SNP markers, only 18 were able to distinguish between 2 parents and 36 hybrids. Notably, SNP4010 and SNP22550 had the highest success rate in distinguishing between parents and hybrids, both at 87.5%. SNP1247, SNP12371, SNP2640, SNP7124, SNP22550, and SNP4010 surpassed a 50% success rate in hybrid identification. Furthermore, SNP12223 and SNP4855 exhibited success rates of less than 10% in identifying hybrids (Figure 3).

3.2. Identification of Hybrid Authenticity

In order to authenticate the authenticity of the hybrid offspring, 451 hybrid offspring, ‘Josapine’, and ‘MD2’ underwent PCR amplification by SNP4010 and SNP22550. The typing results showed that 28 and 17 individual plants exhibiting genotypic consistency with ‘Josapine’ were identified using SNP4010 and SNP22550, with genotypes GG and AA, respectively. Additionally, 19 and 27 individual plants exhibiting genotypic consistency ‘MD2’ were identified using SNP4010 and SNP22550, respectively. Furthermore, a total of 404 and 407 individual plants exhibiting hybrid genotypes GC and AG were identified by SNP4010 and SNP22550, respectively (Figure 4). Combining the two markers, only those that showed both true hybrids were recognized as true hybrids, and a total of 395 true hybrids and 56 false hybrids were identified, with a true hybridization rate of 87.58% (Table 1).

3.3. Sequencing Verification of True Hybrids and Pseudohybrids

In order to verify the accuracy of genotyping, specific primers of the SNP22550 (5578047) position, which is located in chromosome 18, were designed, namely F22550 and R22550. These primers were used to amplify the PCR of the pseudohybrids MJ83, MJ11, MJ227, MJ270, and the true hybrids MJ129, MJ173, respectively, which were identified from genotyping. The PCR products were sent for sequencing by Sangon Biotech (Shanghai, China). These results showed that MJ11 and MJ270 had single peaks at position 5578047 with base A; MJ227 and MJ83 had single peaks with base G; and MJ129 and MJ173 had peak sets with base AG (Figure 5). These results indicated that MJ11, MJ270, MJ83, and MJ227 were pseudohybrids, and that MJ129 and MJ173 were true hybrids. The DNA sequencing results were consistent with the SNP typing results, indicating the stability and credibility of the SNP typing results.

3.4. Genetic Diversity Analysis

A total of 18 SNP markers were used to analyze the genetic diversity of 313 offspring, which remained after we removed the offspring with a high deletion rate (Table S2). The statistical information on genetic diversity revealed that the mean minor allele frequency (MAF) was 0.334, with a minimum of 0.164 (SNP28156), and a maximum of 0.500 (SNP4010) (Table 2). The prevalence of a MAF between 0.20 and 0.30 was 47.06%, followed by from 0.40 to 0.50 and from 0.30 to 0.40 at 35.29% and 11.70%, respectively (Table 2, Figure 6A), indicating a relatively low overall frequency of minor alleles in the markers used. The mean gene diversity index was 0.422, ranging from 0.274 (SNP28156) to 0.500 (SNP4010), with the majority falling between 0.40 and 0.50 (58.82%), followed by 0.30 and 0.40 (35.29%) (Table 2, Figure 6B). This result suggests a relatively high genetic diversity in the markers. The mean observed heterozygosity was 0.429, the maximum value was 0.994 (SNP4010), and the minimum value was 0.191 (SNP30909) (Table 2). Due to the use of SNP markers with two alleles, heterozygosity was primarily distributed between 0.40 and 0.50 (35.29%), followed by 0.20 to 0.30, 0.30 to 0.40, and 0.50 to 0.60, all relatively evenly distributed, and with each accounting for 17.65% (Figure 6C). Polymorphic information content (PIC) was mainly below 0.40, averaging 0.331, and ranging from 0.236 (SNP28156) to 0.750 (SNP5172, SNP20855, and SNP4010) (Table 2). It was evenly distributed in the ranges of 0.30 to 0.35 and 0.35 to 0.40, followed by 0.25 to 0.30 at 17.65% (Figure 6D).

3.5. Clustering Patterns and Class Composition among Genotypes of Parents and Offspring

Here, 18 sets of SNP markers were chosen for a clustering analysis involving the parents and 313 hybrid offspring. The value of the K-means was calculated by using R language (#install.packages (“factoextra”) and #install.packages (“cluster”)). The results have shown that when the number of clusters K was two, the value of the gap statistic K was at its maximum. According to the K-means value, 313 hybrid offspring were clustered into two groups. Cluster I was associated with the parent ‘MD2’, and cluster II was associated with the parent ‘Josapine’ (Figure 7).
The UPGMA clustering revealed that the 313 hybrid offspring were divided into 2 classes, consistent with the results of K-means clustering analysis. Among these, 213 hybrid offspring were grouped with the parent ‘MD2’, representing 68.05% of the total, while 100 hybrid offspring were associated with the parent ‘Josapine’, representing 31.95% of the total (Figure 8). The genetic composition of the hybrid offspring consists of two-thirds maternal genetic information and one-third paternal genetic information.

4. Discussion

The authentication of hybrid offspring plays an important role in maintaining the precision of plant genetic background, advancing gene function research, and facilitating the breeding of new varieties. In pineapple, the artificial cross-pollination process may lead to the generation of pseudohybrids, attributed to different factors, such as an unclear pollen source, a naturally heterogametic pollination, and irregular operations [32]. The presence of pseudohybrids disrupts the accuracy of genetic analysis and gene mapping and reduces breeding efficiency within the population. Usually, the authenticity of the hybrid offspring of pineapples is determined by multi-year and multi-site morphological analysis, which can time-consuming, laborious, and potentially influenced by environmental factors due to certain characteristics, leading to biased results.
Molecular markers are widely used for the authentication of hybrid offspring. In sweet tea, only one pair is required to identify all common-type hybrid offspring by RAPD, regardless of whether co-dominant or pure dominant markers are used [41]. However, there are at least five parental markers to identify true hybrids when heterozygous dominant markers are employed; the hybridization rate is 97% [42]. Eight pairs of highly polymorphic SSR markers were employed to identify sixty-five mango F1 hybrids as true. A total of 62 true hybrids were identified, resulting in a hybridization rate of 95.38%. Although these markers can be used for authenticating offspring, they have fewer detection sites, lower throughput, and are not easily conducive to data sharing [43].
SNP molecular markers, representing the latest generation of molecular markers, can be typed using high-throughput and low-cost technology, are suitable for large-scale high-throughput testing platforms, and significantly shorten the identification time. Previous studies have shown that only one pair of pure co-dominant SNP markers is required to screen pure SNP loci with parental differences for hybrid authenticity [44]. Utilizing competitive allele-specific PCR based on single nucleotide polymorphisms to identify hybrid authenticity in cowpea, KASP-SNP markers detected true hybrids in 72% of the population with a 100% success rate [34]. In this study, we screened for the pure SNP markers SNP4010 and SNP22550, with both exhibiting differences between parents. Subsequently, we utilized these two markers to identify 395 true hybrid plants, resulting in a true hybrid rate of 87.5%.
The absence or nullity of alleles in the maternal locus has been reported to be a cause of pseudohybrids. Our study revealed the detection of 19 and 23 individual plants in pseudohybrids with the same genotype as the maternal plant for SNP4010 and SNP22550, respectively. The probable reason for this absence of the bi-parental locus may stem from abnormal chromosome exchanges, recombination, or mutations due to DNA modification. Similar results were found in mangoes, i.e., the absence of maternal loci was exhibited in the hybrid offspring by 14 AFLP markers [43]. Additionally, it is suggested that there may be interference in the binding of certain sites and individual primers, leading to site deletions [42]. The phenomenon of null alleles also exists in the identification of early poplar hybrid offspring using co-dominant SNP markers, and such hybrid offspring are not included in the final count [45]. Whether the deletion of parental loci can be used as a basis for preliminary determination of hybrids requires more in-depth research. In the present study, combined with field phenotyping, some plants with the same genotype as the parents were indeed not hybrid offspring of ‘Josapine’ and ‘MD2’. Therefore, it is considered that only hybrid offspring with heterozygous genotypes are true hybrids.

5. Conclusions

In this study, 26 homozygous loci were used to develop SNP markers to screen the authentic hybrids offspring of ‘Josapine’ and ‘MD2’. SNP4010 and SNP22550 were successfully used to distinguish between true- and pseudohybrid offspring. After verification of the genotyping and DNA sequencing, the true hybridization rate was 87.58%. Furthermore, these offspring were clustered into two groups, 68.5% of which were related to ‘MD2’, and 31.95% of which were related to ‘Josapine’. These findings serve as a valuable reference for the early discernment of genuine and spurious hybrids in future crossbreeding endeavors involving pineapple and other fruit trees.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14071490/s1, Table S1 The information of 26 primers; Table S2 The information of 18 primers.

Author Contributions

Conceptualization, P.J., S.L. and W.L.; Methodology, W.L. and Q.W.; Software, P.J. and H.Y.; Validation, P.J. and X.X.; Formal analysis, X.Z., W.S. and X.L.; Investigation, S.L. and X.L.; Resources, W.S. and X.L.; Data curation, P.J. and H.Y.; Writing—original draft, P.J. and S.L.; Writing—review and editing, W.L. and Q.W.; Visualization, X.Z. and X.L.; Supervision, W.L., H.Y. and X.X.; Project administration, X.Z. and Q.W.; Funding acquisition, Q.W. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by the Central Public-interest Scientific Institution Basal Re-search Fund (No. 1630062024010); the National Tropical Plants Germplasm Resource Center (NTPGRC2023NTPGRC2024-027); and the National Key Research and Development Special Program of the People’s Republic of China (No. 2019YFD1000505).

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Material, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare that they have no competing interests.

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Figure 1. The fruits of hybrid parents. (A) Josapine, (B) MD2, and (C) Leaf base.
Figure 1. The fruits of hybrid parents. (A) Josapine, (B) MD2, and (C) Leaf base.
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Figure 2. Typing results of some primers. (A) The genotyping of SNP4010, (B) the genotyping of SNP22550, (C) the genotyping of SNP10432, (D) the genotyping of SNP16328, (E) the genotyping of SNP20777, and (F) the genotyping of SNP25886. Note: •  represents the genotype of ‘Josapine’, •  represents the genotype of ‘MD2’, and •  represents the genotype of the F1 generation of the cross.
Figure 2. Typing results of some primers. (A) The genotyping of SNP4010, (B) the genotyping of SNP22550, (C) the genotyping of SNP10432, (D) the genotyping of SNP16328, (E) the genotyping of SNP20777, and (F) the genotyping of SNP25886. Note: •  represents the genotype of ‘Josapine’, •  represents the genotype of ‘MD2’, and •  represents the genotype of the F1 generation of the cross.
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Figure 3. Efficiency of SNP markers in identification of hybrid authenticity.
Figure 3. Efficiency of SNP markers in identification of hybrid authenticity.
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Figure 4. Genotyping results of 451 single plants. (A,B) Genotyping of SNP4010; (C,D) genotyping of SNP22550. Note: • represents the genotype of ‘Josapine’, • represents the genotype of ‘MD2’, and • represents the genotype of the F1 generation of the cross.
Figure 4. Genotyping results of 451 single plants. (A,B) Genotyping of SNP4010; (C,D) genotyping of SNP22550. Note: • represents the genotype of ‘Josapine’, • represents the genotype of ‘MD2’, and • represents the genotype of the F1 generation of the cross.
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Figure 5. Leaf phenotypes and sequencing results of some true- and pseudohybrids. (A) MJ11, (B) MJ270, (C) MJ129, (D) MJ173, (E) MJ227, and (F) MJ83.
Figure 5. Leaf phenotypes and sequencing results of some true- and pseudohybrids. (A) MJ11, (B) MJ270, (C) MJ129, (D) MJ173, (E) MJ227, and (F) MJ83.
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Figure 6. 18 SNP markers for ‘Josapine’ and ‘MD2’ and the distribution of 313 hybrid offsprings’ genetic diversity data. The ordinate represents the proportion of the total number of 18 marks; the abscissa represents the distribution of genetic information content.
Figure 6. 18 SNP markers for ‘Josapine’ and ‘MD2’ and the distribution of 313 hybrid offsprings’ genetic diversity data. The ordinate represents the proportion of the total number of 18 marks; the abscissa represents the distribution of genetic information content.
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Figure 7. K-means clustering of parental and hybrid offspring genotypes (K = 2). The green color represent Cluster I and the blue color represent cluster II. (A) Optimal number of clusters, (B) K-means cluster plot.
Figure 7. K-means clustering of parental and hybrid offspring genotypes (K = 2). The green color represent Cluster I and the blue color represent cluster II. (A) Optimal number of clusters, (B) K-means cluster plot.
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Figure 8. UPGMA tree of parental and hybrid offspring. the red color represent 213 hybrid offspring were grouped with the parent ‘MD2’, the yellow color represent 100 hybrid offspring were grouped with the parent ‘Josapine’.
Figure 8. UPGMA tree of parental and hybrid offspring. the red color represent 213 hybrid offspring were grouped with the parent ‘MD2’, the yellow color represent 100 hybrid offspring were grouped with the parent ‘Josapine’.
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Table 1. Statistical results of ‘MD2’ × ‘Josapine’ hybrid authenticity identification.
Table 1. Statistical results of ‘MD2’ × ‘Josapine’ hybrid authenticity identification.
Hybrid Combination
(♀ × ♂)		Total Number of F1 (Plants)SNP4010
Typing Statistics (Plants)		SNP22550 Typing Statistics (Plants)True Hybrids (Plants)Pseudohybrids (Plants)True Hybrid Rate (%)
‘MD2’ × ‘Josapine’451GG28AA173955687.58
GC404AG407
CC19GG27
Table 2. Genetic diversity statistics for 18 SNP markers in 313 hybrid offspring.
Table 2. Genetic diversity statistics for 18 SNP markers in 313 hybrid offspring.
Statistical InformationMaximum ValueCorresponds to MarkerMinimum ValueCorresponds to MarkerAverage Value
Frequency of secondary effector loci0.500SNP40100.164SNP281560.334
Gene diversity0.500SNP40100.274SNP281560.422
Heterozygosity0.994SNP40100.191SNP309090.429
Polymorphic information content0.375SNP5172, P20855
SNP4010		0.236SNP281560.331
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MDPI and ACS Style

Jia, P.; Liu, S.; Lin, W.; Yu, H.; Zhang, X.; Xiao, X.; Sun, W.; Lu, X.; Wu, Q. Authenticity Identification of F1 Hybrid Offspring and Analysis of Genetic Diversity in Pineapple. Agronomy 2024, 14, 1490. https://doi.org/10.3390/agronomy14071490

AMA Style

Jia P, Liu S, Lin W, Yu H, Zhang X, Xiao X, Sun W, Lu X, Wu Q. Authenticity Identification of F1 Hybrid Offspring and Analysis of Genetic Diversity in Pineapple. Agronomy. 2024; 14(7):1490. https://doi.org/10.3390/agronomy14071490

Chicago/Turabian Style

Jia, Panpan, Shenghui Liu, Wenqiu Lin, Honglin Yu, Xiumei Zhang, Xiou Xiao, Weisheng Sun, Xinhua Lu, and Qingsong Wu. 2024. "Authenticity Identification of F1 Hybrid Offspring and Analysis of Genetic Diversity in Pineapple" Agronomy 14, no. 7: 1490. https://doi.org/10.3390/agronomy14071490

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