Microscopic and Transcriptome Analysis Reveals that the Self-incompatibility in Rabbiteye Blueberry Belongs to the S-RNase-based Gametophytic Type

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Qin Yang College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Yan Fu Qiandongnan National Polytechnic, Kaili 556000, China

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Yalan Liu College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Tingting Zhang College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Shu Peng College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Jie Deng College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Abstract

Berry fruits produced by Vaccinium (Ericaceae) plants are small but have a signature flavor and have become increasingly popular in the 21st century. However, self-incompatibility (SI) results in a relatively low fruit-set ratio and reduced fruit quality in Vaccinium. In this study, using Vaccinium ashei (V. ashei) styles after cross-pollination (CP) and self-pollination (SP) as material, transcriptomics and gene expression analyses were performed using high-throughput RNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, evolutionary analysis and conserved sequences analysis of candidate genes were conducted. Among the 135,324 unigenes, 30,863 were shown to be differentially expressed, and eight randomly selected differentially expressed genes were expressed in the styles at 96 hours after SP and CP. The transcriptomics and qRT-PCR results were significantly correlated, which confirmed the reliability of the differentially expressed genes obtained in our study. Compared with SP96, six differentially expressed ribonuclease T2 family genes were obtained in CP96, which were considered candidates for S-RNase. Additionally, the spatiotemporal and organizational expression trends of six candidates for S-RNase were confirmed by qRT-PCR, and the evolutionary and conservative sequence analysis indicated six candidate S-RNases with the typical S-RNase structure. The spatiotemporal and organizational expression results and evolutionary and conservative sequence analyses of the six candidate S-RNases suggest that SI in V. ashei is likely an S-RNase-mediated gametophytic one. This finding suggests the involvement of novel, previously undiscovered components involved in the V. ashei SI system. These findings help elucidate the molecular mechanisms of SI in rabbiteye blueberry and may also benefit breeding, production, and genomics research in V. ashei and other Vaccinium species.

Largely due to natural selection, ∼60% of higher plants and in at least 100 families have evolved a multitude of mechanisms to avoid inbreeding, encourage outbreeding, and enhance the heterozygosity of their progeny to ensure genetic diversity and maintain heredity and variation (Fujii et al. 2016; Wu et al. 2018). Collectively, these mechanisms are referred to as self-incompatibility (SI), which ensures plant populations’ survival in a complex and changeable environment (Wu et al. 2018). In self-incompatible plants, the style rejects self pollen, leading to the failure of pollen germination on the stigma or the arrest of pollen tube growth; as such, inhibited pollen tubes cannot enter the embryo sac, and plants therefore are unable to complete self-fertilization (He et al. 2021; Yang and Fu 2015). From the genetics perspective, SI is controlled by multiple alleles of the S-locus and accordingly can be classified into two types, heterologous SI and homologous SI (Ma and Qu 2019; Wu et al. 2018). Meanwhile, according to different modes of genetic control of pollen incompatibility phenotypes, homophytic incompatibility has also been classified into sporophytic SI (SSI) and gametophytic SI (GSI) (He et al. 2021; Wu et al. 2018; Yang and Fu 2015). In SSI, the germinated pollen cannot penetrate the stigma mastoid cells of the style, whereas in GSI, the pollen may germinate and grow through the style to the embryo sac. However, if the S-allele of the haploid pollen matches one of the S-alleles of the diploid pistil, the pollen tube growth is arrested in the style, preventing pollen from entering the embryo sac for double fertilization (Abdallah et al. 2020; He et al. 2021; Ma and Qu 2019; Zeng et al. 2019).

Although SI to a large extent ensured the evolutionary success of flowering plants, it presents a great inconvenience to the production and breeding of fruit trees. To obtain stable fruit setting rates and yield, pollinating of trees or artificially assisted pollination is commonly necessary in production. In addition, pollination compatibility between parents needs to be considered for cross-breeding, which is time-consuming and laborious (He et al. 2021; Wu et al. 2018; Yang and Fu 2015). Therefore, to solve these challenges created by SI for fruit trees, a significant amount of research has long been carried out on the pollination process of fruit trees, attempting to understand the cell morphology, physiological biochemistry, molecular biology, and other aspects of SI (Wu et al. 2018). SI studies on fruit trees revealed that peach [Prunus persica (L.) Batsch (Abdallah et al. 2020)], European pear [Pyrus communis (Quinet et al. 2014)], apple [Malus pumila (Li et al. 2012)], plum [Prunus salicina (Nantongo et al. 2016)], loquat [Eriobotrya japonica (Yang et al. 2018b)], pummelo [Citrus maxima (Liang et al. 2020)], and strawberry [Fragaria ×ananassa (Du et al. 2021)] adopt the S-RNase-based GSI. In this GSI type, when the male S-determinant S-locus F-box (SLF)/S-haplotype-specific F-box (SFB), which is encoded and controlled by S-locus alleles, interacts with the female S-determinant S-RNase, which is present in the style tissue, the non-self S-RNase proteins can be recognized and ubiquitinated by SLF in the pollen tube. Through the action of the 26S proteasome, polyubiquitinated S-RNase proteins are subsequently degraded and unable to catalyze the degradation of RNA. By contrast, self S-RNase proteins are not recognized and degraded. Thus, RNA degradation occurs in the pollen tube, arresting its growth and causing SI (Hua and Kao 2006; Meng et al. 2011).

Blueberry (Vaccinium spp.) plants are perennial evergreen or deciduous shrubs that produce small berries and belong to Ericaceae. It is considered one of the fruits with the greatest commercial development potential in the 21st century (Yang et al. 2019). Blueberry farmers have long known that some cultivars of rabbiteye blueberry (Vaccinium ashei), northern highbush blueberry [Vaccinium corymbosum (V. corymbosum)], and southern highbush blueberry (V. corymbosum interspecific hybrids) exhibit SI, and planting with multicultivar mixtures is often practiced in production to improve fruit setting and ensure yield (Chavez and Lyrene 2009; Harrison et al. 1994; Kendall et al. 2020; Moisan-Deserres et al. 2014; Taber and Olmstead 2016; Yang et al. 2015b, 2017). Therefore, to address the negative impact of SI on blueberry production, many researchers have studied pollination compatibility and pollen sensitivity effects among different cultivars, providing reliable knowledge for the selection of suitable pollination cultivars for self-incompatible cultivars (Ehlenfeldt and Kramer 2012; Kendall et al. 2020; Miller et al. 2011; Taber and Olmstead 2016; Yang et al. 2015b, 2017, 2020a). Some scientists have studied the pollination process of self-compatible cultivars, partially self-compatible cultivars, and self-incompatible cultivars of blueberry (Isaacs and Kirk 2010; Kendall et al. 2020; Parrie and Lang 1992), including pollen hydration and germination on the stigma (Parrie and Lang 1992), and the cell morphology of pollen tube growth in the style (Yang et al. 2018a, 2020b). These studies have deepened our understanding of SI in V. ashei, in which the pollen tubes from self-pollination (SP) are inhibited in the base of the style, exhibiting a typical phenotypic characteristic of S-RNase-based GSI (Qu and Drummond 2018; Yang et al. 2018a, 2020b, 2023).

S-RNase-based GSI is controlled by a single multiallelic S locus that is composed of the pistil-S and pollen-S genes. The pistil-S gene encodes a polymorphic ribonuclease (S-RNase) that degrades RNA in the pollen tube during the growth of incompatible pollen (Li et al. 2020). In the S-RNase-based GSI system, if one of the S-haplotypes of the pistil matches with that of the pollen, pollen tube growth in the style is arrested. For example, crossing between cultivars SaSb and ScSd results in cross-compatibility, whereas a cross between cultivars SaSb and SaSb results in cross-incompatibility (Yang and Fu 2015; Yang et al. 2018b). Therefore, S-genotyping and compatibility group pairing are essential for the establishment of productive orchards and efficient breeding programs in fruit trees (Yang et al. 2018b). However, research on the identification of S-genes and mechanisms of SI is lacking in V. ashei. Furthermore, whether there is S-RNase in V. ashei, which is also the key to breakthroughs in the field, requires further verification.

Therefore, in this study, the style of the rabbiteye blueberry ‘Premier’ was subjected to high-throughput transcriptome sequencing analysis after SP and cross-pollination (CP), and candidate S-RNases were screened and validated by their temporal and spatiotemporal and organizational expression with fluorescence quantitative polymerase chain reaction (PCR). Thereafter, evolutionary and conserved sequence analyses were carried out. Our results provide a useful resource for elucidating the molecular mechanism of the SI of blueberry, with important theoretical and practical significance for the breeding and production of blueberry.

Materials and Methods

Plant material.

Eight-year-old rabbiteye blueberry plants (‘Premier’ and ‘Brightwell’) were planted in the Agricultural and Forestry Training Base of Kaili University, Kaili, Guizhou, China (lat. 26°31′N, long. 107°53′E). A total of 60 healthy plants with no disease and pests and of similar growth status were randomly selected as the experimental material.

Pollen collection and pollination.

At the initial flowering stage, robust medium and long fruiting branches (Yang et al. 2015a) outside the crown of each plant were selected. Only the large bud stage flowers were retained for emasculation, and the other flowers were removed. A total of 2880 emasculated flowers were covered with labeled 100-mesh nylon bags. Three days after emasculation, the pollen of ‘Premier’ and ‘Brightwell’ plants was collected following the method previously described (Yang et al. 2020b). A total of 1440 emasculated ‘Premier’ flowers were subjected to SP or to CP with ‘Brightwell’ pollen, which was obtained from 8-year-old ‘Brightwell’ plants planted in the same conditions as ‘Premier’.

Phenotype observation of ‘Premier’ after SP and CP.

Following the methods described in a previous study (Yang et al. 2020b), 60 flowers were collected from self-pollinated and cross-pollinated branches at 2, 24, 48, and 96 h after pollination. The collected flowers were immediately dissected to isolate the styles from the ovaries by cutting the base of the style with a scalpel. The styles were fixed immediately in 10-mL centrifuge tubes filled with FAA [5:5:90 (v/v/v) 38% formaldehyde: acetic acid: 70% (v/v) ethanol] solution to soften them for fluorescence microscopy observation, and the percentage of whole styles traversed by pollen tubes was also surveyed and calculated at 96 h after pollination as described by Yang et al. (2019). Three hundred flowers from self-pollinated and cross-pollinated branches were used to calculate the fruit set and number of seeds per fruit at 30 d after pollination and ripening, with 100 flowers for each treatment and three replicates.

Collection and processing of samples for transcriptome sequencing.

As described previously (Yang et al. 2020b), 300 flowers were collected from self-pollinated and cross-pollinated branches at 24, 48, and 96 h after pollination, immediately frozen and stored in liquid nitrogen, and brought back to the laboratory. The style and ovary were then separated immediately from the base of the style using a scalpel, and the separated styles were immediately placed in a mortar filled with liquid nitrogen. The styles isolated from 100 self-pollinated and 100 cross-pollinated flowers were transferred into 5-mL centrifuge tubes precooled in liquid nitrogen, labeled with the appropriate treatment abbreviation (SP24, SP48, SP96, CP24, CP48, or CP96) and transferred to a −80 °C ultra-low-temperature refrigerator for storage.

Transcriptome sequencing and preprocessing of reads.

The self-pollinated and cross-pollinated styles collected at all time points from ‘Premier’ were sent to Biomarker Technologies (Beijing, China) for RNA extraction using the TRIzol method (Tiangen Biotech, Beijing, China) and treated with RNase-free DNase I (TaKaRa, Dalian, China). RNA degradation and contamination were inspected on 1% agarose gels. The RNA was quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and the quality and integrity were assessed by a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Transcriptome sequencing was performed using the NovaSeq 6000 (Illumina, San Diego, CA, USA) high-throughput sequencing platform at Biomarker Technologies. To ensure the quality and reliability of the sequencing data, clean reads were obtained by removing reads with joints, reads containing N, and low-quality reads from the sequenced raw reads. Meanwhile, Q20, Q30, and guanine and cytosine (GC) contents were calculated for the clean reads. Trinity software was used to assemble clean reads de novo to obtain transcripts or unigenes (Grabherr et al. 2011). Subsequently, the clean reads of each sample were mapped to the assembled transcript or unigene library for subsequent analysis.

Analysis of differentially expressed genes.

Bowtie software [ver. 1.0.0 (Langmead et al. 2009)] was used to align the mapped reads obtained by sequencing to the unigene library. According to the alignment results, RNASeq by Expectation Maximization (Li and Dewey 2011) was used to calculate the values of fragments per kilobase of exon per million mapped fragments [FPKM (Trapnell et al. 2010)], indicating the expression level of the corresponding unigene. DESeq software (Anders and Huber 2010) based on the negative binomial distribution was used to perform statistical analysis of the data and calculate the difference in gene expression. The default threshold P < 0.01 and fold-change >2 were used, and seven comparisons were performed: SP24 vs. SP48, SP48 vs. SP96, SP24 vs. CP24, SP48 vs. CP48, SP96 vs. CP96, CP24 vs. CP48, and CP48 vs. CP96.

Quantitative real-time PCR verification of differentially expressed genes.

To verify further the accuracy of the differentially expressed genes (DEGs) obtained by transcriptome sequencing, quantitative real-time (qRT)-PCR was used to examine eight randomly selected DEGs from the comparison of SP96 vs. CP96. Primer 5.0 (Premier Biosoft International, Palo Alto, CA, USA) was used to design specific amplification primers (Table 1), which were evaluated by Primer-BLAST program (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/) and synthesized by Shengong Bioengineering (Shanghai, China) Co., Ltd. Pistil RNA was isolated using an RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions, and its integrity was detected by agarose gel electrophoresis. The RNA concentration and purity were detected by NanoDrop2000 spectrophotometer (Thermo Fisher Scientific), and RNA with OD260/OD280 ratios between 1.8 and 2.0 was selected. Reverse transcription was performed using a PrimeScript™ first Strand cDNA Synthesis Kit (Takara) and was diluted 20 times and stored at −20 °C for future use. The qRT-PCR was conducted using a LightCycler®96 System (Thermo Fisher Scientific) with the following conditions: 30 s at 95 °C and 40 cycles of 5 s at 95 °C, 30 s at 58–60 °C, followed by 60 °C to 95 °C melting curve detection. Three biological replicates were set up for each sample during the reaction. The Actin gene (c87909.graph_c0) was used as the reference (Zifkin et al. 2012). The expression levels were calculated using the 2−ΔΔCt method. Linear regression analysis of FPKM and qRT-PCR was performed using IBM SPSS 22 software.

Table 1.

Primer sequences of eight differentially expressed genes (DEGs) randomly screened from the comparison of 96 h after self-pollination compared with 96 h after cross-pollination’s styles of rabbiteye blueberry (Vaccinium ashei) cultivar ‘Premier’ for quantitative real-time polymerase chain reaction (qRT-PCR).

Table 1.

Functional annotation and enrichment analysis of DEGs.

BLAST software (Altschul et al. 1997) was used to compare DEG sequences with the Nonredundant [Nr (Deng et al. 2006)], Swiss-Prot (Apweiler et al. 2004), Gene Ontology [GO (Ashburner et al. 2000)], Clusters of Orthologous Groups [COG (Tatusov et al. 2000)], EuKaryotic Orthologous Groups [KOG (Koonin et al. 2004)], eggNOG4.5 (Huerta-Cepas et al. 2016), and Kyoto Encyclopedia of Genes and Genomes [KEGG (Kanehisa et al. 2004)] databases. KOBAS 2.0 (Xie et al. 2011) was used to obtain the KEGG orthology results of the DEGs. After predicting the amino acid sequences of the DEGs, HMMER software (Finn et al. 2011), which is used to search sequence databases for sequence homologs and to make sequence alignments, implementing methods using probabilistic models called profile hidden Markov models (profile HMMs), was employed for comparison with the Pfam database (Finn et al. 2014) to obtain annotations of the DEGs.

Screening of candidate S-RNases and expression verification by qRT-PCR.

The results of fluorescence microscopy observation showed that 96 h after pollination was the critical period of SI, so the comparison of SP96 vs. CP 96 with the most DEGs was selected as screening objects. These were considered candidates for S-RNase for which the results of NR, Swiss-Prot, GO, COG, KOG, eggNOG4.5, and KEGG annotation of DEGs were ribonuclease T2 family genes (Du et al. 2021). Subsequently, the spatiotemporal (at 0, 24, 48, 72, 96, and 120 h after SP and CP) and organizational (buds, leaves, pollen, styles, and filaments) expression of the candidate S-RNases was examined using qRT-PCR as per the method described in the qRT-PCR verification of DEGs. The primers are shown in Table 2. The buds were collected with 5-mL centrifuge tubes precooled in liquid nitrogen before bloom, and the pollen, styles, and filaments isolated from 100 flowers at 3 d after blooming were transferred into 5-mL centrifuge tubes precooled in liquid nitrogen, while the leaves of new shoots in spring were collected with 5-mL centrifuge tubes precooled in liquid nitrogen. All collected tissues were immediately transferred to a −80 °C ultra-low-temperature refrigerator for storage.

Table 2.

Primer sequences of six candidate ribonuclease T2 family genes screened from the comparison of 96 h after self-pollination compared with 96 h after cross-pollination’s styles of rabbiteye blueberry (Vaccinium ashei) cultivar ‘Premier’ for qRT-PCR.

Table 2.

Evolutionary and conservative sequence analysis of candidate S-RNases.

The amino acid sequences of six candidate S-RNases were predicted using Editseq in DNAstar (DNASTAR, Inc., Madison, WI, USA) (Zhang et al. 2016). Using MEGA 7.0 with the neighbor-joining method adopted for cluster analysis and the number of bootstrap replicates set to 1000, these amino acid sequences were subjected to phylogenetic analysis, including another 76 known ribonuclease T2 genes from the 21 genera of Actinidia, Antirrhinum, Arctium, Artemisia, Buddleja, Camellia, Citrus, Coffea, Crataegus, Cynara, Daucus, Diospyros, Lactuca, Malus, Nicotiana, Petunia, Pyrus, Prunus, Rhododendron, Solanum, and Syzygium (Du et al. 2021). According to the results of the evolutionary analysis, the species closely related to the candidate S-RNases of V. ashei and 14 S-RNases from Prunus, Pyrus and Malus (as control), were used to perform conservative structure analysis using DNAMAN (Lynnon Biosoft, San Ramon, CA, USA) (Du et al. 2021; Zhang et al. 2016).

Data analysis.

All statistical analyses were performed using the SPSS 22.0 software. Percentages were subjected to angular transformation to ensure normal distribution before analysis of variance (P ≤ 0.05), followed by the Student–Newman–Keuls multiple range test.

Results and Analysis

Phenotype of ‘Premier’ after SP and CP.

The initiation of pollen germination began 2 h after pollination, as observed in the SP 2 and CP 2 styles. Both SP and CP pollen grains had begun to germinate into the stigma, and a small number of pollen tubes had already penetrated into the style through the mastoid cells (Fig. 1A and B). At 24 h after pollination, the pollen tubes from SP grew more slowly, and no pollen tubes reached the middle of style, reaching only the upper third of style (Fig. 1C), whereas most pollen tubes from CP reached the middle of the style (Fig. 1D). At 48 h after pollination, the pollen tubes from SP just reached the middle of the style, and no pollen tubes from SP reached the base of style (Fig. 1E); a small number of pollen tubes from CP reached the base of style (Fig. 1F). At 96 h after pollination, compared with the 48th h after pollination, the pollen tubes from SP grew extremely slowly and the pollen tubes from SP were enlarged at the apex and stopped growing in the middle or base of the style (Fig. 1G); many of the pollen tubes from CP traversed the base of style (Fig. 1H).

Fig. 1.
Fig. 1.

Squash preparation using 0.1% (w/v) aniline blue for fluorescence microscopy of the pollen tube growth in the styles from self-pollination (SP) and cross-pollination (CP) treatments of the rabbiteye blueberry (Vaccinium ashei) cultivar ‘Premier’. Two hours after SP (A), 2 h after CP (B), 24 h after SP (C), 24 h after CP (D), 48 h after SP (E), 48 h after CP (F), 96 h after SP (G), and 96 h after CP (H).

Citation: J. Amer. Soc. Hort. Sci. 149, 4; 10.21273/JASHS05364-23

As shown in Fig. 2A, at 96 h after pollination, the percentage of styles traversed by pollen tubes was significantly different between the SP and CP treatments of rabbiteye blueberry ‘Premier’. The percentage of styles traversed by pollen tubes of ‘Premier’ SP was only 21.67%, which was significantly lower than that of CP (76.67%). As shown in Fig. 2B, the fruit set of ‘Premier’ SP was only 16.67%, which was significantly lower than that of CP (66.33%). In addition, the number of seeds per fruit of ‘Premier’ SP was only 11.67, which was significantly lower than that of CP (34.33 seeds) (Fig. 2C). The results of pollen tube growth fluorescence microscopy, the percentage of styles traversed by pollen tubes, the fruit setting rate, and the average seed number per fruit ere consistent, which showed that ‘Premier’ was self-incompatible and had cross-compatibility with ‘Brightwell’ via CP.

Fig. 2.
Fig. 2.

Analysis of the difference in the percentage of styles traversed by pollen tubes (A), fruit set (B), and the number of seeds per fruit (C) between the self-pollination (SP) and cross-pollination (CP) treatments of rabbiteye blueberry (Vaccinium ashei) ‘Premier’. The ‘Premier’ flowers were subjected to SP (‘Premier’ × ‘Premier’) or CP (‘Premier’ × ‘Brightwell’) with ‘Brightwell’ pollen at 3 d after emasculation. The data are the means ± SE. Different letters above the bar graph indicate significant differences (P ≤ 0.05) according to the Student–Newman–Keuls test.

Citation: J. Amer. Soc. Hort. Sci. 149, 4; 10.21273/JASHS05364-23

Sequencing data assembly and statistics.

As shown in Table 3, the original sequencing reads were preprocessed based on Illumina high-throughput sequencing, and the base number of ‘Premier’ styles at 24, 48, and 96 h after SP and CP ranged from 5,974,698,736 to 7,329,309,444. The number of clean reads ranged from 40,019,036 to 49,022,592, and the GC (guanine and cytosine) content ranged from 46.09% to 46.53%. The percentages of Q30 bases in the styles at all time periods after SP and CP were higher than 94%, indicating that the sequencing data were highly reliable and could be used for subsequent analysis. Trinity was used to assemble the clean reads, and a total of 229,282 transcripts and 135,324 unigenes were obtained.

Table 3.

Summary of the RNA-seq outcomes in the self-pollinated and cross-pollinated styles of the rabbiteye blueberry rabbiteye blueberry (Vaccinium ashei) cultivar ‘Premier’.

Table 3.

As shown in Table 4, the numbers of the obtained transcripts ranged from 34,392 (15.00%) to 57,860 (25.24%), whereas the numbers of the obtained unigenes ranged from 15,156 (11.20%) to 36,930 (27.29%). The total lengths of transcripts and unigenes were 248,742,668 and 118,137,852 bp, respectively; the mean lengths were 1084.88 and 873.00 bp; and the N50 was 1690 and 1547, indicating high assembly integrity. Sequence alignment of clean data from each sample with the assembled transcript or unigene library yielded 35,187,635 to 43,213,535 mapped reads, with a mapping ratio from 87.54% to 88.34%.

Table 4.

Summary of the transcripts and unigene de novo assembly in the self-pollinated and cross-pollinated styles of the rabbiteye blueberry (Vaccinium ashei) cultivar ‘Premier’.

Table 4.

Differential expression analysis and qRT-PCR verification.

As can be seen from Table 5, in the comparison of SP at different pollination times, 8353 upregulated genes and 3692 downregulated genes were obtained in SP48 compared with SP24, whereas 4111 upregulated genes and 3318 downregulated genes were identified in SP96 compared with SP48. In the CP comparison with different pollination times, 9611 upregulated genes and 5757 downregulated genes were obtained in CP48 compared with CP24, whereas 5727 upregulated genes and 10,715 downregulated genes were obtained in CP96 compared with CP48.

Table 5.

Statistical table of the number of differentially expressed genes in the self-pollinated and cross-pollinated styles of the rabbiteye blueberry (Vaccinium ashei) cultivar ‘Premier’.

Table 5.

In addition, in the comparisons of SP and CP at the same pollination time point, it was found that 2894 upregulated genes and 1474 downregulated genes were identified in CP24 by comparison with SP24, 4347 upregulated genes and 3712 downregulated genes were obtained in CP48 compared with SP48, and 5788 upregulated genes and 10,561 downregulated genes were identified in CP96 compared with SP96. The qRT-PCR verification results (Fig. 3) showed that eight randomly selected DEGs were expressed in the styles at 96 h after SP and CP, and the correlation analysis results of the transcriptional FPKM and qRT-PCR showed a significant correlation (R = 0.864, P = 0.01). This indicated that the qRT-PCR result was highly consistent with the FPKM results of the transcriptome analysis, demonstrating the reliability of the DEGs obtained by high-throughput sequencing analysis in this study.

Fig. 3.
Fig. 3.

Quantitative real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed genes: gene expression patterns of randomly selected genes in both self-pollinated blueberry styles and cross-pollinated ones 96 h after pollination of rabbiteye blueberry (Vaccinium ashei) ‘Premier’. (A–H) Panels present the expression profiles of maker-VaccDscaff3-snap-gene-101.24, maker-VaccDscaff6-augustus-gene-341.38, maker-VaccDscaff791-augustus-gene-0.4, augustus_masked-VaccDscaff3-processed-gene-100.6, maker-VaccDscaff18-augustus-gene-271.38, maker-VaccDscaff22-augustus-gene-8.25, maker-VaccDscaff11-augustus-gene-137.25, and maker-VaccDscaff21-snap-gene-378.53, respectively. The relative transcription level was calculated using the 2−ΔΔCT method with actin reference genes as the control. The error bars represent the standard error of three biological replicates.

Citation: J. Amer. Soc. Hort. Sci. 149, 4; 10.21273/JASHS05364-23

Screening of candidate S-RNases and qRT-PCR validation.

In the comparison of SP and CP at the same pollination time, the number of DEGs in SP96 vs. CP96 was the largest with 16,349 DEGs (Table 5), and BLAST software was used to obtain information for these DEGs using the Nr, Swiss-Prot, GO, COG, KOG, eggNOG4.5, and KEGG databases. The biological annotations of these DEGs showed that compared with those in SP96, six downregulated differentially expressed ribonuclease T2 family genes [snap_masked-VaccDscaff14-processed-gene-260.11 (S 260.11), maker-VaccDscaff25-augustus-gene-300.46 (M 300.46), maker-VaccDscaff2-augustus-gene-306.35 (M 306.35), maker-VaccDscaff3-augustus-gene-108.33 (M 108.33), and maker-VaccDscaff22-snap-gene-307.39 (M 307.39), and maker-VaccDscaff18-snap-gene-262.45 (M 262.45)] were obtained in CP96.

The COG_class_annotation function of the six downregulated genes was associated with translation, ribosomal structure, and biogenesis functions. The GO_annotation function indicated that the six downregulated genes were related to RNA binding and ribonuclease T2 activity function. The KEGG_annotation function annotation indicated that the six downregulated genes were related to ribonuclease T2 and intracellular ribonuclease LX-like, whereas the KOG_class_annotation and eggNOG_class_annotation suggested that the six downregulated genes were related to RNA processing and modification. The Swiss-Prot_annotation function showed that S 260.11, M 300.46, M 306.35, and M 108.33 were intracellular ribonucleases, while M 307.39 was ribonuclease 2. The NR_annotation showed that S 260.11, M 300.46, M 306.35, and M 108.33 were SI–associated ribonucleases, while M 307.39 was ribonuclease 2-like. The GO_annotation function of M 262.45 showed that it had ribonuclease T2 activity and could bind to RNA, while the KEGG_annotation showed that it was a ribonuclease T2 family protein. The Swiss-Prot_annotation function of M 262.45 showed that it was homologous to the ribonuclease S-6 of Nicotiana alata, while the Nr_annotation showed it was homologous to the ribonuclease S-6-like of Camellia sinensis.

The results of the qRT-PCR verification (Fig. 4) showed that the six DEGs of the ribonuclease T2 family exhibited temporal expression characteristics of S-RNases in the styles of ‘Premier’ at 0, 24, 48, 72, 96, and 120 h after SP and CP. The expression levels of S 260.11, M 300.46, M 306.35, M 108.33, M 307.39, and M 262.45 in the self-pollinated and cross-pollinated styles first increased and then decreased. It is worth noting that the expression of genes tested in the self-pollinated styles gradually increased, and the expression level was the highest at 96 h after pollination, whereas the expression level in the cross-pollinated styles was the highest at 48 h after pollination. Interestingly, the pollen tube growth in SP stopped growing in the middle or base of the style at 96 h after pollination (Fig. 1G). Moreover, at 72 h to 120 h after pollination, the expression of the six DEGs of the ribonuclease T2 family tested in the self-pollinated styles was much greater than that in the cross-pollinated styles. qRT-PCR of the six DEGs of the ribonuclease T2 family tested in different blueberry tissues (Fig. 5) confirmed that these genes were expressed at much higher levels in styles than in other tissues, such as buds, leaves, pollen, and filaments. This suggests that the six DEGs of the ribonuclease T2 family play an important role in the SI reaction.

Fig. 4.
Fig. 4.

Quantitative real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed genes: gene expression patterns of selected ribonuclease T2 family genes in both self-pollinated blueberry styles and cross-pollinated styles 96 h after pollination of rabbiteye blueberry (Vaccinium ashei) ‘Premier’ at 0, 24, 48, 72, 96, and 120 h in the styles after pollination. (A–F) Panels present the expression profiles of snap_masked-VaccDscaff14-processed-gene-260.11, maker-VaccDscaff25-augustus-gene-300.46, maker-VaccDscaff2-augustus-gene-306.35, maker-VaccDscaff3-augustus-gene-108.33, maker-VaccDscaff22-snap-gene-307.39, and maker-VaccDscaff18-snap-gene-262.45, respectively. The relative transcription level was calculated according to the 2−ΔΔCT method with actin reference genes as the control. The error bars represent the standard error of three biological replicates.

Citation: J. Amer. Soc. Hort. Sci. 149, 4; 10.21273/JASHS05364-23

Fig. 5.
Fig. 5.

Quantitative real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed genes: gene expression patterns of selected ribonuclease T2 family genes in buds, leaves, pollen, styles, and filaments of rabbiteye blueberry (Vaccinium ashei) ‘Premier’. (A–F) Panels present the expression profiles of snap_masked-VaccDscaff14-processed-gene-260.11, maker-VaccDscaff25-augustus-gene-300.46, maker-VaccDscaff2-augustus-gene-306.35, maker-VaccDscaff3-augustus-gene-108.33, maker-VaccDscaff22-snap-gene-307.39, and maker-VaccDscaff18-snap-gene-262.45, respectively. The relative transcription level was calculated according to the 2−ΔΔCT method with actin reference genes as the control. The error bars represent the standard error of three biological replicates.

Citation: J. Amer. Soc. Hort. Sci. 149, 4; 10.21273/JASHS05364-23

Evolutionary and conservative sequence analysis of candidate S-RNases.

A phylogenetic tree (Fig. 6) based on the amino acid sequences of the aforementioned 82 ribonuclease T2 genes revealed three main groups. These included 20 ribonuclease T2 genes from Malus, Pyrus, and Crataegus, which were clustered in one subfamily group, and then 19 ribonuclease T2 genes from Prunus, which clustered into one main group. The 19 ribonuclease T2 genes from Rhododendron, Camellia, Buddleja, Artemisia, Lactuca, Cynara, Arctium, Daucus, Diospyros, Actinidia, Solanum, Syzygium, Antirrhinum, and Citrus were clustered together with the five candidate S-RNases (S 260.11, M 300.46, M 306.35, M 108.33, and M 307.39) screened in this study, whereas the remaining 18 ribonuclease T2 genes from Solanum, Petunia, Nicotiana, Antirrhinum, and Coffea were clustered in another main group with the candidate gene S-RNase (M 262.45).

Fig. 6.
Fig. 6.

The phylogenetic tree of the putative S-RNase genes in the study from rabbiteye blueberry (Vaccinium ashei) ‘Premier’ and RNase T2 gene family from 21 genera, including Actinidia, Antirrhinum, Arctium, Artemisia, Buddleja, Camellia, Citrus, Coffea, Crataegus, Cynara, Daucus, Diospyros, Lactuca, Malus, Nicotiana, Petunia, Pyrus, Prunus, Rhododendron, Solanum, and Syzygium, using the neighbor-joining method. Bootstrapping was performed with 1000 replicates.

Citation: J. Amer. Soc. Hort. Sci. 149, 4; 10.21273/JASHS05364-23

The aforementioned evolutionary analyses showed that the six candidate S-RNases screened in this study display significant homology to ribonuclease T2 genes from the other 21 genera. S 260.11, M 306.35, M 108.33, and M 300.46 were most closely related to ribonuclease T2 genes from Rhododendron molle and C. sinensis. Furthermore, M 307.39 was most closely related to ribonuclease T2 genes from Citrus reticulata and Antirrhinum mollissimum, whereas M 262.45 was most closely related to ribonuclease T2 genes from Coffea arabica, Coffea eugenioides, and Coffea canephora. Therefore, the evolutionary analysis indicated that the candidate S-RNases S 260.11, M 300.46, M 306.35, M 108.33, M 307.39, and M 262.45 experienced evolutionary trajectories relatively similar to those of S-RNases of Malus, Pyrus, and Prunus and that S-allele divergence in V. ashei predated speciation.

According to the results of the evolutionary analysis, the six candidate S-RNases in V. ashei were closely related to the genera Coffea and Camellia. Therefore, a total of 33 known S-RNases were selected for analyzing the conserved S-RNase structure in V. ashei, including six and 10 from Coffea and Camellia, respectively; four, three, and four from Prunus, Pyrus, and Malus (as control); and six candidate S-RNases screened in this study. On the basis of the S-RNase characteristics of Rosaceae, the conserved S-RNase structure in V. ashei was divided further into C1–C3, RC4, and C5, three conserved regions, and a hypervariable region (RHV) (Fig. 7), which showed that the six candidate S-RNases had the typical S-RNase structure.

Fig. 7.
Fig. 7.

Multiple alignment of amino acid sequences of candidate S-RNases in the rabbiteye blueberry (Vaccinium ashei) ‘Premier’. The six candidate S-RNases in V. ashei were closely related to Coffea and Camellia, and it is known that the conserved structure of S-RNases in Rosaceae. Therefore, 27 Coffea, Camellia, Prunus, Pyrus, and Malus S-RNases were used to compare and analyze the conserved S-RNase structure in V. ashei. On the basis of the S-RNase characteristics of Rosaceae, the conserved S-RNase structure in V. ashei was divided further into C1–C3, RC4, and C5 as conserved regions and RHV as a hypervariable region. These regions are marked with thick lines.

Citation: J. Amer. Soc. Hort. Sci. 149, 4; 10.21273/JASHS05364-23

Discussion

In the present study, the pollens obtained from both CP and SP germinated at 2 h after pollination, at which point SP and CP pollen grains had begun to germinate into the stigma, and a small number of pollen tubes had already penetrated into the style through the mastoid cells (Fig. 1A and B). However, self-pollen tubes grew slower than cross-pollen tubes from 24 to 96 h after pollination, which was consistent with previous studies that also reported reduced pollen tube growth from an SI cross (Yang et al. 2018a, 2020b, 2023). In addition, consistent with previous reports (Yang et al. 2018a, 2020b, 2023), the pollen tubes from SP were enlarged at the apex and stopped growing in the middle or base of the style at 96 h after pollination (Fig. 1G), whereas a large number of pollen tubes from CP traversed the base of style (Fig. 1H). This is a typical phenotypic characteristic of S-RNase-based GSI, a type of pollen–pistil interaction in which pollen grains germinate and grow normally on the stigma, and the rejection of self-pollen tube is mainly determined by the style (Du et al. 2022).

The majority of the V. ashei pollen tubes from SP were arrested in the style at 96 h after pollination (Fig. 1G), implying that certain time-specific genes (96 h after pollination) regulating pollen tube growth are the key targets of the V. ashei SI system. The previous study showed that S-RNase-based GSI is controlled by a single multiallelic S locus that is composed of the pistil-S and pollen-S genes, among which the pistil-S gene encodes a polymorphic ribonuclease (S-RNase) that degrades RNA in the pollen tube during the growth of incompatible pollen (Li et al. 2020). However, research on the identification of S-genes and the mechanisms of SI is lacking in V. ashei, and whether there is S-RNase in V. ashei remains unknown, which is the key to breakthroughs in the SI of V. ashei. Transcriptome sequencing technology is powerful because it can capture nearly all the expressed transcripts in a particular tissue sample at one or specific developmental stages and/or treatments. With technological advances and reductions in sequencing costs, transcriptome sequencing has become more effective at identifying and tracking candidate genes in various plant processes (Hou et al. 2022). In recent years, the comprehensive application of transcriptome analysis and qRT-PCR has been widely used to study SI in plants, and the efficacy of this method has been rigorously demonstrated in the identification of many critical genes associated with SI responses (Du et al. 2021, 2022; Gómez et al. 2019; He et al. 2020; Hou et al. 2022; Li et al. 2020; Shi et al. 2017; Zhang et al. 2016).

The present study identified numerous significant DEGs after rabbiteye blueberry ‘Premier’ underwent self-incompatible and cross-compatible pollination events. The total number of gene changes demonstrate that SP or CP is a complex process. These findings are consistent with other plant pollination studies (Du et al. 2021, 2022; Gómez et al. 2019; He et al. 2020; Hou et al. 2022; Li et al. 2020). Importantly, the present study not only identified several common up- and downregulated genes in comparison group SP96 vs. CP96, in which the number of DEGs was the largest among the comparisons of SP and CP at the same pollination time, showing that 96 h after pollination was the critical time point for the self-pollen tubes to stop growing in the style, but also predicted candidate genes at the S-locus. Compared with those in SP96, six differentially expressed Ribonuclease T2 family genes were obtained in CP96 from 16,349 DEGs, and their expressions were confirmed by qRT-PCR. Previous studies have shown that S-RNase belongs to Class III in the RNase T2 protein family (Du et al. 2021; Takayama and Isogai 2005), and therefore the six differentially expressed ribonuclease T2 family genes screened were further assessed using evolutionary and conservative sequence analysis as candidate S-RNases.

A phylogenetic tree based on the amino acid sequences of the candidate S-RNases and known S-RNases or RNase T2 family members from the other 21 genera (Fig. 6) revealed three main groups. The six candidate S-RNases, and the S-RNAses or RNAse T2 family members from Rhododendron, Camellia, Coffea, and Citrus exhibited higher levels of amino acid homology and were distributed across different groups in the phylogenetic tree. The candidate S-RNases S 260.11, M 306.35, M 108.33, and M 300.46 were more closely associated with those of Rhododendron and Camellia, whereas the candidate S-RNases M 307.39 and M 262.45 were more closely associated with those of Citrus and Coffea, respectively. The results indicated that some of the candidate S-RNases of V. ashei displayed higher homology to the S-RNases of other genera and also indicated that the S-RNases of V. ashei, Rhododendron, Camellia, Citrus, and Coffea are paralogous, implying that S-allele divergence in V. ashei predated speciation, a phenomenon consistent with the results on Prunus, Malus, and Eriobotrya japonica (Gu et al. 2015; Yang et al. 2018b). Moreover, using Coffea, Camellia, Prunus, Pyrus, and Malus S-RNases as a reference, the comparison of the deduced amino acids showed that the six candidate S-RNases possessed five characteristic conserved S-RNase regions (C1, C2, C3, RC4, and C5) and the RHV (Fig. 7), which are the typical structural features of S-RNases in the Rosaceae family (He et al. 2021; Yang et al. 2018b).

Previous studies have shown that S-RNase not only has five characteristic conserved S-RNase regions (C1, C2, C3, RC4, and C5) and an RHV but also has introns (He et al. 2021) in which the sequence can influence the expression of S-RNase (Sanzol 2009). However, in this study, the intron sequence of the identified six candidate S-RNases was not obtained by transcriptomic sequencing. To advance research on the molecular mechanism of SI of blueberry, the next step should be to design intron-specific primers at the intron–exon boundary of the six candidate S-RNases identified here. The full-length sequences of S-RNases will be obtained using the segmented cloning approach (Du et al. 2021), and the structure of S-RNases in blueberry will be clarified. In addition, the S-genotyping of incompatible cultivars needs to be identified in V. ashei. Previous studies have shown that allele-specific PCR (AS-PCR) is a rapid, simple, and reliable method for the identification of S-RNases in which the degenerate primers are designed based on the conserved sequences of S-RNases. This method has been widely used in loquat, apple, pear, sweet cherry, and other fruit trees (Yang et al. 2013, 2018b). Therefore, the establishment and optimization of the AS-PCR system for blueberry will be established based on the six candidate S-RNases, and its application in the identification of the S-genotype in blueberry is of great significance for promoting the use of blueberry resources and guiding the selection of pollination cultivars.

In addition, qRT-PCR verification showed that six candidate S-RNases exhibited temporal and spatial expression characteristics in the styles of ‘Premier’ at 0, 24, 48, 72, 96, and 120 h after SP and CP, similar to the spatiotemporal expression of S-RNases in Rosaceae (Du et al. 2021; He et al. 2021). Additionally, the expression levels of M 300.46, M 306.35, M 108.33, M 307.39, and S 260.11 in the self-pollinated styles were much higher than those in the cross-pollinated styles at 96 h after pollination, correlating with the degree of pollen tube inhibition, and the highest levels of expression were observed 96 h after SP (Yang et al. 2018a, 2020b, 2023). This may be attributed to the fact that after the S-RNases secreted by the style entered the pollen tube, the non-self S-RNases could be recognized by SLF, labeled by ubiquitination, and degraded under the action of the 26 S proteasome, thereby losing nuclease activity, and failing to reduce RNA. By contrast, the self S-RNase is not recognized and still shows nuclease activity, degrading the RNA and then inhibiting pollen tube growth, and such an inhibitory effect is positively correlated with the S-RNase concentration (He et al. 2021).

In addition, outside of the S-RNase-based GSI in Solanaceae, Plantaginaceae, and Rosaceae, the signal transduction-based GSI cannot be ignored in Papaveraceae, in which a Ca2+ signaling cascade leads to programmed cell death (Du et al. 2021; Zhang et al. 2016). An increasing number of studies have shown that Ca2+ signal transduction is also involved in S-RNase-mediated pollen tube signal transduction in pear; that is, during the self-incompatible reaction, S-RNase is transported into the pollen tubes, triggers RNA and nuclei degradation and mitochondria collapse, and specifically disrupts tip-localized reactive oxygen species of incompatible pollen tubes. Thus, the pollen tube plasma membrane Ca2+ current decreases. It can also depolymerize the actin cytoskeleton, finally causing the pollen tubes’ programmed cell death. At the same time, the pollen tubes initiate a defense mechanism to delay the process of programmed cell death by increasing phosphatidic acid (He et al. 2021; Wang et al. 2009). Therefore, it is of great value to elucidate the molecular mechanism of SI in V. ashei to investigate the effects of S-RNase on the RNA, nucleus, mitochondria, actin cytoskeleton, and reactive oxygen species gradient in pollen tubes in the signal transduction pathways underlying the S-RNase-based inhibition of self-pollen tubes next.

Notably, the fruit set of ‘Premier’ SP was only 16.67%, significantly lower than that of ‘Premier’ CP (66.33%) with ‘Brightwell’, which showed weak SI. At this level, it would be difficult to ensure the fruit output of ‘Premier’ SP, and the fruit set would not meet the demand of producers. Differences in SI strength are relatively widespread in plants with SI systems, such as pear (Hiratsuka et al. 2012) and Solanum carolinense (Mena-Alí et al. 2009), and the strength of SI is divided into three categories—strong, moderate, and weak SI—according to the ratio of styles traversed by the self-pollen tubes. However, it should be noted that the pollen tube growth of different varieties was inhibited at different locations in the SI reaction, which may have been caused by spatiotemporal differences in the expression of S-RNase. S-RNase is expressed specifically in the styles and is maintained at higher levels after SP, which is critical for the development of the SI phenotype. If the concentration of S-RNase is reduced, the intensity of SI may be reduced, showing weak self-compatibility. This could also be due to competitive effects (Ma and Qu 2019), which the polyploidy of rabbiteye blueberry and the duplication of the S gene in pollen can also cause the gametophyte SI to be broken, resulting in the phenomenon of SI, and additional research is needed to clarify its mechanism.

Conclusion

For the first time, transcriptome and gene expression analysis of the styles after CP and SP in V. ashei identified six candidate S-RNases, which are RNase T2 family members and involved in SI. The spatiotemporal and organizational expression and evolutionary and conservative sequence analysis results of the six candidate S-RNases suggest that SI in V. ashei belongs to S-RNase-based GSI. Our study offers novel insights into the SI in V. ashei. These findings not only assist in elucidating the associated molecular mechanisms but are also useful for breeding programs, production improvement, and genomics research in V. ashei and other Vaccinium plants.

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  • Fig. 1.

    Squash preparation using 0.1% (w/v) aniline blue for fluorescence microscopy of the pollen tube growth in the styles from self-pollination (SP) and cross-pollination (CP) treatments of the rabbiteye blueberry (Vaccinium ashei) cultivar ‘Premier’. Two hours after SP (A), 2 h after CP (B), 24 h after SP (C), 24 h after CP (D), 48 h after SP (E), 48 h after CP (F), 96 h after SP (G), and 96 h after CP (H).

  • Fig. 2.

    Analysis of the difference in the percentage of styles traversed by pollen tubes (A), fruit set (B), and the number of seeds per fruit (C) between the self-pollination (SP) and cross-pollination (CP) treatments of rabbiteye blueberry (Vaccinium ashei) ‘Premier’. The ‘Premier’ flowers were subjected to SP (‘Premier’ × ‘Premier’) or CP (‘Premier’ × ‘Brightwell’) with ‘Brightwell’ pollen at 3 d after emasculation. The data are the means ± SE. Different letters above the bar graph indicate significant differences (P ≤ 0.05) according to the Student–Newman–Keuls test.

  • Fig. 3.

    Quantitative real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed genes: gene expression patterns of randomly selected genes in both self-pollinated blueberry styles and cross-pollinated ones 96 h after pollination of rabbiteye blueberry (Vaccinium ashei) ‘Premier’. (A–H) Panels present the expression profiles of maker-VaccDscaff3-snap-gene-101.24, maker-VaccDscaff6-augustus-gene-341.38, maker-VaccDscaff791-augustus-gene-0.4, augustus_masked-VaccDscaff3-processed-gene-100.6, maker-VaccDscaff18-augustus-gene-271.38, maker-VaccDscaff22-augustus-gene-8.25, maker-VaccDscaff11-augustus-gene-137.25, and maker-VaccDscaff21-snap-gene-378.53, respectively. The relative transcription level was calculated using the 2−ΔΔCT method with actin reference genes as the control. The error bars represent the standard error of three biological replicates.

  • Fig. 4.

    Quantitative real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed genes: gene expression patterns of selected ribonuclease T2 family genes in both self-pollinated blueberry styles and cross-pollinated styles 96 h after pollination of rabbiteye blueberry (Vaccinium ashei) ‘Premier’ at 0, 24, 48, 72, 96, and 120 h in the styles after pollination. (A–F) Panels present the expression profiles of snap_masked-VaccDscaff14-processed-gene-260.11, maker-VaccDscaff25-augustus-gene-300.46, maker-VaccDscaff2-augustus-gene-306.35, maker-VaccDscaff3-augustus-gene-108.33, maker-VaccDscaff22-snap-gene-307.39, and maker-VaccDscaff18-snap-gene-262.45, respectively. The relative transcription level was calculated according to the 2−ΔΔCT method with actin reference genes as the control. The error bars represent the standard error of three biological replicates.

  • Fig. 5.

    Quantitative real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed genes: gene expression patterns of selected ribonuclease T2 family genes in buds, leaves, pollen, styles, and filaments of rabbiteye blueberry (Vaccinium ashei) ‘Premier’. (A–F) Panels present the expression profiles of snap_masked-VaccDscaff14-processed-gene-260.11, maker-VaccDscaff25-augustus-gene-300.46, maker-VaccDscaff2-augustus-gene-306.35, maker-VaccDscaff3-augustus-gene-108.33, maker-VaccDscaff22-snap-gene-307.39, and maker-VaccDscaff18-snap-gene-262.45, respectively. The relative transcription level was calculated according to the 2−ΔΔCT method with actin reference genes as the control. The error bars represent the standard error of three biological replicates.

  • Fig. 6.

    The phylogenetic tree of the putative S-RNase genes in the study from rabbiteye blueberry (Vaccinium ashei) ‘Premier’ and RNase T2 gene family from 21 genera, including Actinidia, Antirrhinum, Arctium, Artemisia, Buddleja, Camellia, Citrus, Coffea, Crataegus, Cynara, Daucus, Diospyros, Lactuca, Malus, Nicotiana, Petunia, Pyrus, Prunus, Rhododendron, Solanum, and Syzygium, using the neighbor-joining method. Bootstrapping was performed with 1000 replicates.

  • Fig. 7.

    Multiple alignment of amino acid sequences of candidate S-RNases in the rabbiteye blueberry (Vaccinium ashei) ‘Premier’. The six candidate S-RNases in V. ashei were closely related to Coffea and Camellia, and it is known that the conserved structure of S-RNases in Rosaceae. Therefore, 27 Coffea, Camellia, Prunus, Pyrus, and Malus S-RNases were used to compare and analyze the conserved S-RNase structure in V. ashei. On the basis of the S-RNase characteristics of Rosaceae, the conserved S-RNase structure in V. ashei was divided further into C1–C3, RC4, and C5 as conserved regions and RHV as a hypervariable region. These regions are marked with thick lines.

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Qin Yang College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Yan Fu Qiandongnan National Polytechnic, Kaili 556000, China

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Yalan Liu College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Tingting Zhang College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Shu Peng College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Jie Deng College of Life and Health Science, Kaili University, Kaili, Guizhou 556000, China; and Key Laboratory of Molecular Breeding and Variety Creation of Horticultural Plants for Mountain Features in Guizhou province, Kaili, Guizhou 556000, China

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Contributor Notes

This work was supported by the Key Projects of Science and Technology Research Scheme of Guizhou Province [grant no. Guizhou Science Basic (2019) 1443]; the “Thousand” Level Innovative Talent Training Project of Guizhou Province [Qian Thousand Layer Talent (2021) 201402]; the Supporting Scheme of the Guizhou Provincial Department of Education for the Top-notch Talents [grant no. Guizhou Education (KY 2018) 076]; the National Natural Science Foundation of China (grant no. 31860546); and the First-class Discipline of Kaili University (Horticulture) (grant no. 202102). We thank LetPub (www.letpub.com) for linguistic assistance and presubmission expert review.

Q.Y. is the corresponding author. E-mail: yangqin1028518@126.com.

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  • Fig. 1.

    Squash preparation using 0.1% (w/v) aniline blue for fluorescence microscopy of the pollen tube growth in the styles from self-pollination (SP) and cross-pollination (CP) treatments of the rabbiteye blueberry (Vaccinium ashei) cultivar ‘Premier’. Two hours after SP (A), 2 h after CP (B), 24 h after SP (C), 24 h after CP (D), 48 h after SP (E), 48 h after CP (F), 96 h after SP (G), and 96 h after CP (H).

  • Fig. 2.

    Analysis of the difference in the percentage of styles traversed by pollen tubes (A), fruit set (B), and the number of seeds per fruit (C) between the self-pollination (SP) and cross-pollination (CP) treatments of rabbiteye blueberry (Vaccinium ashei) ‘Premier’. The ‘Premier’ flowers were subjected to SP (‘Premier’ × ‘Premier’) or CP (‘Premier’ × ‘Brightwell’) with ‘Brightwell’ pollen at 3 d after emascul