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Article

Identification and Characterization of HS4-Mediated Hybrid Seed Shattering in Rice

by
Daiqi Wang
1,2,3,4,
Wantong Xie
3,4,
Hong Chen
3,
Tifeng Yang
5,
Ziqiang Liu
3,4,
Ying Ruan
1 and
Chunlin Liu
1,2,*
1
Key Laboratory of Hunan Provincial on Crop Epigenetic Regulation and Development, Hunan Agricultural University, Changsha 410128, China
2
College of Agronomy, Hunan Agricultural University, Changsha 410128, China
3
Guangdong Provincial Key Laboratory of Plant Molecular Breeding, College of Agriculture, South China Agricultural University, Guangzhou 510642, China
4
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China
5
Guangdong Provincial Key Laboratory of New Technology in Rice Breeding, Rice Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(6), 1218; https://doi.org/10.3390/agronomy14061218
Submission received: 11 April 2024 / Revised: 29 May 2024 / Accepted: 4 June 2024 / Published: 5 June 2024
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
Versions Notes

Abstract

:
Seed shattering is an adaptive feature of seed dispersal in wild rice, and it is also an important agronomic trait affecting yield. Reduced seed shattering was a significant progress during rice domestication. However, the evolutionary pathway and molecular mechanism of hybrid seed shattering remain largely unknown. In order to gain a deeper understanding of the regulation of hybrid seed shattering, HS4, a locus conferring hybrid seed shattering between Oryza sativa and Oryza glaberrima, was identified and fine mapped to a 13.5-kb genomic region containing two putative genes during the development of chromosomal segment substitution lines (CSSLs). Expression analysis indicated that the hybrid seed shattering was not related to the expression of HS4. Preliminary research on the molecular mechanism of HS4-mediated hybrid seed shattering indicated that HS4HJX74 and HS4HP61 may form a multimer in heterozygotes, achieving the original function of a trihelix transcription factor through protein interaction. The identification and characterization of HS4 in this study not only provides new insights into the molecular mechanisms underlying hybrid seed shattering, but also provides a potential target for genome editing to reduce the difficulty of hybridization between the two species, facilitating hybrid breeding and increasing yield in rice.

1. Introduction

During the process of crop domestication, one of the most significant changes that occurred was the loss of shattering because plants with non-shattering habits were easier to cultivate. The selection of crops with non-shattering may have been unconscious at first. However, due to the favoritism of artificial selection, the frequency of the non-shattering allele type increased, eventually replacing the shattering allele type [1]. The shattering trait at the maturity of wild species is an adaptation that allows for the dispersal of more offspring into the surrounding environment, contributing to their reproduction and population maintenance [2,3,4]. However, seed shattering is a disadvantage for agricultural production in the case of rice. It is unsuitable for mechanical harvesting and reduces rice yield [5]. To promote effective seed collection at maturity and avoid production loss caused by seed shattering, rice varieties that are non-shattering but suitable for mechanical harvesting are preferred in modern breeding [6].
Seed shattering is related to the formation and degradation of the abscission zone (AZ). The AZ is composed of multiple layers of cells adjacent to the abscission location. These cells can differentiate into one or more layers of parenchyma cells, forming the abscission layer (AL). The AL cells respond to the signals that promote abscission, resulting in the degradation of the AL cells and ultimately leading to seed shattering [7]. Seed shattering in rice is a quantitative genetic trait that is co-regulated by several major and minor genes. During the domestication of rice, qSH1 and SH4/SHA1/GL4 are two major genes that are mainly responsible for the transition away from seed shattering [1,8,9,10,11]. A single nucleotide polymorphism (SNP) in the 5′ regulatory region of qSH1, a BEL1-type homeobox gene, results in non-shattering in Asian cultivated rice due to the absence of AL [10]. SH4, SHA1 and GL4 are alleles encoding trihelix transcription factors. The wild-type gene product carrying an extended third trihelix was identified as a protein that specifically binds to GT elements and promotes the complete development and function of the abscission layer in mature seed pedicels [12]. These GT elements are DNA sequences that occur in tandem repeats within the promoter regions of plant genes, notable for their high concentration of T and A nucleotides. A ‘G237T’ mutation in SH4 results in a single amino acid change in the trihelix DNA-binding domain, which causes the loss of seed shattering due to the incomplete AZ formation in Asian cultivated rice [1]. However, a ‘C760T’ mutation in GL4 results in reduced seed shattering due to premature termination of the GL4 protein in African cultivated rice [8,11]. SHAT1 encodes a transcription factor APETALA2 [13], and SH5 is highly homologous to qSH1 [14]. These two seed shattering-related genes are required for proper AZ formation. In addition, SHAT1 and SH4 are induced by SH5 [14]. OsCPL1 encodes a C-terminal domain phosphatase-like protein and inhibits the differentiation of the AZ during panicle development [15]. OgSH11 encodes a MYB transcription factor that inhibits the expression of genes involved in lignin biosynthesis and the deposition of lignin by binding to the GH2 promoter [16]. Some research has been conducted to establish a regulatory network among genes related to seed shattering [13,17]. These findings have aided our comprehension of the evolutionary pathways and molecular mechanisms that underlie seed shattering in rice.
Within the AA genome group, there exist two cultivated rice species: Asian cultivated rice (O. sativa) and African cultivated rice (O. glaberrima). Previous studies have demonstrated that O. sativa evolved from O. rufipogon, while O. glaberrima evolved from O. barthii [18,19]. During the process of rice domestication, the O. sativa and O. glaberrima were formed with non-shattering seed habits due to environmental adaptation and the artificial selection of ancestors with seed shattering habits. This indicates that seed non-shattering was the direction of evolution and selection. In future breeding, distant hybridization can be an effective way to improve rice yield. However, the difficulty arises despite the significant heterosis resulting from their genetic distances. The hybridizations of O. sativa and O. glaberrima are challenging due to hybrid seed shattering, in addition to hybrid sterility. In this study, we identified a locus HS4, which confers hybrid seed shattering between O. sativa and O. glaberrima during the development of CSSLs using an O. glaberrima accession, HP61, as the donor parent. The analysis of putative genes revealed that HS4 is allelic to SH4/GL4. Although the mutation sites of HS4HJX74 and HS4HP61 in the mapping parents are identical to those of SH4 and GL4, respectively, HS4 induces seed shattering in the heterozygous materials, in contrast to SH4/GL4-mediated seed shattering in the homozygous materials. Moreover, the correlation between seed shattering and the expression levels of SH4/GL4 at the late stage of grain maturation has not previously been studied. In order to ascertain the cause of hybrid seed shattering, we observed cytological differences in AZ cells, and explored the expression pattern of HS4 and the molecular mechanism of HS4-mediated hybrid seed shattering. The potential exists to target HS4 for genome editing in order to reduce the difficulty of hybridization between O. sativa and O. glaberrima, thereby facilitating hybrid rice breeding. This research offers new insights into the molecular mechanisms underlying hybrid seed shattering in rice.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The rice materials used for fine mapping and functional analysis in this study included Huajingxian74 (HJX74) and its chromosomal segment substitution line CSSL-HS4, as well as their hybrid progeny. HJX74 is an elite indica cultivar that was bred by the South China Agricultural University. CSSL-HS4 is a chromosomal segment substitution line that exhibits the phenotype of hybrid seed shattering. It was developed from a hybrid progeny between the recipient parent HJX74 and the donor parent O. glaberrima (HP61) (Supplementary Figure S1). All plants were grown in the paddy field in Guangzhou, China (23°07′ N, 113°15′ E).

2.2. Evaluation of Shattering

The seed shattering ratio of HJX74, CSSL-HS4 and HJX74/CSSL-HS4 hybrids was examined on the main panicles of 20 individuals at rice maturity using the dropping method [20]. Pedicel breaking tensile strength (BTS) was measured on a total of 100 spikelets or grains from 5 main panicles for each material using a digital force gauge (FGP-1, Nidec-Shimpo, Glendale Heights, IL, USA). The maximum tensile strength measured at the moment when the spikelet separated from the pedicel was recorded as BTS, and the unit was gram-force (gf).

2.3. Cytological Analysis

To examine the structure of the abscission zone (AZ), ten spikelets from HJX74, CSSL-HS4 and HJX74/CSSL-HS4 hybrids were collected at the flowering stage. A longitudinal section was made with hand cutting at the junction between the floret and pedicel. The sections were then stained with 0.01% (w/v) acridine orange in the dark for 10 min, and then rinsed with distilled water three times. Finally, the sections were observed using a laser scanning microscope (LSM7810, Zeiss, Oberkochen, Germany) with a 488 nm and a 543 nm laser line.
For scanning electron microscopy (SEM) observation, the pedicel junctions were pre-fixed in 0.1 M sodium phosphate buffer containing 4% glutaraldehyde (pH 7.0) for 8 to 10 h at 4 °C after the detachment of mature seeds for each material. Pre-fixed samples were treated with 1.5% osmium tetroxide in PBS buffer (pH 7.0), gold plated, and then scanned using a scanning electron microscope (EVO MA 15, ZEISS, Oberkochen, Germany) with an acceleration voltage of 10 kV.

2.4. Fine Mapping and Sequencing Analysis

An F3 population was created from the cross between HJX74 and CSSL-HS4 for the purpose of fine mapping of HS4. Crude DNA was extracted from fresh leaves using a previously reported quick extraction method [21]. PCR was performed using a total volume of 20 µL containing 10 ng of template DNA, 0.2 µM of each primer, and 10 µL of 2 × Taq PCR StarMix (GenStar, Beijing, China). The PCR program consisted of 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, preceded by an initial step of 94 °C for 4 min and followed by a final extension step of 72 °C for 5 min. The PCR products were separated on a 6% nondenaturing polyacrylamide gel and detected using the silver staining method [22]. A linkage map was constructed based on the genetic linkage between the genotype of SSR or InDel markers and the seed shattering phenotype in the F3 population.
For sequencing analysis, genomic DNA was extracted from fresh leaves using the CTAB method. Genomic DNA sequences of the HS4 candidate genes were amplified from HJX74 and CSSL-HS4 using long-length PCR with KOD FX Neo DNA polymerase (TOYOBO, Osaka, Japan). The PCR program consisted of 30 cycles at 98 °C for 10 s and 68 °C for 1 min, preceded by an initial step at 94 °C for 3 min and followed by a final extension at 68 °C for 5 min. The PCR products were separated in 1% agarose gels. All primers for fine mapping and sequencing analysis are listed in Table S1.

2.5. RNA Isolation and qRT-PCR Analysis

Total RNA was extracted from various rice tissues using Trizol reagent (Invitrogen, Waltham, MA, USA) following the manufacturer’s instructions. First-strand cDNA was then reverse transcribed using the Hifair® III 1st Strand cDNA Synthesis Kit (YEASEN, Shanghai, China) following the manufacturer’s instructions. For qRT-PCR analysis, cDNA from the junction between floret and pedicel after flowering of HJX74, CSSL-HS4 and their hybrids was obtained. QRT-PCR was performed using the SYBR Green Master Mix (Life Technologies, Carlsbad, CA, USA). The qRT-PCR program consisted of an initial incubation at 94 °C for 3 min, followed by 40 amplification cycles (94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s). The rice housekeeping gene UBQ5 was used as the internal control, and each experiment was repeated at least three times. The qRT-PCR primer list is available in Table S1.

2.6. Subcellular Localization

To determine the subcellular localization of HS4, we cloned the full coding regions of HS4HJX74 and HS4HP61 without stop codon into the pRTVcGFP vector using the Hieff Clone® Plus One Step Cloning Kit (Yeasen, Shanghai, China). The plasmids were then collected using the FinePure EndoFree Plasmid Mini Kit (GENFINE, Shanghai, China) following the manufacturer’s instructions. Plasmid constructs containing empty green fluorescent protein (GFP), HS4HJX74 and HS4HP61 were co-transformed with a nucleus marker into rice protoplasts using the polyethylene glycol method [23]. After incubation at 28 °C for 14 to 20 h in the dark, GFP and mCherry fluorescence was examined using confocal microscopy (LSM7810, Zeiss, Oberkochen, Germany) with 488 nm and 543 nm laser lines. The primers used for vector construction are listed in Table S1.

2.7. Yeast Two-Hybrid Analysis

For yeast two-hybrid analysis, the full coding regions of HS4HJX74 and HS4HP61 were cloned into the pGADT7 and pGBKT7 vectors, respectively, using the Hieff Clone® Plus One Step Cloning Kit (Yeasen, Shanghai, China). The plasmids were prepared using the Plasmid Mini Kit (Biomed, Beijing, China) according to the manufacturer’s instructions. The two plasmid types were co-transformed into the competent AH109 yeast strain (Coolaber, Beijing, China) according to the manufacturer’s instructions. Transformants were grown on screening medium plates (SD/–Trp–Leu) for 3 to 4 days, and then incubated on test plates without Trp, Leu, His and Ade at 30 °C for 3 to 4 days. The primers used for vector construction are listed in Table S1.

3. Results

3.1. Identification of HS4 Controlling Hybrid Seed Shattering

During the development of CSSLs, CSSL-HS4 was found to have two substituted segments from HP61 on chromosome 4 and chromosome 6 in the indica cultivar HJX74 genetic background (Supplementary Figure S1). Notably, when CSSL-HS4 was backcrossed with HJX74 to produce F1, the resulting hybrid displayed seed shattering at maturity, whereas its parents did not. The results of phenotypic observation indicate that the F1 hybrid had a higher seed shattering ratio at rice maturity and a weaker pedicel breaking tensile strength (BTS) compared to CSSL-HS4 and HJX74 (Figure 1). To investigate possible cytological mechanisms of the hybrid seed shattering phenomenon in the F1 hybrid, the seed bases were observed using acridine orange staining and scanning electron microscopy (SEM). It can be observed that the F1 hybrid has a complete and continuous abscission layer, while HJX74 has an irregular and partially formed abscission layer (Figure 2). CSSL-HS4, on the other hand, does not have an abscission layer (Figure 2). SEM analysis revealed that the seed bases of the F1 hybrid had smoother surfaces compared to those of its parents, and the seed bases of HJX74 were smoother than those of CSSL-HS4 (Figure 2). These results indicate that the complete abscission layer in the F1 hybrid does not provide strong support to the seed, which increases seed shattering at rice maturity.

3.2. Fine Mapping and Candidate Gene Analysis of HS4

The presence of the interspecific hybrid sterile gene S1 in the substitution fragment on chromosome 6 of CSSL-HS4 makes it challenging to eliminate this fragment using HP61 as the donor during the development of CSSLs [24,25,26]. However, the homozygosity of this fragment does not impact the phenotype of other fragments. Therefore, we selected plants with a homozygous substitution fragment on chromosome 6 and a heterozygous substitution fragment on chromosome 4 in the F2 population to develop the F3 population, which was used for the fine mapping of HS4. The distribution of seed shattering ratio in 318 HJX74/CSSL-HS4 F3 plants showed a visible bimodal pattern with an apparent valley at 30–40% (Figure 3a). Taking 30–40% as the cut-off region between seed shattering and no-shattering, the population segregated for shattering (>40%) and no-shattering (<30%) at a ratio of 1:1 (χ2 = 0.113 < χ20.05, 1 = 3.84), suggesting a monogenic inheritance of the trait. This result showed that hybrid seed shattering in the F3 population was determined using a single gene on the substituted segment of chromosome 4 (named as HS4). The same F3 population containing 318 plants was used for the primary mapping of HS4 locus. The linkage between seed shattering and SSR markers was analyzed, and the HS4 locus was pinpointed to an interval between PSM361 and PSM382, which is a ~412.8-kb region at the end of Chr. 4 (Figure 3b). The high-resolution mapping of HS4 using 5529 F3 individuals and five new markers in the marker interval of PSM361-PSM382 identified six recombinants (Figure 3c). Three markers in this interval were developed and used to genotype these six recombinants. The HS4 locus was ultimately narrowed down to an approximately 13.5-kb interval between markers RM7314 and H2, including two putative genes based on the Nipponbare sequence (Figure 3d).
The sequence analysis of two putative genes, ORF1 and ORF2, revealed two SNPs in the coding sequence between ORF2HJX74 and ORF2CSSL-HS4, while causing no substitution in the amino acid sequence (Supplementary Figure S2). The sequencing results of ORF2 amplified in HJX74 and CSSL-HS4 genomes are consistent with the reference genome, thus excluding the possibility of ORF2 as the candidate gene. ORF1 is a different allele of the major seed shattering genes SH4 and GL4 in rice [1,8]. Therefore, it is considered that ORF1 is the hybrid seed shattering gene HS4, and ORF1 in HJX74 and CSSL-HS4 are named HS4HJX74 and HS4HP61, respectively. HS4HJX74, which encodes a protein containing 390-aa, has two exons, with the second exon containing a predicted nuclear localization signal (NLS). Compared to HS4HJX74, HS4HP61 has a 7-aa deletion, 5-aa substitutions and premature termination due to 13 SNPs and five deletions (Figure 4 and Supplementary Figure S3). Therefore, HS4HP61 has only one exon without NLS and encodes a 253-aa protein. Both HS4HJX74 and HS4HP61 contain a trihelix DNA-binding domain and are predicted to encode trihelix transcription factor family proteins. However, HS4HJX74 contains an additional coiled-coil domain at the C-terminal end, which is absent in HS4HP61 (Figure 4).

3.3. Expression Analysis and Subcellular Localization of HS4

In order to study the molecular mechanism of hybrid seed shattering caused by HS4, we analyzed the expression of HS4 in HJX74, CSSL-HS4 and their F1 hybrid. The qRT-PCR results showed that HS4 was equally expressed among HJX74, CSSL-HS4 and the F1 hybrid. The expression of HS4 in the junctions between the floret and the pedicel initially increased and then decreased after flowering, reaching its highest level on the 15th day after flowering among the three genotypes (Figure 5a). There was no significant difference in the expression pattern of HS4 after flowering among the three genotypes, indicating that the hybrid seed shattering is not related to the expression of HS4.
To investigate the subcellular localization of HS4, HS4HJX74 and HS4HP61 were fused with the GFP coding sequence and co-expressed with a nuclear marker in rice protoplasts using the polyethylene glycol method. The results indicated that the 35S::HS4HJX74-GFP fluorescence signal was exclusively distributed in the nucleus, whereas the 35S::HS4HP61-GFP fluorescence signal was observed in both the nucleus and cytoplasm (Figure 5b). These findings suggest that HS4HP61 is unable to fully enter the nucleus due to the absence of the nuclear localization signal resulting from premature termination.

3.4. Preliminary Research on the Molecular Mechanism of HS4-Mediated Hybrid Seed Shattering

Based on the subcellular localization results, both HS4HJX74 and HS4HP61 were found to have a nucleus localization signal, indicating the possibility of interaction between the two proteins. To investigate this interaction, we cloned the full-length HS4HJX74 and HS4HP61 into the pGADT7 and pGBKT7 vectors, respectively. The yeast two-hybrid analysis confirmed the physical interaction between HS4HJX74 and HS4HP61 (Figure 6). Furthermore, in the yeast two-hybrid system, HS4HJX74 demonstrated the ability to physically interact with itself, whereas HS4HP61 did not (Figure 6). This could be attributed to the presence of a coiled-coil domain in HS4HJX74, which typically forms a dimer or multimer, whereas HS4HP61 lacks such a domain.

4. Discussion

Crop domestication is a process of remolding wild species to suit human needs and facilitate planting [4,27]. A crucial step in this process was the elimination of seed shattering. Both O. sativa and O. glaberrima, two cultivated species that evolved independently in Asia and Africa, lost this trait [28,29].
This study reports an atavism phenomenon where the F1 hybrid, resulting from the cross between the HJX74 and CSSL-HS4, restored the seed shattering trait, which was absent in its parents. Previous study in cultivated barley has also identified a similar phenomenon. The molecular evolution analysis of BTR1 and BTR2 revealed that the nonbrittle btr1 and btr2 are two closely linked genes, and the F1 hybrid derived from the cross between the btr1-type (btr1/btr1; Btr2/Btr2) and the btr2-type (Btr1/Btr1; btr2/btr2) displayed the brittle phenotype due to the nonallelic interaction in cultivated barley [30]. However, the molecular mechanism behind this phenomenon remains largely unknown. This study investigates the molecular mechanism of hybrid seed shattering mediated by HS4.
Previous research has shown that a ‘G237T’ mutation in SH4 leads to a substitution of Lys with Asn in the trihelix DNA-binding domain, resulting in reduced seed shattering in Asian cultivated rice [1]. Similarly, a ‘C760T’ mutation in GL4 results in decreased seed shattering due to a premature stop codon in African cultivated rice [8]. In this study, the mutation sites of HS4HJX74 and HS4HP61 are the same as SH4 and GL4, respectively (Figure 4 and Supplementary Figure S3). Therefore, HJX74 and CSSL-HS4 exhibit a non-shattering phenotype. The question remains: what caused the HJX74/CSSL-HS4 F1 hybrid to exhibit the seed shattering phenotype? We propose two hypotheses, one at the post-transcriptional level and the other at the protein level. The first hypothesis regarding post-transcription is that two pre-mRNAs transcribed from HS4HJX74 and HS4HP61 combine through trans-splicing to create a chimeric transcript. Trans-splicing is a post-transcriptional process that two or more pre-mRNA molecules are spliced to form a chimeric mRNA [31,32]. The cDNA of the HJX74/CSSL-HS4 F1 hybrid in the AZ at 15 days after flowering was amplified for HS4. The PCR products were then connected to the T-vector, and sequencing did not detect any trans-splicing products. In contrast, only two types of fragments, HS4HJX74-type and HS4HP6-type, were shown in the transcript sequencing results of HJX74/CSSL-HS4 heterozygote materials. Therefore, there is no direct evidence to support the first conjecture. Another hypothesis at the protein level is that HS4HJX74 and HS4HP61 cause seed shattering through protein interaction (Figure S4b). HS4 belongs to the trihelix transcription factor family protein and usually binds cis-elements in the form of dimers or multimers [12,33]. Previous studies have demonstrated that alleles of the same gene can compensate for each other, even if both alleles produce a faulty gene product, through intragenic complementation [34]. In heterozygotes, two alleles with independent mutations in different domains complement each other’s defects by forming multimers of two mutated proteins [35]. HS4HJX74 possesses an abnormal trihelix DNA-binding domain as a result of an amino acid substitution (Supplementary Figure S4a). HS4HP61, on the other hand, loses its NLS and coiled-coil domain due to premature termination of protein translation (Supplementary Figure S4a). These findings suggest that HS4HP61 is unable to fully enter the nucleus and form a dimer, which is consistent with our results of subcellular localization and yeast two-hybrid assays (Figure 5b and Figure 6). Thus, it is hypothesized that HS4HJX74 and HS4HP61 could form a multimer in heterozygotes to achieve the original function of a trihelix transcription factor through protein interaction. Based on this conjecture, a model was proposed for the molecular genetic basis of HS4 mediated hybrid seed shattering (Supplementary Figure S4b). The yeast system was used to demonstrate that HS4HJX74 physically interacts with HS4HP61 (Figure 6). The results of the protein interaction experiment in vitro provide preliminary verification of our previous conjecture. Although further verification is required through more detailed protein interaction experiments in vivo, this study provides new insights into the molecular mechanisms underlying hybrid seed shattering in rice.
Currently, breeders cultivate hybrid rice mainly through intraspecific hybridization, but the utilization of heterosis is limited due to the small genetic difference between parents [36]. To overcome this limitation, distant heterosis can be employed as an effective way to expand genetic variation, create new germplasm resources, and solve the narrow genetic basis in future breeding process [37,38]. However, the phenomenon of hybrid disadvantage in rice seriously limits the application of distant hybridization breeding and the improvement of rice yield. Although O. sativa and O. glaberrima have a long genetic distance and exhibit huge heterosis, the difficulty of distant hybridization between them is mainly due to hybrid seed shattering, in addition to interspecific hybrid sterility [39,40]. In this study, we identified a new hybrid seed shattering locus HS4 between O. sativa and O. glaberrima. The potential exists to target HS4 for genome editing in order to reduce the difficulty of hybridization between O. sativa and O. glaberrima, thereby facilitating hybrid rice breeding.

5. Conclusions

In conclusion, we identified a locus HS4 that confers hybrid seed shattering between O. sativa and O. glaberrima during the development of CSSLs using an O. glaberrima accession, HP61, as the donor parent. In order to ascertain the cause of HS4-mediated hybrid seed shattering, further experiments were conducted. Cytological analysis indicates that the HJX74/CSSL-HS4 hybrids have a complete and continuous abscission layer, which does not provide strong support to the seed and thus increases shattering after seed ripening. HS4 was finally fine-mapped to a 13.5-kb genomic region based on the sequence of Nipponbare, containing two putative genes. The analysis of putative genes revealed that ORF1 is the hybrid seed shattering gene HS4. Expression analysis indicated that the hybrid seed shattering was not related to the expression of HS4. Subcellular localization results indicated that the HS4HJX74 was located in the nucleus, whereas the HS4HP61 was located in both the nucleus and cytoplasm. Preliminary research on the molecular mechanism of HS4 mediated hybrid seed shattering suggested that HS4HJX74 and HS4HP61 could form a multimer in heterozygotes to achieve the original function of a trihelix transcription factor through protein interaction according to the yeast two-hybrid assay. Some limitations need to be noted regarding the present study. Direct genetic evidence, such as genome editing or complementary transformation, is lacking in this study to verify the function of HS4. Additionally, the results of the protein interaction experiment in vitro provide preliminary verification of our conjecture. More detailed protein interaction experiments in vivo are needed for further verification in the future. The identification and characterization of HS4-mediated hybrid seed shattering in this study provides new insights into the molecular mechanisms underlying hybrid seed shattering in rice. Additionally, HS4 can be targeted for genome editing to reduce the difficulty of hybridization between the two species, facilitating hybrid rice breeding.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14061218/s1. Table S1: Primers used in this research; Figure S1: Development of CSSL-HS4; Figure S2: Sequence alignment between ORF2HJX74 and ORF2HP61; Figure S3: Coding sequence alignment between HS4HJX74 and HS4HP61; Figure S4: Molecular mechanism of HS4 mediated hybrid seed shattering.

Author Contributions

Conceived and designed the experiment, Z.L.; performed the field experiments and fine-mapping, D.W., H.C. and W.X.; performed the molecular experiments, D.W.; writing—original draft preparation, D.W.; writing—review and editing, T.Y., Z.L. and C.L.; supervision, C.L. and Y.R.; project administration, C.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Guangdong Provincial Key Laboratory of New Technology in Rice Breeding (2023B1212060042 to Z.L.) and Guangdong Provincial Natural Science Foundation (Grants No. 2021A1515010446 to Z.L.).

Data Availability Statement

The data supporting this study are included in the main text and the Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Seed shattering in HJX74/CSSL-HS4 F1 hybrid. (a) Panicles of HJX74, CSSL-HS4 and HJX74/CSSL-HS4 F1 hybrid at maturity. Scale bars = 3 cm. (b) Comparison of seed shattering ratios among three genotypes. (c) Comparison of breaking tensile strength among three genotypes at different days after flowering. ** and *** indicate significance at the level of 0.01 and 0.001, respectively, as determined using Student’s t-test.
Figure 1. Seed shattering in HJX74/CSSL-HS4 F1 hybrid. (a) Panicles of HJX74, CSSL-HS4 and HJX74/CSSL-HS4 F1 hybrid at maturity. Scale bars = 3 cm. (b) Comparison of seed shattering ratios among three genotypes. (c) Comparison of breaking tensile strength among three genotypes at different days after flowering. ** and *** indicate significance at the level of 0.01 and 0.001, respectively, as determined using Student’s t-test.
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Figure 2. AZ morphology of HJX74, CSSL-HS4 and HJX74/CSSL-HS4 F1 hybrid. (a–c) Characterization of AZ morphology in HJX74 (a), HJX74/CSSL-HS4 F1 hybrid (b) and CSSL-HS4 (c). (a1–c1) show the spikelets of the three materials. The white boxes indicate the junction between floret and pedicel and are enlarged in (a2–c2). (a3–c3) show the fluorescence images of longitudinal sections across the floret and pedicel junction. Enlargements of the white boxes in (a3–c3) are shown in (a4), (b4) and (c4), respectively. Arrows point to the AZ of HJX74, HJX74/CSSL-HS4 F1 hybrid or CSSL-HS4. (a5–c5) show SEM photographs of the seed bases where the pedicels attach. (a6–c6) are magnifications of the white boxes in (a5), (b5) and (c5), respectively. Bars = 2 mm in panels (1), 500 μm in panels (2), 200 μm in panels (3), 100 μm in panels (4) and (5), and 10 μm in panels (6).
Figure 2. AZ morphology of HJX74, CSSL-HS4 and HJX74/CSSL-HS4 F1 hybrid. (a–c) Characterization of AZ morphology in HJX74 (a), HJX74/CSSL-HS4 F1 hybrid (b) and CSSL-HS4 (c). (a1–c1) show the spikelets of the three materials. The white boxes indicate the junction between floret and pedicel and are enlarged in (a2–c2). (a3–c3) show the fluorescence images of longitudinal sections across the floret and pedicel junction. Enlargements of the white boxes in (a3–c3) are shown in (a4), (b4) and (c4), respectively. Arrows point to the AZ of HJX74, HJX74/CSSL-HS4 F1 hybrid or CSSL-HS4. (a5–c5) show SEM photographs of the seed bases where the pedicels attach. (a6–c6) are magnifications of the white boxes in (a5), (b5) and (c5), respectively. Bars = 2 mm in panels (1), 500 μm in panels (2), 200 μm in panels (3), 100 μm in panels (4) and (5), and 10 μm in panels (6).
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Figure 3. Fine mapping of HS4. (a) Segregation analysis of HS4. (b) HS4 was mapped to a 412.8 kb region flanked by PSM361 and PSM382 using 318 HJX74/CSSL-HS4 F3 plants. (c) HS4 was fine-mapped to the region between RM7314 and RIDM80541 using 5,529 HJX74/CSSL-HS4 F4 plants. (d) Six key recombinants defined HS4 to the region between RM7314 and H2. There were two genes, ORF1 and ORF2, in the 13.5 kb region between RM7314 and H2 referring to Nipponbare genome. The white bar indicates the genotype of HJX74, the gray bar indicates the genotype of HJX74/CSSL-HS4 hybrid, and the black bar indicates the genotype of CSSL-HS4.
Figure 3. Fine mapping of HS4. (a) Segregation analysis of HS4. (b) HS4 was mapped to a 412.8 kb region flanked by PSM361 and PSM382 using 318 HJX74/CSSL-HS4 F3 plants. (c) HS4 was fine-mapped to the region between RM7314 and RIDM80541 using 5,529 HJX74/CSSL-HS4 F4 plants. (d) Six key recombinants defined HS4 to the region between RM7314 and H2. There were two genes, ORF1 and ORF2, in the 13.5 kb region between RM7314 and H2 referring to Nipponbare genome. The white bar indicates the genotype of HJX74, the gray bar indicates the genotype of HJX74/CSSL-HS4 hybrid, and the black bar indicates the genotype of CSSL-HS4.
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Figure 4. Difference in the gene structure and amino acid sequence between HS4HJX74 and HS4HP61. The gray bar indicates trihelix DNA-binding domain, the red bar indicates nuclear localization signal (NLS), the green bar indicates coiled-coil domain, the hyphen indicates deletion of amino acid and the asterisk indicates stop codon.
Figure 4. Difference in the gene structure and amino acid sequence between HS4HJX74 and HS4HP61. The gray bar indicates trihelix DNA-binding domain, the red bar indicates nuclear localization signal (NLS), the green bar indicates coiled-coil domain, the hyphen indicates deletion of amino acid and the asterisk indicates stop codon.
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Figure 5. Expression analysis and subcellular localization of HS4. (a) Expression analysis of HS4 in HJX74, CSSL-HS4 and HJX74/CSSL-HS4 F1 hybrid. (b) Subcellular localization of HS4HJX74 and HS4HP61.
Figure 5. Expression analysis and subcellular localization of HS4. (a) Expression analysis of HS4 in HJX74, CSSL-HS4 and HJX74/CSSL-HS4 F1 hybrid. (b) Subcellular localization of HS4HJX74 and HS4HP61.
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Figure 6. Yeast two−hybrid assay for testing the interaction between HS4HJX74 and HS4HP61. AD and BD represent the activation domain in pGADT7 and the binding domain in pGBKT7, respectively. Interaction between T−AD and 53−BD was used as positive control, and interactions of T−AD + Lam−BD, HS4HJX74−AD + BD, HS4HJX74−BD + AD, HS4HP61−AD + BD and HS4HP61−BD + AD were used as negative controls.
Figure 6. Yeast two−hybrid assay for testing the interaction between HS4HJX74 and HS4HP61. AD and BD represent the activation domain in pGADT7 and the binding domain in pGBKT7, respectively. Interaction between T−AD and 53−BD was used as positive control, and interactions of T−AD + Lam−BD, HS4HJX74−AD + BD, HS4HJX74−BD + AD, HS4HP61−AD + BD and HS4HP61−BD + AD were used as negative controls.
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MDPI and ACS Style

Wang, D.; Xie, W.; Chen, H.; Yang, T.; Liu, Z.; Ruan, Y.; Liu, C. Identification and Characterization of HS4-Mediated Hybrid Seed Shattering in Rice. Agronomy 2024, 14, 1218. https://doi.org/10.3390/agronomy14061218

AMA Style

Wang D, Xie W, Chen H, Yang T, Liu Z, Ruan Y, Liu C. Identification and Characterization of HS4-Mediated Hybrid Seed Shattering in Rice. Agronomy. 2024; 14(6):1218. https://doi.org/10.3390/agronomy14061218

Chicago/Turabian Style

Wang, Daiqi, Wantong Xie, Hong Chen, Tifeng Yang, Ziqiang Liu, Ying Ruan, and Chunlin Liu. 2024. "Identification and Characterization of HS4-Mediated Hybrid Seed Shattering in Rice" Agronomy 14, no. 6: 1218. https://doi.org/10.3390/agronomy14061218

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