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Plant Commun
Plant Commun
Plant Communications
2590-3462
Elsevier

S2590-3462(24)00210-4
10.1016/j.xplc.2024.100940
100940
Correspondence
A rare inter-haplotypic recombination at the S-locus contributed to the loss of self-incompatibility in trifoliate orange
Hu Jianbing hujianbing@mail.hzau.edu.cn
12∗
Guo Furong 1
Du Zezhen 12
Chen Peng 3
Shi Chunmei 1
Zhang Jinzhi 1
Ye Junli 1
Deng Xiuxin 12
Larkin Robert M. 12
Jiao Wenbiao 12
Lin Zongcheng 12
Bosch Maurice 4
Chai Lijun chailijun@mail.hzau.edu.cn
12∗∗
1 National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, P.R. China
2 Hubei Hongshan Laboratory, Wuhan 430070, P.R. China
3 Horticultural Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, P.R. China
4 Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Aberystwyth SY23 3FL, UK
∗ Corresponding author hujianbing@mail.hzau.edu.cn
∗∗ Corresponding author chailijun@mail.hzau.edu.cn
07 5 2024
09 9 2024
07 5 2024
5 9 10094028 12 2023
20 1 2024
2 5 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Published: May 7, 2024
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pmcDear Editor,

In trans-specific S-haplotypes, crossover recombination at the S-locus, which controls self-incompatibility, has been thought to be highly suppressed. Here, we discovered a previously unreported super S haplotype, derived from a rare inter-haplotypic recombination event, that contained two complete suites of functional S-RNase and SLF genes and could break the SI response in trifoliate orange through “self-recognition” within the same pollen.

Self-incompatibility (SI) is an important prezygotic mechanism that is widespread in angiosperms and helps to guard against the negative consequences of inbreeding and species degradation (Kubo et al., 2010). Each SI system depends on a complex genetic mechanism and is mostly controlled by a single S-locus that contains two types (male and female) of tightly linked S-determinant genes (Fujii et al., 2016). Once the linkage between these two types of genes is disconnected or undergoes a disruptive mutation, SI breaks down, which ultimately leads to a species that is capable of selfing (Zhao et al., 2022).

We have a critical need to understand these complex genetic mechanisms because the loss of SI is the primary and key step required for the evolution of selfing (Li et al., 2023). Tight linkage of functional male and female S-determinant genes is required to maintain the SI system. Although evidence for intragenic recombination-based genetic exchanges at the Petunia S-locus has been reported, crossover recombination has always been thought to be highly suppressed (Kubo et al., 2015). Indeed, the loss of SI due to crossover recombination at the S locus is theoretically possible, but there have been no confirmed reports to date.

Previously, we demonstrated that citrus utilizes the S-RNase-based SI system, which has frequently been lost during intergeneric evolution (Liang et al., 2020; Hu et al., 2024). The mechanisms underlying such losses are not fully understood. Poncirus, an ancient branch of true citrus, is a genus in the Aurantioideae of the Rutaceae family. The reproductive characteristics of Poncirus are poorly understood.

To confirm that trifoliate orange (Poncirus trifoliata) is self-compatible (SC), we selected styles 1 day before anthesis for self-pollination (Figure 1A). Aniline blue staining of self-pollinated styles showed that the pollen tubes of P. trifoliata could penetrate the stigma and reach the ovary. These data demonstrated that P. trifoliata is SC (Supplemental Figure 1A). An analysis of the S-RNase genotype indicated that, surprisingly, P. trifoliata has three S alleles. Sequence alignment confirmed that these three S-alleles belong to the Class III Ribonuclease T2 family; they were specifically expressed in the style tissue and were highly homologous to the previously identified S10-RNase, S30-RNase, and S31-RNase alleles from other citrus accessions. They were therefore named PtrS10-RNase, PtrS30-RNase, and PtrS31-RNase (Figure 1B; Supplemental Figures 2–4). Flow cytometry analysis indicated that P. trifoliata containing these three S-alleles was diploid (Supplemental Figure 5). The results of genotype identification demonstrated that most P. trifoliata accessions contained three S alleles and that the S10-RNase and S30-RNase genotypes were most abundant (104/113 and 103/113) in 113 P. trifoliata accessions (Supplemental Figure 1B; Supplemental Table 1). Aniline blue staining demonstrated that P. trifoliata accessions that contained three S-alleles were SC (Supplemental Figure 6).Figure 1 Annotation and recombination analysis of a super S-haplotype in Poncirus trifoliata.

(A) Morphology of P. trifoliata floral organs and pistils. Flowers 1 day before anthesis (left) were selected for pollination. Scale bar, 1 cm.

(B) Semi-quantitative PCR was used to compare the expression levels of three S alleles in different tissues of P. trifoliata.

(C) Fluorescence images of pollen tubes in pistils from a cross-pollination population (Citrus clementina × P. trifoliata ‘precocious’). Representative images are shown. The accession names and genotypes are indicated at the bottom; scale bars, 500 μm.

(D) Segregation of S-RNases in the F1 progeny of a cross-pollinated population (C. clementina × P. trifoliata ‘precocious’). Genotyping of the F1 progeny using PCR with S7-, S10-, S11-, S30-, and S31-RNase specific primers revealed that the S10- and S30-alleles were always found in linkage disequilibrium in the same individual progeny (as shown in the red boxes).

(E) Isolation analysis of S-haplotypes in F1 progeny of P. trifoliata ‘little flower’ × Citrus reticulata. The Sm genotype was present in all the individual F1 progeny produced by the pistils from P. trifoliata ‘little flower’ (as shown in the red boxes). The primers used are listed in Supplemental Table 9.

(F) Identification of self-compatibility after cross-pollination. Fluorescence images of pollen tubes in pistils of P. trifoliata ‘little flower’ after cross-pollination with C. reticulata. The accession names and their respective S genotypes are indicated on the left. A representative image is shown; scale bar, 500 μm.

(G) Segregation analysis of S-haplotypes in F1 progeny from two crosses (C. clementina × P. trifoliata ‘precocious’ and P. trifoliata ‘little flower’ × C. reticulata). aThe red underlines indicate the observed genotypes. bThe S genotype ratios observed in all of the progeny. cThe upper segregation ratio is that expected from a gametophytic self-incompatibility (GSI) system, whereas the lower segregation ratio is that expected from simple Mendelian inheritance. All crosses with parents sharing an S-RNase haplotype showed a result consistent with GSI, with a non-significant chi squared value for this prediction and a highly significant difference (∗∗P < 0.001) for the lower segregation ratio.

(H) Percentage similarity of the recombination site in the PtrS10-S30-locus with the left and right flanking regions of the S-locus. The recombination breakpoint in the candidate recombination region (PtrS10-S30-locus: 313 483–314 400) was predicted. The color code used for each fragment from different S-loci is shown on the right.

(I) Schematic diagram for the gene annotation and collinearity analysis of a super S-haplotype in P. trifoliata and two other common S-haplotypes in C. reticulata. The bright blue and orange lines indicate the left and right flanking conserved regions of the S-locus, respectively. The cyan lines indicate the syntenic sequences among the S-alleles and SLF genes at the three S-locus. The colored boxes represent different genes associated with the PtrS10-S30-locus, CreS10-locus, and CreS30-locus. The red boxes represent S-RNase, and the green boxes represent the SLF genes. The red dotted box in the middle of the PtrS10-S30-locus indicates the candidate recombination region (PtrS10-S30-locus: 313 483–314 400). A red cross between the two S-locus (CreS10-locus and CreS30-locus) indicates the occurrence of a crossover recombination event.

(J) Neighbor-joining phylogenetic trees of deduced amino acid sequences of SLFs designated type 7 (i) and type 8 (ii) predict the SLF–S-RNase interaction. The SLF types shown here have a diverged or deleted SLF (indicated in orange); several have duplicate copies, indicated by a and b. The S-RNases (red characters) that are cognate to the diverged or deleted SLFs are predicted to interact with the conserved SLFs within the two-headed brackets under the non-self-recognition model (Kubo et al., 2010, 2015; Fujii et al., 2016). The blue characters (PtrS30/PtrS10-SLF) are predicted to be targeted by the PtrS10-RNase and PtrS30-RNase in the PtrS10-S30-locus, respectively. Gray dotted lines indicate negative interactions, which do not lead to pollen acceptance. Light gray characters indicate inactivated SLFs (lacking F-box or F-box-associated domain).

To investigate the genetic linkage between the three S-alleles and the loss of SI in Poncirus, we used two previously constructed hybrid populations derived from crosses between Citrus clementina (SI diploid line; S7S11) × P. trifoliata ‘precocious’ (SC diploid line; S10S30S31) and P. trifoliata ‘little flower’ (SC, S10S30S31) × C. reticulata (SC, S10Sm) that included 158 and 89 stable, flowering F1 progeny, respectively. The results of genotype analysis showed that gametes containing the S10- and S30-alleles were always found in the same individual (Figure 1C–1E). In the F1 progeny of C. clementina × P. trifoliata ‘precocious’, four genotypes were detected (S7S10S30:S7S31:S11S10S30:S11S31; the underline indicates linkage between two S-alleles), with a ratio of approximately 1:1:1:1 (χ2 = 0.84, P = 0.84), which is consistent with Mendelian inheritance (Figure 1C, 1D, and 1G). Aniline blue staining of styles showed that individuals with the S7S10S30 and S11S10S30 genotypes were SC and that haplotypes containing the female S7- and S11-alleles were SI (Figure 1C). In addition, segregation analysis of S-alleles in the F1 progeny of P. trifoliata ‘little flower’ × C. reticulata showed that only two genotypes (S10S30Sm and S31Sm) were detected, with a ratio of approximately 1:1 (χ2 = 0.91, P = 0.34) (Figure 1E and 1G). The Sm-allele existed in all of the progeny produced by the pistils of P. trifoliata (Figure 1E). The S10S10S30 and S10S31 genotypes, which were expected outcomes of this cross, were absent from the F1 progeny (Figure 1E and 1G). The results of aniline blue staining also confirmed that the pollen tubes containing S10- and Sm-alleles showed half-compatibility in the styles of P. trifoliata after cross-pollination (Figure 1F). These results suggest that the functional components required for the SI downstream response are present in the pistils of P. trifoliata, resulting in a partial ability to reject pollen from C. reticulata. Taken together, our data provide compelling evidence that the non-self-recognition model in the self-pollination of P. trifoliata is working well.

To understand the linkage between the two S-alleles (PtrS10-RNase and PtrS30-RNase), we sequenced and assembled the genome of P. trifoliata (Supplementary Table 2). Two complete S-locus sequences (PtrS10-S30-locus and PtrS31-locus) without gaps were obtained (Supplemental Figure 7; Supplementary Table 3). The annotation results showed that the PtrS10-S30-locus contains 2 S-RNase genes (PtrS10-RNase and PtrS30-RNase) and 28 S-locus F-Box (SLF) genes, showing linked genetic characteristics (Supplementary Tables 4–6). A phylogenetic analysis indicated that these PtrSLFs can be divided into 14 families (Supplemental Figures 8 and 9). Expression analysis showed that the PtrSLFs at the PtrS31-locus and the PtrS10-S30-locus were specifically expressed only in the anther (Supplemental Figure 10B–10D). These results indicated that the super S-haplotype (PtrS10-S30) contained two complete suites of functional S-RNase and SLF genes.

Synteny results indicated that the left side of the super S-haplotype was highly collinear with the CreS30-locus and that the right side was highly collinear with the CreS10-locus (Supplemental Figure 11A). The results of whole-sequence alignment and recombination detection confirmed that the right flanking regions of different S-loci were highly consistent with the left side of the query sequence (PtrS10-S30-locus: 313 483–314 400). By contrast, the left flanking regions were highly consistent with the right side of the query. A cross between these two types of fragments was apparent in the middle of the query sequence, indicating that there was an obvious recombination breakpoint (Figure 1H; Supplemental Figure 11B and 11C). Gene collinearity results also provided evidence that genes near the candidate recombination region had high collinearity with the right and left flanking regions of different S-loci. These data indicated the occurrence of crossover recombination events (Figure 1I). The remapping results of short and long reads also demonstrated the presence of the recombination site and the accuracy of the S-locus assembly (Supplemental Figure 12).

The collaborative non-self-recognition model (Fujii et al., 2016; Kubo et al., 2010; Kubo et al., 2015) predicts that “self-recognition” within the super SC haplotype is possible between the two complete suites of functional S-RNases and SLFs. Pairwise sequence identities and neighbor-joining phylogenetic analysis of the deduced SLF amino acid sequences predicted the SLF–S-RNase interaction (Supplemental Figure 13; Supplemental Table 7). In the type 7 and type 8 SLF groups, S10-SLF7 and S30-SLF8 are the most diverged, so S10-RNase and S30-RNase are predicted to be the targets of the more conserved SLF7 and SLF8 proteins, respectively (Figure 1J).

In fact, the majority of Poncirus accessions contained three S-alleles, and two of these alleles were closely linked in the super S-haplotype. The other S-allele (PtrS31-RNase) was present in the common S-haplotype (PtrS31), which was SC, and also had a fairly high genotype frequency in the Poncirus population (Supplemental Figure 1B). The promoter region of the S31-allele contained a 315-bp miniature inverted-repeat transposable element insertion, which may have inhibited its expression (Hu et al., 2024) (Figure 1B and 1C; Supplemental Figure 10A). Therefore, the Poncirus genus contains two SC haplotypes, which explains the overwhelming preponderance of these three S-alleles in Poncirus.

Under the collaborative non-self-recognition model, there are multiple routes for the loss of S-RNase-based SI (Zhao et al., 2022). Nonetheless, the deletion, inactivation, or silencing of the female S-determinant gene (Huang et al., 2008; Liang et al., 2020; Hu et al., 2024) and the duplication of the male S-determinant gene at the S-locus (Tsukamoto et al., 2005) are the most common. Here, we found a previously unreported route for the loss of SI, in which a rare inter-haplotypic recombination at the S-locus breaks the restriction of non-self-recognition, resulting in the creation of “self-recognition” in the same pollen that breaks SI. This is similar to the scenarios of tetraploidization of individuals, duplication of SLF, and exogenous introduction of non-self SLF to break SI by “competitive interactions” (Entani et al., 1999; Sijacic et al., 2004; Tsukamoto et al., 2005).

LTR retroelements may have been involved in the genetic exchange between SLF genes during the evolution of the S-locus (Wu et al., 2020). In this study, sequence insertions were also detected in the right and left flanking regions of the S-locus associated with a recombination breakpoint (Supplemental Figure 14; Supplemental Table 8) and therefore might be one of the important driving forces for this rare crossover event. This haplotypic recombination occurred only in Poncirus, possibly resulting from a recent random event, and was fixed during the propagation of the SC haplotype accompanied by apomixis (Liang et al., 2020).

In summary, there are three S-alleles in Poncirus, and two of the functional S-alleles are closely linked owing to cross-recombination between S-haplotypes, resulting in a previously unreported super S haplotype that contains two complete suites of functional S-RNases and SLFs. Our results demonstrated that the super S-haplotype produced by inter-haplotype recombination, which could create “self-recognition” within the same pollen, resulted in the loss of SI. The findings described here not only deepen our understanding of the genetic basis of SI systems but also provide fascinating insights that will stimulate investigations into the origin of SI and the complex molecular evolution of the S-locus.

Data and code availability

All sequence data have been deposited in the China National GeneBank Sequence Archive (CNSA) associated with project no. CNP0004675. The genome assembly has been deposited at the CNSA with accession no. CNP0003278. Other data are available in the source data file or will be made available upon request. Source data are provided with this paper. Detailed information on the S-locus in different intergeneric hybrids of citrus is provided in Supplemental Table 3. S-RNase and SLF sequence data from this article can be found in the GenBank data libraries associated with particular accession numbers (Supplemental Tables 4 and 5).

Funding

This research was financially supported by the 10.13039/501100001809 National Natural Science Foundation of China (grants 32122075 , 32302489 , and 32072523 ), the 10.13039/501100003819 Hubei Provincial Natural Science Foundation of China (grant 2023AFB094 ), and the 10.13039/501100012453 China Agricultural Research System (grant CARS-26 ). The work was also supported by the Biotechnology and Biological Sciences Research Council (10.13039/501100000268 BBSRC ) grant BB/T00486X/1 to M.B. and Vernonica E. Franklin-Tong.

Author contributions

J.H. conceived the project. J.H. performed transcriptomics, genomic sequencing, and data acquisition. J.H., F.G., and C.S. performed PCR, ploidy assays, and recombination breakpoint analysis. Z.D. performed the genome assembly and annotation. F.G. and P.C. collected the plant samples and performed pollination assays. X.D. and L.C. supervised the project and revised the manuscript. J.H. wrote the manuscript with the help of J.Y., Z.L., M.B., and W.J. All authors contributed to the article and approved the manuscript before submission.

Supplemental information

Document S1. Figures S1–S14 and Tables S1–S9

Document S2. Article plus supplemental information

Acknowledgments

We thank Prof. Chungeng Hu for providing genetic population materials. No conflict of interest is declared.

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.
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