Genome-wide identification and characterization of SRLK gene family reveal their roles in self-incompatibility of Erigeron breviscapus
BMC Genomics volume 24, Article number: 402 (2023)
Self-incompatibility (SI) is a reproductive protection mechanism that plants acquired during evolution to prevent self-recession. As the female determinant of SI specificity, SRK has been shown to be the only recognized gene on the stigma and plays important roles in SI response. Asteraceae is the largest family of dicotyledonous plants, many of which exhibit self-incompatibility. However, systematic studies on SRK gene family in Asteraceae are still limited due to lack of high-quality genomic data. In this study, we performed the first systematic genome-wide identification of S-locus receptor like kinases (SRLKs) in the self-incompatible Asteraceae species, Erigeron breviscapus, which is also a widely used perennial medicinal plant endemic to China.52 SRLK genes were identified in the E. breviscapus genome. Structural analysis revealed that the EbSRLK proteins in E. breviscapus are conserved. SRLK proteins from E. breviscapus and other SI plants are clustered into 7 clades, and the majority of the EbSRLK proteins are distributed in Clade I. Chromosomal and duplication analyses indicate that 65% of the EbSRLK genes belong to tandem repeats and could be divided into six tandem gene clusters. Gene expression patterns obtained in E. breviscapus multiple-tissue RNA-Seq data revealed differential temporal and spatial features of EbSRLK genes. Among these, two EbSRLK genes having high expression levels in tongue flowers were cloned. Subcellular localization assay demonstrated that both of their fused proteins are localized on the plasma membrane. All these results indicated that EbSRLK genes possibly involved in SI response in E. breviscapus. This comprehensive genome-wide study of the SRLK gene family in E. breviscapus provides valuable information for understanding the mechanism of SSI in Asteraceae.
Self-incompatibility (SI) is a reproductive mechanism in angiosperm plants to prevent inbreeding by inhibiting self-pollination [1, 2]. SI has been widely observed and documented in plant families such as Brassicaceae , Solanaceae , Rosaceae , Plantaginaceae , Scrophulariaceae , Poaceae , and Rutaceae . Based on the genetic patterns of pollen incompatibility phenotypes, SI is classified as gametophytic self-incompatibility (GSI) and sporophytic self-incompatibility (SSI) . In SSI, the molecular mechanism of SI is well elucidated in detail in the Brassica genus of the Brassicaceae family [10, 11]. SSI represented by Brassica is controlled by a multi-allelic S locus, which has multiple genes. Among them, there are three key multiple alleles in controlling SI, namely the S-locus receptor kinases (SRKs), the S-locus glycoprotein (SLG) and the S-locus cysteine-rich protein (SCR).
SRKs belong to the family of plant receptor protein kinases and are generally localized on the cell membrane with functions of signal perception and recognition. The SRK protein is specifically expressed in the papillary cells of the stigma. The SRK family comprises of 10 subfamilies, which are reported to play important roles in SI of different species . Typical SRKs have three domains: an N terminal extracellular domain, a transmembrane domain, and a C terminal cytoplasmic kinase domain exhibiting serine/threonine protein kinase activity. The extracellular domain of SRKs shares extensive sequence identity with SLG, thus also called SLG-like ectodomain (eSRK) . eSRK consists of two lectin domains with 12 conserved cysteine residues, six in the EGF-like domain, and six in the PAN domain [14, 15]. SRK is a highly polymorphic protein among the haplotypes, with three hypervariable (HVI–III) regions, which are important for perception specificity in SI response [15, 16].
Functional complementation confirms the participation of SLGs and SRKs in SI response . By transforming self-incompatible plants of Brassica napa with an SRK28 and an SLG28 transgene separately, Takasaki et al. (2000) confirmed that expression of SRK28 alone, but not SLG28 alone, conferred the ability to reject self (S28)-pollen on the transgenic plants. They also showed that the ability of SRK28 to reject S28 pollen was enhanced by SLG28, suggesting that SRK alone determines the S haplotype specificity of the stigma, and that SLG promotes a full manifestation of the self-incompatibility response . The role of SRK as the female determinant of SSI is further supported by the functional verification of SRK in Brassica oleracea L. . In addition to the above mentioned components, other proteins such as Arm repeat-containing protein 1 (ARC1), thioredoxin H-Like proteins (THL1/THL2), Aquaporin, Exo70A1 and M-Locus protein kinase (MLPK) are also involved in SSI [17,18,19].
Genetic studies of SI in Asteraceae started with species Crepis foetida L. , guayule , and annual chrysanthemum  in the 1950s and 1960s. A literature survey on the breeding system of 571 species in the Asteraceae family showed that most (63%) of the Asteraceae plants are self-incompatible . Senecio squalidus L. is a model species for studying SI in Asteraceae. Studies on S. squalidus indicate that SI in Asteraceae belongs to the SSI system and is controlled by an S locus [24,25,26,27,28]. Three SRK-like (SRLKs) cDNAs were amplified from the stigma of the heterozygous S (1) and S (2) S alleles of S. squalidus. However, the expression patterns of these SRLKs are different from the patterns of typical SSI in Cruciferae, suggesting that the S. squalidus SRLKs unlikely directly participate in SI or pollen-stigma interaction . The results of cellular and molecular studies on S. squalidus revealed that although they share the same SSI genetic system, the molecular mechanisms of Cruciferae and Asteraceae are different . Despite some explorations have been carried out in S. squalidus, the SI mechanism in Asteraceae is still elusive.
Erigeron breviscapus (Vaniot) Hand.-Mazz., which belongs to the genus Erigeron of the Asteraceae family, is a genuine medicinal plant in southwest China. In a previous study, we performed a comparative transcriptomic analysis in the capitulum of E. breviscapus plants under different pollination treatments. In the three cDNA libraries (non-pollinated, self-pollinated and cross-pollinated of capitulum of E. breviscapus), approximately 230 differentially expressed genes that may be related to the SI response were identified. For example, SRLK and its downstream signaling factor MLPK are up-regulated in self-pollinated flowers, but down-regulated in cross-pollinated flowers . Based on the transcriptomic data, using RACE techniques, we cloned the full-length EbSRLK1 gene in E. breviscapus. In addition, quantitative real-time PCR (qRT-PCR) analysis showed that the EbSRLK1 gene is lowly expressed in roots, stems, and leaves, but highly expressed in flowers, especially in the flowers on the day before opening. This study provides preliminary information for investigating the SI mechanism in E. breviscapus and Asteraceae .
In this study, in order to explore the roles of SRLK in controlling SI in Asteraceae, we systematically identified the SRLK gene family in the E. breviscapus genome and selected the SRLK genes from the typical GSI plant Pyrus spp. communis (Rosaceae family) and from the typical SSI plant B.oleracea (Brassicaceae family) for phylogenetic comparison. The conserved domain analysis of EbSRLKs was performed and the expression patterns of EbSRLKs in different tissues/organs of E. breviscapus and at different flowering stages or pollination conditions were compared. This study provides valuable information for future studies to understand the SI mechanism in E. breviscapus and more broadly in Asteraceae.
Materials and methods
Plant materials and growth conditions
The Erigeron breviscapus seedlings used in this study were provided by Professor Shenchao Yang at Yunnan Agricultural University. Since 2020, seedlings were cultivated in the greenhouse of Honghe University under a photoperiod of 14 h light (25℃) and 10 h dark (16℃), with 200 ~ 600 umol/m2/s photosynthetic photon flux density and 65 ~ 80% humidity for 2 months, and then transferred to a photoperiod of 10 h light (25℃) and 14 h dark (16℃) to induce flowering. Based on the flower morphology, the entire flowering process was divided into 6 developmental stages (Fs1 to Fs6).
Identification of the SRK family members in Erigeron breviscapus
Because the Arabidopsis SRKs are well documented, they were used as reference sequences. All the sequences of A. thaliana SRK proteins (AtSRKs) were downloaded from The Arabidopsis thaliana (L.) Heynh. Information Resource (TAIR). The whole genome sequence of E. breviscapus was downloaded from the GigaScience Database (http://gigadb.org/). For identification purposes, AtSRKs were used as query sequences, and BLASTP was performed (using the NCBI-BLAST-2.7.1 + program) against the E. breviscapus genome. The e-value was set to 1E−5. For the correct identification, more than 60% of similar sequences were included. These identified candidates were further filtered through domain analysis to ensure that the selected sequences are non-redundant sequences to determine the true SRLK family members. For the domain analysis, the standard HMM profiles of the modular S-domain, which includes the B_lectin (PF01453), PAN (PF08276) and SLG (PF00954), were downloaded from the Pfam database (http://pfam.xfam.org/) and then searched against the local protein database of E. breviscapus. All candidate members should contain one of the Pfam domain model. Lastly, one or more transmembrane helices (TMHs) should be present to ultimately identify SRLK family members. The candidate SRLKs were confirmed by the SMART web server (http://smart.emblheidelberg.del) and the CD-search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).
Multiple sequence alignment and construction of phylogenetic tree
To evaluate the phylogenetic relationship of EbSRLK with SRK genes from other self-incompatible species, we chose the typical sporophytic self-incompatibility species B. oleracea and the typical gametophytic self-incompatibility species Pyrus spp. communis. The annotated protein sequences in the sequenced genomes of B. oleracea and Pyrus spp. communis were downloaded from the Ensembl Genomes Database (http://plants.ensembl.org/index.html) and the Rosaceae Genome Database (https://www.rosaceae.org), respectively. The same methods that were used for identifying EbSRLKs were used to identify the SRLKs in B. oleracea (BoSRLK) and P. spp communis (PsSRLK). The full-length SRLK protein sequences of EbSRLK, BoSRLK and PsSRLK were aligned using the ClustalX 2.0 program with the default parameters . The phylogenetic tree was constructed using the Maximum-Likelihood (ML) method and JTT substitution model of the MEGA7.0 software with 1000 bootstrap replicates.
Characterization of EbSRLK genes and proteins
The CDS and protein sequences of EbSRLKs were extracted using the TBtools . To illustrate the structures of EbSRLK genes, the coding sequence of each SRLK gene was aligned with its genomic sequence using the Gene Structure Display Server (GSDS) program (http://gsds.gao-lab.org/). The protein isoelectric point (pI) and molecular weight (MW) of EbSRLK proteins were predicted using the ExPASy proteomics server database (https://www.expasy.org/) and the protein sequence identities of the SRLKs were analyzed using BioEdit . Motifs were identified using the MEME program (http://meme-suite.org/). The parameters were set as zero or one occurrence (of a contributing motif site) per sequence, and the numbers of motif were chosen as five motifs; the motif width was set to 6 to 50. Putative transmembrane helices in proteins were predicted using TMHMM 2.0 web server (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0).
Chromosomal distribution and duplication events of EbSRLK genes
The chromosomal location of all putative EbSRLK genes were analyzed using a local BLAST program  and visualized using the OmicCircos R package . The gene density and the syntenic relationship in the E. breviscapus genome were analyzed using the RIdeogram R package  and MCScanX, respectively . Gene duplication events were analyzed manually, and tandem duplications were characterized as multiple members in one family occurring within the same intergenic region or in neighboring intergenic regions . Major criteria used for analyzing potential gene tandem duplication included the length of aligned sequence coverage > 75% of the longer gene and the similarity of aligned region > 75% . Ka and Ks value were calculated using KaKs Calculator .
Expression analysis of EbSRLKs under different pollination treatments and in different tissues
The expression profiles of EbSRLKs were obtained by analyzing RNA-Seq data downloaded from the Erigeron breviscapus genome database (http://gigadb.org/dataset/100290), including sequences data from multiple tissues. The RNA-Seq data for self-pollinated and cross-pollinated flowers were downloaded from NCBI (SRR1867750). The gene expression levels were estimated by the TopHat/Cufflinks pipeline described in the previous reports with FPKM (fragments per kilobase of exon per million fragments mapped) values. The heat maps for expression profiles in different tissues and at different flowering stages were generated with the OmicShare Tools (http://www.omicshare.com/tools/Home/Index/index.html).
For the expression analysis of EbSRLK genes by quantitative real-time PCR (qRT-PCR), total RNA was isolated from indicated E. breviscapus tissues using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using a PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Japan). qRT-PCR was carried out using an ABI 7500 Real-time PCR System (Bio-Rad, USA) with SYBR® Premix Ex Taq™ (TaKaRa, Japan). The Beacon Designer 8.14 program was used to design the primers (Premier Biosoft International, Palo Alto, CA). The E. breviscapus ACTIN gene was used as the reference control. Primers used in qRT-PCR were listed in Supplementary Table S6. Changes in gene expression were calculated using the 2ΔΔCt method . For each qRT-PCR experiment, three biological replicates of each tissue sampled were used. For each biological replicate, three technical replicates were used.
Subcellular localization prediction and confirmation
The subcellular localization of the 52 EbSRLK proteins were predicted using the online predictor server CELLO v2.5 (http://cello.life.nctu.edu.tw/). The full length CDS sequences (without stop codon) of EbSRLK23 and EbSRLK43, which exhibit high expression levels in most tissues and flowering stages, were amplified. The PCR products were ligated to the pEGOEP35S-eGFP plasmid by T4 DNA ligase (TaKaRa, Japan) to generate fusion expression vectors. The Arabidopsis thaliana (L.) Heynh protoplasts were prepared following the method described by Abel and Theologis  and the 35S::EbSRLKs::eGFP and 35S::eGFP plasmids were introduced into protoplasts using the PEG-mediated method . To further confirm the subcellular localization results generated in protoplasts, transient transformation of epidermal cells of tobacco leaves was employed . The Agrobacterium tumefaciens (strain GV3101) suspension that contains one of the above recombinant vectors was infiltrated into 5-week-old tobacco leaves. Seventy-two hours later, tobacco leaves were removed from the plant and the GFP fluorescence was visualized using the LSM800 confocal microscopy imaging system (Zeiss Co., Oberkochen, Germany).
Identification of SRLKs and phylogenetic analysis
To evaluate the phylogenetic relationship of EbSRLKs with SRLK genes in other self-incompatible species, we chose the typical sporophytic self-incompatibility species B.oleracea and the typical gametophytic self-incompatibility species Pyrus communis for SRLK identification. The S domain (SD), which includes three subdomains with a configuration of B_lectin-SLG-PAN, is the key feature of SRLKs and was used for SRLK identification. Using standard HMM profiles, 50 sequences from B.oleracea, 63 sequences from Pyrus communis and 53 sequences from E. breviscapus with the typical SD domain architecture were identified (Supplementary Table S1). Proteins with stand-alone SLG and PAN domains were rare, whereas the B_lectin domain was more abundant in the genomes of the three species (Supplementary Fig. 1a). The sequences identified based on SD were then uploaded to the Pfam database to search for kinase domains (KD). After excluding 22 (B.oleracea), 15 (Pyrus communis) and 1 (Erigeron breviscapus) sequences that had no kinase domains, the final identified SRLKs in the three species were 28 BoSRLKs, 48 PsSRLKs and 52 EbSRLKs (Supplementary Fig. 1a). The domain architectures were classified into 26 types based on combinations of B_lectin, SLG, PAN and KD domains. Among these types, B_lectin-SLG-PAN-PK_tyr-ser-Thr was the dominant type of architecture in the three species (Supplementary Fig. 1b).
In order to understand the evolutionary relationships between EbSRLKs and SRLKs from other two self-incompatibility species, the amino acid sequences of 28 putative BoSRLKs, 48 putative PsSRLKs and 52 putative EbSRLKs were aligned to construct an unrooted neighbor-joining phylogenetic tree. The results indicated that the SRLKs from the three species were classified into seven clades (Clade I to VII) (Fig. 1). SRLKs from different species were extremely unevenly distributed in different clades. For example, Clade I was the largest group with 41 members, 38 of them being EbSRLKs. Clade VII was formed predominantly by 17 BoSRLKs. Clade V and VI were formed predominantly by 11 and 17 PsSRLKs, respectively. Clade III had only 7 PsSRLKs, no other SRLKs from E. breviscapus or B. oleracea. The remaining 14 EbSRLKs were scattered in Clade IV, V, VI and VII. Similarly, 3, 6, 1, and 1 BoSRLKs were distributed in Clade II, IV, V and VI, respectively; and 3, 2, 3, and 5 PsSRLKs were scattered in Clade I, II, IV and VII, respectively. The results revealed considerable interspecific conservation and intraspecific diversification in different SRLK gene families.
EbSRLK gene features, structures and conserved motifs analysis of EbSRLK proteins
Phylogenetic analysis was carried out using the amino acid sequences of the 52 EbSRLK proteins. The relationship among the 52 EbSRLKs proteins in the new phylogenetic tree (Fig. 2a) was basically similar to the results in the phylogenetic tree constructed in multiple species (Fig. 1). Levels of conservation among EbSRLKs proteins were analyzed via a heat map analysis of amino acid similarity. The results showed that amino acid similarities were consistent with the results of phylogenetic analysis (Fig. 2b). Using the coding sequence (CDS) information of 52 EbSRLKs, the gene structures were analyzed (Fig. 2c). The results indicated that the exon numbers of the EbSRLK genes ranged from 2 (EbSRLK51) to 20 (EbSRLK48) (Supplementary Table S2), with most EbSRLK genes containing 6–9 exons but a few genes containing more than 15 exons, such as EbSRLK35, EbSRLK45, EbSRLK46 and EbSRLK48. The CDSs of the 52 EbSRLK genes ranged from 1,392 bp (EbSRLK29) to 5,052 bp (EbSRLK37), with a mean length of 2,644 bp. The genomic regions of most EbSRLK genes (43 genes) were less than 8.5 kb, while eight of the EbSRLK genes had genomic regions between 20 to 43 kb Exceptionally, EbSRLK37 had a genomic region of 192 kb (Fig. 2c). Combined with the phylogenetic tree and protein similarity data, the results of gene structures indicated that the exon/intron structures were not highly conserved in each clade. The theoretical pI of the 52 EbSRLKs proteins ranged from 5.18 to 8.69. Subcellular localization prediction showed that 25, 11, 6, 5 and 4 of 52 EbSRLK proteins were predicted to localize on the plasma membrane, chloroplast, nucleus, vacuolar membrane and extracellular matrix, respectively (Supplementary Table S2).
Based on studies from Brassicaceae, SRKs have an extracellular S domain, which is responsible for ligand binding, and an intracellular kinase domain, which is responsible for transducing signals into cellular responses by phosphorylating the Arm Repeat Containing (ARC) proteins . The S domain and the kinase domain is separated by one or more transmembrane domains (TMs). Therefore, the SRLKs in Erigeron breviscapus should contain a TM. To confirm the presence of TMs in 52 EbSRLKs, the protein sequences were submitted to the TMHMM Server for TMs prediction. The results showed that 46 of the 52 EbSRLKs contained at least 1 TMs, 6 of the 52 EbSRLKs (EbSRLK2, 4, 5, 35, 36, 40) had no TMs (Supplementary Table S2, Supplementary Figure S2).
The conserved motifs of the 52 EbSRLK proteins were analyzed using the online MEME software. A total of eight conserved motifs were identified (Supplementary Figure S3). Among these motifs, Motif 1 was found in the B_lectin domain region, and Motif 2 was found in the SLG domains, while Motif 4 and Motif 5 were found in the PAN domain. Basically, all 52 EbSRLK proteins contained at least 5 of the 8 conserved motifs, while a few proteins lacking some of the conserved motifs, for example, EbSRLK48 lacking Motif 2, EbSRLK41 lacking Motif 4, EbSRLK40 lacking Motif 8, EbSRLK29 and EbSRLK12 lacking Motif 6, Motif 7 and Motif 8.
Chromosomal distribution and duplication events of EbSRLK genes
The 52 EbSRLK genes were unevenly distributed in the nine Erigeron breviscapus chromosomes, ranging from 1 to 20 genes per chromosome (Fig. 3, Supplementary Table S2). Chromosome 1 and 6 harbored the most number of EbSRLK genes (19 and 20 genes, respectively), whereas Chromosome 4, 5, 7, and 8 had only one EbSRLK gene on each chromosome.
Whole genome duplication (WGD), tandem and segmental duplication gave rise to gene families during biological evolution [45, 46]. To investigate the possible reason of EbSRLK gene expansion, we re-analyzed the E. breviscapus genome using MCScanX. The whole genome analysis did not yield significant evidence of whole-genome duplication but plenty of syntenic blocks were detected (Fig. 3, Supplementary Table S3). To confirm the effect of tandem duplication in EbSRLK gene family expansion, the Ka and Ks values of the gene pairs that are distributed in the same intergenic region or in the neighboring intergenic region were calculated. The analysis results indicated that 65% (34/52) of the EbSRLK genes belong to tandem repeats (Fig. 3, Supplementary Table S4). Based on the locations of these genes on chromosomes, the tandem repeat genes were divided into six tandem-gene clusters, with the largest cluster detected on Chromosome 6 with 13 EbSRLK genes (Fig. 3, Supplementary Table S2).
By integrating the chromosomal distribution of EbSRLKs and the syntenic block information, three pairs of segmental duplications were identified, including the EbSRLK33 and EbSRLK28 gene cluster, the EbSRLK40 and EbSRLK44 gene cluster, and the EbSRLK41 and EbSRLK44 gene cluster (Fig. 3, Supplementary Table S3).
Expression profiles of EbSRLK genes
To explore the spatiotemporal expression patterns of each EbSRLK gene, the FPKM values of the identified EbSRLK genes were extracted from our previously reported E. breviscapus Expression Database . The expression levels (FPKM values) in different tissues and at different flowering stages were presented as heat maps (Fig. 4a). Based on this analysis, some genes, such as EbSRLK44, EbSRLK35, EbSRLK31, EbSRLK28 and EbSRLK18, showed low expression levels in all tissues examined. In contrast, other genes, such as EbSRLK48, EbSRLK43, EbSRLK21, EbSRLK6 and EbSRLK3, were constitutively expressed in every tissue or every flowering stage investigated (Fig. 4a). Compared with other flowering stages, EbSRLK43 and EbSRLK48 had high expression levels at Fs2; EbSRLK29, EbSRLK45 and EbSRLK46 had high expression levels at Fs3; and EbSRLK40 and EbSRLK51 had high expression levels at Fs4 (Fig. 4a, d).
As a typical Asteraceae family species, E. breviscapus plants have dense rosette leaves, tubular flowers and tongue flowers in the floral organs (Fig. 4b, 4c). Since SRK genes are considered to be key genes that control the recognition and initiation of the self-incompatibility response in typical SSI species (such as the Brassicaceae family), in this study, we expected to identify EbSRLK genes that show high expression levels in E. breviscapus floral organs relative to other vegetative organs. However, we did not find a single EbSRLK gene with a significantly elevated expression level in floral organs based on the expression profile of 52 EbSRLKs (Fig. 4a), except EbSRLK23 and EbSRLK6, which showed a slightly higher expression level in tongue flowers. By comparing the transcriptomic data of self-pollination and cross-pollination samples, a total of 10 EbSRLKs genes (EbSRLK13, 16, 17, 29, 37, 40, 45, 46, 47, and 52) were identified with higher expression levels in self-pollination than in cross-pollination samples. Interestingly, most of these 10 EbSRLK genes had low expression levels in flowers at different stages (Fig. 4a).
To further confirm the expression patterns of EbSRLK genes from transcriptomic profiling data, 16 EbSRLK genes with high expression levels from the transcriptional profiles in different tissues were selected and analyzed using qRT-PCR, with β-ACTIN as the reference gene (Supplementary Figure S4, Supplementary Table S5). Totally, 9 EbSRLK genes were successfully amplified. EbSRLK3, EbSRLK12 and EbSRLK26 showed high expression in roots. EbSRLK34 and EbSRLK48 showed high expression in leaves and relatively low expression in floral organs. EbSRLK6 and EbSRLK23 showed high expression levels in tongue flowers and showed increased expression level as flowers developed. At 6 flowering stages, both EbSRLK26 and EbSRLK43 showed a peak expression level at Fs2 (Supplementary Figure S4). In general, the qRT-PCR results confirmed the expression profiles of EbSRLK genes from the transcriptomic data.
Subcellular location of EbSRLK genes
To further confirm the predicted subcellular localization of EbSRLK proteins, the coding sequence (without stop codon) of two EbSRLK genes, EbSRLK23 and EbSRLK43, which showed high expression levels in tongue flowers and Fs2 flowers, respectively, were amplified and ligated to fuse with the reporter gene eGFP under the control of the CaMV35S promoter. Subcellular localization in transfected Arabidopsis thaliana (L.) Heynh protoplasts showed that both EbSRLK23 and EbSRLK43 were detected on the plasma membrane (Fig. 5a). To confirm the results in protoplasts, transient expression of the two constructs was carried out in epidermal cells of tobacco leaves. Results showed that strong fluorescent signals for both EbSRLK23 and EbSRLK43 were detected on the plasma membrane (Fig. 5b).
Previous studies demonstrate that SRKs are crucial in SSI. SRK cloning and functional characterization have been reported in numerous Brassicaceae plants. Furthermore, identification of SRK gene families has been reported in A.thaliana, Brassica napa and B.oleracea. Though SI extensively exits in Asteraceae, except sporadic reports on SRK gene cloning, no systematic characterization of SRK gene families has been reported in Asteraceae. In a previous transcriptomic comparative study of E. breviscapus, we identified 230 differentially expressed genes potentially involved in SI. RNA-Seq analysis revealed that some genes (such as SRLK, MLPK and KAPP) are upregulated in self-pollinated flowers but not in cross-pollinated flowers. In contrast, other genes (such as THL and CaM) exhibit an opposite pattern. Still some genes (such as Exo70A1) have little or no significant changes in different pollinated flowers . We further cloned SRLK1 gene using RACE and analyzed the expression pattern of SRLK1 . Based on the high quality genome assembly of E. breviscapus, we systematically analyzed and characterized the SRLK gene family in this study.
SRLK gene family in E. breviscapus
Self-incompatibility prevents inbreeding depression caused by self-pollination, thus is very important in the inheritance and evolution of flowering plants. Understanding the mechanism of self-incompatibility is of great significance in plant breeding and crop production . In most SSI plants, SRK is specifically expressed on the stigma and is the only important female determinant [51, 52]. Previous studies have identified the SRK genes in B. oleracea , A. thaliana (Cruciferae) , Capsella grandiflora , P. spp (Rosaceae) , Ipomoea trifida (Convolvulaceae) , S.squalidus (Asteraceae) [24,25,26,27,28,29] and Corylus heophylla × Corylus avellana (Betulaceae) . In this study, we systematically identified the SRLK genes in the Asteraceae plant E. breviscapus. Fifty-two EbSRLK genes were identified in the E. breviscapus genome. This is the first systematic analysis and comparison of the SRLK gene family in E. breviscapus and in Asteraceae.
Expression data analysis shows possible involvement of EbSRLKs in E. breviscapus SI response
Our analysis showed marked polymorphism in the EbSRLK genes (Fig. 3). The polymorphism may be due to the strong negative frequency-dependent selection occurred at the S-locus [59, 60]. In many plants, the S-locus alleles are highly polymorphic [61,62,63,64], such as in A. halleri and A. lyrate , and B. neustriaca . In Brassica genus, the sequences of the SRK gene family are complex . The diversity of SRK alleles in Brassica plants is as high as 35% . SRK genes can maintain at such a high diversity level in the population for a long period of time, which could be beneficial for finely regulated self-incompatibility.
SRKs are membrane proteins, containing three distinctive domains: an extracellular domain (S-domain), a transmembrane domain and an intracellular domain with kinase activity . This allows the SRK proteins to play important roles in the extracellular recognition events between pollen and pistil and the intracellular signal transduction pathway that initiates self-incompatibility [70, 71]. Most EbSRLKs contain at least one transmembrane domain (Supplementary Table S2). This means that most EbSRLKs can be positioned on the cell membrane to exert their functions. The SRK proteins in Brassica contain five subdomains. Among them, S_locus_glycoprotein (SLG, PF00954) is a necessary domain for SSI . The 52 EbSRLK genes contain part or all of the five structural or functional domains. For example, EbSRLK2 contains all five structural domains, and EbSRLK11 contains only three structural domains, but the SLG domain is a structural domain in all EbSRLKs.
Phylogenetic analyses of the SRLK sequences in E. breviscapus and two other self-incompatibility species indicate that like in B. oleracea and Pyrus communis, majority of SRLKs in E. breviscapus form an independent group. However, there are also 14 EbSRLKs that are scattered in different clades, predominantly clustered with PsSRLKs or BoSRLKs. Both A. thaliana and B. oleracea are SSI species in the Cruciferae , and P. communis is a GSI species in the Rosaceae . Therefore, although some researchers used dyeing methods to observe the phenotypes and believed that E. breviscapus is an SSI species , we still need further solid evidence to determine whether it is indeed an SSI species.
Tissue- or organ-specific expression patterns usually reflect the corresponding biological functions of genes. In B. oleracea, reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of SRK family members in stigma, leaf and root tissues and the SRK expression was found to be stigma-specific . In our experiments, except that the expression levels of EbSRLK5 and EbSRLK9 are too low to be detected, other EbSRLKs are expressed in roots, stems, leaves and flowers, indicating that EbSRLKs may have other functions besides involving in self-incompatibility. In Corylus heterophylla × Corylus avellana, the ChaSRK gene was also found to be expressed in non-flower tissues, with the highest expression level in root tips . Twenty-four hours after pollination, 10 EbSRLKs genes exhibit higher expression levels in self-pollinated flowers than in cross-pollinated flowers, suggesting these EbSRLK genes may play a role in self-incompatibility. Other EbSRLKs genes do not exhibit differential expressions between self- and cross-pollinated flowers, suggesting they may play roles in other aspects of growth and development in E. breviscapus. Our study provides clues for future research on deciphering the complex functions of SRK genes in E. breviscapus.
Evolutionary expansion of the E. breviscapus SRLK gene family
Phylogenetic comparison indicates that EbSRLKs account for the majority (38 out of 41) of Clade I, while Clade III only contains SRLKs from P. communis. The SRLKs from B. oleracea constitute 68% of Clade VII members. This phenomenon is possibly due to the differences among the three species. Gene expansion could be caused by whole genome duplication (WGD), tandem and segmental duplication at the chromosomal level, and transposon-mediated duplication. The main mechanism of gene duplication was segmental duplication and tandem duplication, The genes in duplicate pairs may go through positive selection and gene conversion after the duplication [76, 77]. In previous studies, several rounds of whole genome duplication in rice genome were reported and this resulted in many duplicated genes [78, 79]. On a genome scale it was found that the rice and poplar TLP family should have expanded mainly through segmental duplication events, rather than through tandem duplication and replicative transposition events [79, 80]. Asteraceae is estimated to originate at ~ 83 Mya. During the evolution, 41 WGDs have been detected in Asteraceae species, accompanied by morphological changes including herbaceousness and capitulescence with multiple flower-like capitula, often with distinct florets and scaly pappus/receptacular bracts. Multiple WGDs might have contributed to the survival of early Asteraceae species by providing new genetic pools to support morphological adaptation. The resulting competitive advantage for adapting to different niches would have increased biodiversity in Asteraceae, making it one of the largest flowering plant families . Duplication events, especially segment type, could increase the gene family members in plants and mutations in the regulatory regions such as promoter site can modify the expression levels and function of new members [81, 82]. Our results indicate that 92.68% of EbSRLK are clustered in Clade I. This biased distribution leads us to speculate whether one or more gene duplication events occurred during the evolution of E. breviscapus. Chromosomal positioning analysis suggests multiple tandem duplications of EbSRLK gene clusters. Transposon-mediated duplication might also lead to EbSRLK gene expansion as exemplified by EbSRLK22 and EbSRLK14. In addition, chromosomal segmental shift is another factor in gene duplication. For example, EbSRLK33 and EbSRLK28 share apparent co-linearity. Similar pattern can be found between the gene cluster of EbSRLK44/48/45/37 and EbSRLK40 and EbSRLK41.
To understand the exact functions of the EbSRLK genes, we will determine the diverse functionality of the EbSRLK genes by constructing overexpression constructs of individual EbSRLK genes and generate transgenic plants, or using the CRISPR/Cas9 system for EbSRLK function validation.
A total of 52 EbSRLKs genes were identifed and their distribution on chromosomes, gene structure, conserved motif, evolutionary relationships and Subcellular location were analyzed. The results show that EbSRLKs are highly conserved and contains a conserved S-locus glycoprotein domain, which is the special sequences protein structure of the SRLKs family. EbSRLKs with higher expression level in self-pollinated sample than in cross-pollinated sample. which deserve further attention and research, and these results implyed that EbSRLKs genes may involved in the SI reaction of E.breviscapus. This study provides useful information for further study of the function of the EbSRLKs genes and SI mechanism in Asteraceae.
Availability of data and materials
The supporting data such as the high-quality reads produced in this study have been deposited in the NCBI SRA database, an open access repository (accession number: PRJNA 379607, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA806708. & accession number PRJNA277583, https://www.ncbi.nlm.nih.gov/bioproject/PRJNA277583).
Takayama S, Isogai A. Self-incompatibility in plants. Annu Rev Plant Biol. 2005;56:467–89.
Silva NF, Goring DR. Mechanisms of self-incompatibility in flowering plants. Cell Mol Life Sci. 2001;58:1988–2007.
Nasrallah JB. Self-incompatibility in the Brassicaceae: regulation and mechanism of self-recognition. Curr Top Dev Biol. 2019;131:435–52.
Richman AD, Kohn JR. Evolutionary genetics of self-incompatibility in the Solanaceae. Plant Mol Biol. 2000;42:169–79.
Sassa H. Molecular mechanism of the S-RNase-based gametophytic self-incompatibility in fruit trees of Rosaceae. Breed Sci. 2016;66:116–21.
McClure B, Cruz-García F, Romero C. Compatibility and incompatibility in S-RNase-based systems. Ann Bot. 2011;108:647–58.
Wang Y, Wang X, Skirpan AL, Kao TH. S-RNase-mediated self-incompatibility. J Exp Bot. 2003;54:115–22.
Lu Y, Moran Lauter AN, Makkena S, Scott MP, Evans MMS. Insights into the molecular control of cross-incompatibility in Zea mays. Plant Reprod. 2020;33:117–28.
Zhang S, Liang M, Wang N, Xu Q, Deng X, Chai L. Reproduction in woody perennial Citrus: an update on nucellar embryony and self-incompatibility. Plant Reprod. 2018;31:43–57.
Kachroo A, Schopfer CR, Nasrallah ME, Nasrallah JB. Allele-specific receptor-ligand interactions in Brassica self-incompatibility. Science. 2001;293:1824–6.
Takayama S, Shimosato H, Shiba H, Funato M, Che FS, Watanabe M, et al. Direct ligand-receptor complex interaction controls Brassica self-incompatibility. Nature. 2001;413:534–8.
Shiu SH, Karlowski WM, Pan R. Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell. 2004;16:1220–34.
Kusaba M, Nishio T, Satta Y. Striking sequence similarity in inter-and intraspecific comparisons of class I SLG alleles from Brassica oleracea and B. campestris: implications for the evolution and recognition mechanism. Proc Natl Acad Sci USA. 1997;94(14):7673–8.
Shiu SH, Bleecker AB. Plant receptor-like kinase gene family: diversity, function, and signaling. Sci STKE. 2001; 20–22.
Naithani S, Chookajorn T, Ripoll DR, Nasrallah JB. Structural modules for receptor dimerization in the S-locus receptor kinase extracellular domain. Proc Natl Acad Sci USA. 2007;104:12211–6.
Sato K, Nishio T, Kimura R. Coevolution of the S-locus genes SRK, SLG and SP11/SCR in Brassica oleracea and B. rapa. Genetics. 2002;162:931–40.
Takasaki T, Hatakeyama K, Suzuki G, Watanabe M, Isogai A, Hinata K. The S receptor kinase determines self-incompatibility in Brassica stigma. Nature. 2000;403:913–6.
Conner J, Tantikanjana T, Stein JC, Kandasamy MK, Nasrallah JB, Nasrallah ME. Transgene induced silencing of S-locus genes and related genes in Brassica. Plant J. 1997;11:809–23.
Nishio T, Kusaba M. Sequence diversity of SLG and SRK in Brassica oleracea L. Ann Bot. 2008;85:141–6.
Hughes MB, Babcock EB. Self-incompatibility in Crepis foetida (L.) subsp. rhoeadifolia (bieb.) Schinz et Keller. Genetics. 1950;35:570–88.
Gerstel DU. Self-incompatibility studies in guayule; inheritance. Genetics. 1950;35:482–506.
Jain HK, Gupta SB. Genetic nature of self-incompatibility in annual chrysanthemum. Experientia. 1960;16:364–5.
Ferrer MM, Good-Avila SV. Macrophylogenetic analyses of the gain and loss of self-incompatibility in the Asteraceae. New Phytol. 2007;173:401–14.
Hiscock SJ. Genetic control of self-incompatibility in Senecio squalidus L. (Asteraceae): a successful colonizing species. Heredity. 2000;85:10–9.
Brennan AC, Harris SA, Tabah DA, Hiscock SJ. The population genetics of sporophytic self-incompatibility in Senecio squalidus . (Asteraceae) I: S allele diversity in a natural population. Heredity. 2002;89:430–8.
Brennan AC, Harris SA, Hiscock SJ. The population genetics of sporophytic self-incompatibility in Senecio squalidus L. (Asteraceae): avoidance of mating constraints imposed by low S-allele number. Philos Trans R Soc Lond B Biol Sci. 2003;358:1047–50.
Brennan AC, Harris SA, Hiscock SJ. Population genetics of sporophytic self-incompatibility in Senecio squalidus L. (Asteraceae) II: a spatial autocorrelation approach to determining mating behaviour in the presence of low S allele diversity. Heredity. 2003;91:502–9.
Brennan AC, Tabah DA, Harris SA, Hiscock SJ. Sporophytic self-incompatibility in Senecio squalidus (Asteraceae): S allele dominance interactions and modifiers of cross-compatibility and selfing rates. Heredity. 2011;106:113–23.
Hiscock SJ, McInnis SM, Tabah DA, Henderson CA, Brennan AC. Sporophytic self-incompatibility in Senecio squalidus L. (Asteraceae)--the search for S. J Exp Bot. 2003;54:169–74.
Zhang W, Wei X, Meng HL, Ma CH, Jiang NH, Zhang GH, et al. Transcriptomic comparison of the self-pollinated and cross-pollinated flowers of Erigeron breviscapus to analyze candidate self-incompatibility-associated genes. BMC Plant Biol. 2015;15:248.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–8.
Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13:1194–202.
Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser. 1999;41:95–8.
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421.
Hu Y, Yan C, Hsu CH, Chen QR, Niu K, Komatsoulis GA, et al. OmicCircos: a simple-to-use R package for the circular visualization of multidimensional omics data. Cancer Inform. 2014;13:13–20.
Hao Z, Lv D, Ge Y, Shi J, Weijers D, Yu G, et al. RIdeogram: drawing SVG graphics to visualize and map genome-wide data on the idiograms. PeerJ Comput Sci. 2020;6: e251.
Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40: e49.
Ramamoorthy R, Jiang SY, Kumar N, Venkatesh PN, Ramachandran SA. comprehensive transcriptional profiling of the WRKY gene family in rice under various abiotic and phytohormone treatments. Plant Cell Physiol. 2008;49:865–79.
Gu Z, Cavalcanti A, Chen FC, Bouman P, Li WH. Extent of gene duplication in the genomes of Drosophila, nematode, and yeast. Mol Biol Evol. 2002;19:256–62.
Zhang Z, Li J, Zhao XQ, Wang J, Wong GK, Yu J. KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genomics Proteomics Bioinformatics. 2006;4:259–63.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–8.
Abel S, Theologis A. Transient gene expression in protoplasts of Arabidopsis thaliana. Methods Mol Biol. 1998;82:209–17.
Cao J, Yao D, Lin F, Jiang M. PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts: a valuable tool for signal transduction study in maize. Acta Physiol Plant. 2014;36:1271–81.
Matsuo K, Fukuzawa N, Matsumura T. A simple agroinfiltration method for transient gene expression in plant leaf discs. J Biosci Bioeng. 2016;122:351–6.
Ha M, Kim ED, Chen ZJ. Duplicate genes increase expression diversity in closely related species and allopolyploids. Proc Natl Acad Sci USA. 2009;106:2295–300.
Vollger MR, Dishuck PC, Sorensen M, Welch AE, Dang V, Dougherty ML, et al. Long-read sequence and assembly of segmental duplications. Nat Methods. 2019;16:88–94.
Yang J, Zhang G, Zhang J, Liu H, Chen W, Wang X, et al. Hybrid de novo genome assembly of the Chinese herbal fleabane Erigeron breviscapus. GigaScience. 2017;6:1–7.
Zhang W, Chen M, Meng HL, Yang J, Wei X, Yang SC. Molecular cloning and expression analysis of SRLK1 gene in self-incompatible Asteraceae species Erigeron breviscapus. Mol Biol Rep. 2019;46(3):3157–65.
Zhang C, Huang CH, Liu M, Hu Y, Panero Jose L, Luebert F, et al. Phylotranscriptomic insights into Asteraceae diversity, polyploidy, and morphological innovation. J Integr Plant Biol. 2021;63:1273–93.
Muñoz-Sanz JV, Zuriaga E, Cruz-García F, McClure B, Romero C. Self-(In)compatibility systems: target traits for crop-production, plant breeding, and biotechnology. Front Plant Sci. 2020;11:195.
McCubbin AG, Kao T. Molecular recognition and response in pollen and pistil interactions. Annu Rev Cell Dev Biol. 2000;16:333–64.
Nasrallah JB, Nasrallah ME. S-locus receptor kinase signalling. Biochem Soc Trans. 2014;42:313–9.
Stein JC, Dixit R, Nasrallah ME, Nasrallah JB. SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco. Plant Cell. 1996;8:429–45.
Sherman-Broyles S, Boggs N, Farkas A, Liu P, Vrebalov J, Nasrallah ME, et al. S locus genes and the evolution of self-fertility in Arabidopsis thaliana. Plant Cell. 2007;19:94–106.
Paetsch M, Mayland-Quellhorst S, Neuffer B. Evolution of the self-incompatibility system in the Brassicaceae: identification of S-locus receptor kinase (SRK) in self-incompatible Capsella grandiflora. Heredity. 2006;97:283–90.
Jung S, Ficklin SP, Lee T, Cheng CH, Blenda A, Zheng P,et al. The Genome Database for Rosaceae (GDR): year 10 update. Nucleic Acids Res. 2014;42 (Database issue), D1237–1244.
Kowyama Y, Kakeda K, Nakano R, Hattori T. SLG/SRK-like genes are expressed in the reproductive tissues of Ipomoea trifida. Sex Plant Reprod. 1995;8:333–8.
Li Q, Zhao T, Liang L, Hou S, Wang G, Ma Q. Molecular cloning and expression analysis of hybrid hazelnut (Corylus heterophylla × Corylus avellana) ChaSRK1/2 genes and their homologs from other cultivars and species. Gene. 2020;756: 144917.
Schierup MH, Vekemans X, Christiansen FB. Allelic genealogies in sporophytic self-incompatibility systems in plants. Genetics. 1998;150:1187–98.
Busch JW, Sharma J, Schoen DJ. Molecular characterization of Lal2, an SRK-like gene linked to the S-locus in the wild mustard Leavenworthia alabamica. Genetics. 2008;178:2055–67.
Boyes DC, Nasrallah JB. Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea. Mol Gen Genet. 1993;236:369–73.
Zhang X, Wang L, Yuan Y, Tian D, Yang S. Rapid copy number expansion and recent recruitment of domains in S-receptor kinase-like genes contribute to the origin of self-incompatibility. FEBS J. 2011;278:4323–37.
Kusaba M, Nishio T, Satta Y, Hinata K, Ockendon D. Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: implications for the evolution and recognition mechanism. Proc Natl Acad Sci USA. 1997;94:7673–8.
Sato K, Nishio T, Kimura R, Kusaba M, Suzuki T, Hatakeyama K,et al. Coevolution of the S-locus genes SRK, SLG and SP11/SCR in Brassica oleracea and B. rapa. Genetics. 2002; 162, 931–940.
Castric V, Vekemans X. Evolution under strong balancing selection: how many codons determine specificity at the female self-incompatibility gene SRK in Brassicaceae? BMC Evol Biol. 2007;7:132.
Leduc JB, Gosset CC, Gries R, Calin K, Schmitt E, Castric V, et al. Self-incompatibility in Brassicaceae: identification and characterization of SRK-like sequences linked to the S-locus in the tribe Biscutelleae. G3 (Bethesda). 2014;G3(4):983–92.
Kumar V, Trick M. Sequence complexity of the S receptor kinase gene family in Brassica. Mol Gen Genet. 1993;241:440–6.
Stein JC, Howlett B, Boyes DC, Nasrallah ME, Nasrallah JB. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea. Proc Natl Acad Sci USA. 1991;88:8816–20.
Goring DR, Rothstein SJ. The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase. Plant Cell. 1992;4:1273–81.
Delorme V, Giranton JL, Hatzfeld Y, Friry A, Heizmann P, Ariza MJ, et al. Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene. Plant J. 1995;7:429–40.
Giranton J, Ariza MJ, Dumas C, Cock JM, Gaude T. The S locus receptor kinase gene encodes a soluble glycoprotein corresponding to the SKR extracellular domain in Brassica oleracea. Plant J. 1995;8:827–34.
Takayama S, Isogai A. Molecular mechanism of self-recognition in Brassica self-incompatibility. J Exp Bot. 2003;54:149–56.
Claessen H, Keulemans W, Van de Poel B, De Storme N. Finding a compatible partner: self-incompatibility in European Pear (Pyrus communis); molecular control, genetic determination, and impact on fertilization and fruit set. Front Plant Sci. 2019;10:407.
Chen M, Fan W, Hao B, Zhang W, Yan M, Zhao Y, et al. EbARC1, an E3 Ubiquitin Ligase Gene in Erigeron breviscapus, Confers Self-Incompatibility inTransgenic Arabidopsis thaliana. Int J Mol Sci. 2020;21:1458.
Kumar V, Trick M. Expression of the S-locus receptor kinase multigene family in Brassica oleracea. Plant J. 1994;6:807–13.
Raes J, Vandepoele K, Simillon C, Saeys Y, Van de Peer Y. Investigating ancient duplication event in the Arabidopsis genome. J Struct Funct Genomics. 2003;3(1–4):117–29.
Hurles M. Gene duplicaion: the genomic trade in spare parts. PloS Biol. 2004;2(7):E206.
Guyot R, Keller B. Ancestral genome duplication in rice. Genome. 2004;47(3):610–4.
Yu J, Wang J, Lin W, Li S, Zhou J, Ni P, et al. The Genomes of Oryza sativa: a history of duplication. Plos Biol. 2005;3(2): e38.
Yang X, Tuskan GA, Cheng MZ. Divergence of the Dof gene families in poplar, Arabidopsis, and rice suggests multiple modes of the gene evolution after duplication. Plant Physiol. 2006;142(3):820–30.
Heidari P, Puresmaeli F, Mora-Poblete F. Genome-wide identification and molecular evolution of the magnesium transporter (MGT) gene family in Citrullus lanatus and Cucumis sativus. Agronomy. 2022;12(10):2253–64.
Faraji, S.; Heidari, P.; Amouei, H.; Filiz, E.; Abdullah; Poczai, P. Investigation and Computational Analysis of the Sulfotransferase (SOT) Gene Family in Potato (Solanum tuberosum): Insights into Sulfur Adjustment for Proper Development and Stimuli Responses. Plants. 2021;10(12): 2597–2610.
This work was supported by National Natural Science Foundation of China (NSFC) (Grant No: 81960684); The Yunnan Major Science and Technology Special Program (202102AA310045) and the special fund project for basic research of local universities in Yunnan Province (Grant No: 2018FH001-011); The open foundation of Key Laboratory of Ethnomedicine KLEM-KF2020GD03 (Minzu University of China), Ministry of Education; The program of Innovation guidance and science and technology enterprises of Yunnan Province(202204BP090011).
Ethics approval and consent to participate
The seedling of Erigeron breviscapus were obtained from Yunnan Zesheng Biotechnology Co., Ltd. The use of plant parts in present study compiles with international, national and/or institutional guidelines.
Consent for publication
All the authors declare that they have no competing of interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Xiang, C., Tao, H., Wang, T. et al. Genome-wide identification and characterization of SRLK gene family reveal their roles in self-incompatibility of Erigeron breviscapus. BMC Genomics 24, 402 (2023). https://doi.org/10.1186/s12864-023-09485-0