Genome‐wide identification, gene expression and haplotype analysis of the rhomboid‐like gene family in wheat (Triticum aestivum L.)

The rhomboid‐like (RBL) gene encodes serine protease, which plays an important role in the response to cell development and diverse stresses. However, genome‐wide identification, expression profiles, and haplotype analysis of the RBL family genes have not been performed in wheat (Triticum aestivum L.). This study investigated the phylogeny and diversity of the RBL family genes in the wheat genome through various approaches, including gene structure analysis, evolutionary relationship analysis, promoter cis‐acting element analysis, expression pattern analysis, and haplotype analysis. The 41 TaRBL genes were identified and divided into five subfamilies in the wheat genome. RBL family genes were expanded through segmented duplication and purification selection. The cis‐element analysis revealed their involvement in various stress responses and plant development. The results of RNA‐seq and quantitative real‐time‐PCR showed that TaRBL genes displayed higher expression levels in developing spike/grain and were differentially regulated under polyethylene glycol, NaCl, and abscisic acid treatments, indicating their roles in grain development and abiotic stress response. A kompetitive allele‐specific PCR molecular marker was developed to confirm the single nucleotide polymorphism of TaRBL14a gene in 263 wheat accessions. We found that the elite haplotype TaRBL14a‐Hap2 showed a significantly higher 1000‐grain weight than TaRBL14a‐Hap11 in at least three environments, and the TaRBL14a‐Hap2 was positively selected in wheat breeding. The findings will provide a good insight into the evolutionary and functional characteristics of the TaRBL genes family in wheat and lay the foundation for future exploration of the regulatory mechanisms of TaRBL genes in plant growth and development, as well as their response to abiotic stresses.

The RBL protein was first discovered in Drosophila (Drosophila melanogaster) and named Rho-1, which directly cleaved transforming growth factor α (TGFα) Spitz within its transmembrane domain and activated the epidermal growth factor receptor (EGFR) pathway in cell (Urban et al., 2001).Then, ubiquitin-binding RBL4 was found to play a vital role in identifying and cleavage of unstable substrates in ER to promote their degradation (Fleig et al., 2012;.J. D. Knopf et al., 2020;Li et al., 2015).
In Arabidopsis, a total of seven RBL genes have been isolated and named AtRBL1-AtRBL7 (Kanaoka et al., 2005).AtRBL2 was found to cleave the Drosophila ligands Spitz and Keren, when expressed in mammalian cells, indicating that the proteolytic activity and substrate specificity of this family also conserved in plants (Kanaoka et al., 2005).Comparative proteomic analysis using double-knockout plants lacking both chloroplast RBL proteins AtRBL8 and AtRBL9 showed a decreased amount of several chloroplast envelope proteins compared with the wild-type plants (R. R. Knopf et al., 2012).The RBL10 gene has been reported to be necessary for the processes of root growth, floral development, fertility, and photoprotection (Lavell et al., 2021;Thompson et al., 2012).A recent study showed that KOM, a Rhomboid serine protease protein, was involved in the regulation of meiocyte-specific callose accumulation and pollen wall formation (Kanaoka et al., 2022).In addition, ER-associated degradation factor Derlin-1, as a rhomboid protease, was required for the dislocation of the mutant α-1 antitrypsin from the ER (Greenblatt et al., 2011).The inhibition of the rice (Oryza sativa L.) OsDER1 gene can activate the unfolded protein responses and the hypersensitivity to ER stress, resulting in powderiness and shrinkage of seeds (Qian et al., 2018).Several members of the RBL genes were also found to be involved in the abiotic stress responses.For example, the ER-bound ANAC013 factor was cleaved by the RBL2 in the initial response to hypoxia in Arabidopsis (Arabidopsis thaliana) (Eysholdt-Derzsó et al., 2023).A genome-wide association analysis study reported that candidate genes for osmotic adjustment and drought traits included RBL, DREB1, and SWEET genes in durum wheat, suggesting that RBL genes could be involved in the regulation of the osmotic adjustment and drought stress in plants (Condorelli et al., 2022).
Wheat (Triticum aestivum L.) provides nearly 20% of the protein and calories in the human diet, so increasing wheat production is a priority for global food security (Calderini et al., 2021;Foulkes et al., 2011).RBL gene has been reported to be involved in plant growth and development, especially in influencing seed morphology in Arabidopsis and rice (Bölter et al., 2006;Qian et al., 2018;Thompson et al., 2012).However, the members and biological functions of the wheat RBL gene are poorly understood.In this study, the members of the RBL family genes were identified at the wheat genome-wide level, and their physicochemical properties, gene structure, conserved domains, gene duplication, and expression patterns were analyzed.Among the wheat RBL gene family, a kompetitive allele-specific PCR (KASP) molecular marker was developed for the TaRBL14a with a high expression level in spike/grain and the favorable haplotype, and temporal and spatial distribution was analyzed in the wheat breeding process.These results provide insights for further exploration of the function of RBL genes.

Identification of TaRBL genes
The wheat genome data file was acquired from the Ensembl Plants database (http://plants.ensembl.org/info/website/ftp/index.html).The RBL conserved domain HMMER file (PF01694) as the query sequence downloaded from the Browse-InterPro (http://ebi.ac.uk) database (Paysan-Lafosse et al., 2023).The HMM search 3.0 software program was used to search for the whole genome protein sequence of wheat (threshold E < 1e-5) to obtain the TaRBL candidate genes (Eddy, 2011).The reported members of the Rhomboid family in Arabidopsis, rice, and maize (Zea mays L.) were blasted as the reference species (Li et al., 2015).The redundant sequence in the HMM search and the BLAST results were removed.Subsequently, the NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and SMART databases (https://smart.embl.de/)were used to confirm these protein sequences (Letunic et al., 2021;Marchler-Bauer et al., 2005).Based on the RBL phylogenetic clustering in Arabidopsis and rice, the identified wheat RBL gene nomenclature was consistent with the description in previous reports (Li et al., 2015).Using the ExPASY server (https://web.expasy.org/compute_pi/) to calculate the molecular weight (MW) and theoretical isoelectric point (pI) of proteins in the wheat TaRBL genes family.The subcellular localization was predicted using the WoLFPSORT website (

Core Ideas
• A total of 41 TaRBL genes were identified in the wheat genome.• The wheat RBL family genes were expanded through segmented duplication and purification selection.• TaRBL genes were generally highly expressed in panicles/grains.• A kompetitive allele-specific PCR molecular marker was developed for TaRBL14a.• The haplotype TaRBL14a-Hap2 was favorable for improving grain weight.
and CCTOP (https://cctop.ttk.hu/job/submit)databases were used to predict transmembrane helixes.There were false positive or ambiguous results in the algorithm applied to predict the transmembrane helix, so we adopted the "TM≥6" principle to provide a reasonable annotation for transmembrane prediction.

Sequence alignment and phylogenetic analysis of TaRBL genes
The MUSCLE method in soft MEGA 11.0 was applied to multiplex the sequence alignment of RBL protein sequences in Arabidopsis, rice, maize, and wheat.The neighbor joining method was used to construct a phylogenetic tree with 1000 bootstrap replications (Tamura et al., 2013).Finally, the tree was visualized with the online tool iTOL (https://itol.embl.de/upload.cgi).According to this tree, RBL families can be divided into five subgroups and named as RhoA, RhoB, RhoC, RhoD, and RAPL (Li et al., 2015).

Gene structure, motif, and domain analysis of TaRBL members
The GSDS server (http://gsds.gao-lab.org/)was used to compare the genomes of TaRBL genes and their corresponding CDS sequences and to analyze the structural schematic diagram of the TaRBL genes.Using the conserved motif to identify the TaRBL protein via the MEME v5.5.3 (MEME-Submission form (meme-suite.org)) with the motif number set to 20 (Table S1) (Bailey et al., 2009).The function domain of TaRBL proteins was identified using the SMART website (SMART: Main Page (embl.de)).The visual plotting was constructed by the TBtools software (Chen et al., 2020).

Chromosome location, gene duplication, and collinearity analysis of TaRBL genes
The physical location of the TaRBL genes was obtained from the wheat genome annotation information (gff3), and the TaRBL genes were mapped on the wheat chromosome.The segmental duplication and tandem repeat events for TaRBL genes family were analyzed through MCScanX program in the TBtools software.The Multiple Synteny Plot module of TBtools was employed to conduct the collinearity analysis of RBL genes in wheat, Arabidopsis, rice, and maize.The synonymous substitution rates (K s ) and the non-synonymous substitution rates (K a ) of duplicate gene pairs were calculated using the TBtools software (Table S2).Here, the K a /K s ratio = 1 means neutral mutation, K a /K s < 1 represents purifying selection, and K a /K s > 1 represents the trend evolution accelerator with positive selection (Zhang et al., 2006).

Analysis of the cis-regulatory elements in the promoter of TaRBL genes
The DNA sequence of TaRBL genes initiation codon (ATG) upstream 2000 bp was extracted from the whole wheat genome sequence through the TBtools software.They were submitted to the online database PlantCARE (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/)for analyzing cis-acting elements.

Expression profiles analysis of TaRBL genes
The RNA-Seq data of TaRBL genes in different tissues/organs of the wheat variety of Chinese spring at different developmental periods were downloaded from the WheatOmics1.0(http://202.194.139.32/expression/wheat.html).The differential expression of TaRBL family genes was analyzed, and then the data were visualized and mapped based on the TBtools software.

Plant growth and abiotic stress treatments
The seeds of the winter wheat variety Jinmai 47 were cultured in Hoagland nutrient solution after disinfection with sodium hypochlorite and rinsing with distilled water, with a photoperiod of 16 h light/8 h darkness and a temperature of 25˚C.The Hoagland nutrient solution was changed every 3 days to ensure a consistent supply of nutrients.When the seedlings had grown to 12 days, they were treated with 20% polyethylene glycol (PEG) 6000, 200 mM NaCl, and 100 μM abscisic acid (ABA).The first leaves of wheat seedlings were taken at 0, 6, 12, 24, and 48 h after stressed treatments, placed in the liquid nitrogen for quick freezing, and then stored in a −80˚C freezer.Each treatment was replicated three times.
The wheat seedlings, roots, stems, leaves, and young spikes at the booting stage, grains at 5, 10, 15, 20, 25, and 30 days post anthesis (DPA), were collected for RNA extraction and tissue expression analysis.
The 263 wheat accessions (Table S3) used for the KASP marker development and association analysis were grown in Tongwei farm station (35˚11′N, 105˚19′E, altitude 1750 m) in 2021, Tongwei and Zhuanglang farm station (35˚21′N, 105˚58′E, altitude 2110 m) in 2022, and Tianshui farm station (34˚34′N, 105˚53′E, altitude 1550 m) and Zhuanglang in 2023, respectively.All wheat accessions were sown in late September and harvested in early July of the following year.A randomized complete block design was conducted with three replications, where the row length of each plot was 1 m and the row spacing was 20 cm, and 30 seeds were sown in each row.Local wheat cultivation practices were considered in field management.After harvesting, two hundred seeds for each cultivar were used to measure grain length (GL), grain width (GW), and 1000-grain weight (TGW) by an image analysis system using the SC-G wheat grain appearance quality (Hangzhou WSeen Detection Technology Co., Ltd.).All measurements were conducted with three biological replicates.

Quantitative real-time-PCR (qRT-PCR) analysis of TaRBL genes
The TIANGEN Plant Tissue RNA Rapid Extraction Kit was used to extract total RNA from collected samples and determine RNA concentration by ultra microphotometer.The synthesis of the first strand of cDNA using FastKing gDNA Dispelling RT SuperMix.FastReal qPCR PreMix (SYBR Green) was used to detect the changes of the relative expression of six genes in different tissues and under different stress treatments by qRT-PCR analysis.Wheat TaGADPH was used as a reference gene for wheat tissue development expression analysis, and wheat TaActin was used as reference gene for wheat stress expression analysis (L.Guo et al., 2022;Mei et al., 2022).The primers for qRT-PCR were listed in Table S4.The relative expression levels of six TaRBL genes were calculated using the 2 −ΔΔCT method (Schmittgen & Livak, 2008).All quantifications were performed on three biological replicates.
The PCR conditions were as follows: 94˚C for 5 min, 35 cycles of 94˚C for 30 s, 58˚C for 30 s, 72˚C for 1 min, and a final extension of 72˚C for 5 min.The PCR products were checked on agarose gel (1%), and the desired bands were purified using the TIANgel Purification Kit (TIANGEN) before gene sequencing (Sangon Biotech).

KASP marker development and association analysis
The SNP (C/G) at position −779 bp in the promoter of TaRBL14a was converted into KASP marker for genotyping of 260 wheat accessions from different provinces in China and three American wheat accessions (Table S3).Two reverse primers and one forward primer were designed for the TaRBL14a-KASP marker using the WheatOmics database (Table S4).The KASP assays were tested in a 96-well plate with 4 μL reaction volume containing 2 μL of 2× KASP master mix, 1 μL of SNP primer mix (4×), and 1 μL of DNA template.The PCR conditions were as follows: hot start at 94˚C for 15 min, followed by 10 touchdown cycles (94˚C for 20 s; touchdown at 61˚C initially and decreasing by −0.6˚C per cycle for 40 s), followed by 35 cycles of annealing (94˚C for 20 s and 55˚C for 45 s).The fluorescence signal was detected and read using Kluster Caller software (v. 3.4.1.36;https://www.biosearchtech.com).
The grain-related phenotypic data of 111 wheat accessions from the published study (Ma et al., 2016), 260 wheat accessions from different provinces in China, and three American accessions in this study were used for association analysis using TASSEL5.1 software (Tables S5 and S6).A one-way analysis of variance was performed to analyze significant differences using SPSS 22.0 (IBM Corporation).The geographical distribution of the two haplotypes of TaRBL14a in China was analyzed with 301 wheat accessions from the WheatUnion database and 224 wheat accessions collected from different provinces in China (Table S7).The selection of the two haplotypes of TaRBL14a in breeding history in China was analyzed using 117 wheat accessions from the Whea-tUnion database and 204 wheat accessions collected from different provinces in China (Table S8).

Identification of RBL members in wheat
In this study, a total of 41 wheat TaRBL genes were identified from the wheat genome.They were renamed as TaRBL1a to TaPARL6 in order according to their position on the chromosome and the Arabidopsis nomenclature.The analysis of the physicochemical properties of TaRBL proteins showed that the protein length ranged from 192 to 401 aa, the protein MW ranged from 20.71 to 44.16 KDa, and the pI varied from 5.45 to 11.62.In addition, the subcellular prediction showed that TaRBL proteins were distributed in the plasma membrane, chloroplasts, ER, or vacuoles, indicating that TaRBL genes could play different roles in different biological processes (Table 1).

Phylogenetic analysis of TaRBL genes
The construction of the phylogenetic tree can further study the conservation of genes in the process of evolution.To analyze the evolutionary relationship of the RBL genes in wheat, we conducted a Neighbor-Joining analysis with the RBL protein sequences encoded by RBL genes.The results showed that RBL proteins were divided into five subfamilies: RhoA, RhoB, RhoC, RhoD, and RAPL (Figure 1).In addition, the gene duplication and missing were identified mainly in the RhoA subfamily.

Gene structure and conserved motif analysis of TaRBL family members
The exon-intron structure of TaRBL family members was analyzed using GSDS to gain further insights into the RBL gene family.The results revealed that the TaRBL genes consist of 2-13 exons, with most members within the same subfamily exhibiting similarities in terms of exon length and intron number (Figure 2).Furthermore, the MEME software was applied to detect the motif composition, and the SMART database was employed to analyze the functional domain of RBL proteins in wheat.At least 20 motifs and four domains were identified successfully (Figure 2; Table S1).Among them, the Rhomboid domain is the main domain of RBL protein, which was identified in all TaRBL proteins.TaRBL proteins in the same subfamily had similar motifs and distributions, indicating functional similarity among members of the same subfamily.During evolution, different subfamilies could undergo divergence.For instance, TaRBL14s had an RBZ/RanBP-type zinc finger domain at its carboxyl terminus, and TaRBL15s had a ubiquitin-binding correlation motif (UBA).Therefore, the zinc finger structure of TaRBL14s could enable it to interact with other proteins or DNA, while TaRBL15s likely played a role in ubiquitinated degradation.All RBL family members comprised six or seven conserved transmembrane helices (TMHS).Of these, the histidine residues and GxxxG motifs in TMH6, which played an important role in protein structure and function (Figure S1).The serine in TMH4 and histidine in TMH6 were crucial for the enzymatically active sites required for proteolytic activity and were conserved in most RBL proteins, indicating their potential proteolytic activity (Figure S1).

Chromosome localization, gene duplication, and collinearity analysis of TaRBL family genes
The 41 TaRBL genes were unevenly localized in 20 chromosomes in wheat, except for 4D (Figure S2).The gene duplication was found in the genome expansion and function acquisition during the wheat polyploidization.Here, 32 segmental duplication gene pairs were identified in the wheat TaRBL family genes.Most of them belonged to the RhoA subfamily without the tandem duplication (Figure 3A).To explore the collinearity between the TaRBL genes of wheat and other species, we used the MCScanX software to analyze the collinear relationship of RBL genes between wheat and Arabidopsis (Figure 3B), rice (Figure 3C), and maize (Figure 3D).The results showed that wheat TaRBL had six homology genes with Arabidopsis, 49 homology genes with rice, and 52 homology genes with maize.These results were highly consistent with the phylogenetic analysis using the protein sequences from wheat, rice, and Arabidopsis, with collinear gene pairs belonging to the same subfamily.This indicated that most TaRBL genes were conservative among plants.The K a /K s ratio of duplicated genes was calculated as a marker of selection pressure.The K a /K s results showed that duplicated gene pairs in the TaRBL family were all less than 1, except for one pair (TaPARL1 and TaPARL3), indicating that the TaRBL genes were mainly driven by purification selection pressure after duplicated events (Table S2).

Cis-acting regulatory elements in the promoters of TaRBL genes
The 2000 bp upstream sequences of 41 TaRBL genes were extracted by the TBtools software and then submitted to the PlantCARE platform for the cis-acting element prediction.As a result, 104 cis-acting elements were identified in the promoter regions on TaRBL genes, including lightresponsive, development-related, phytohormone-responsive, stress-responsive elements, and other elements with unknown function (Figure S3).Among them, MYC/MYB, CAT-box, CCGTCC-box, and ABRE elements related to the responses The Plant Genome to plant development and abiotic stresses were found in almost all TaRBL genes (Figures S4-S6).These elements had been widely reported to be involved in the control of circadian rhythms, flowering time, hypocotyl elongation, and stress tolerance, suggesting that TaRBL genes could be involved in plant growth, development, and abiotic stress responses.

Analysis of the expression patterns of TaRBL genes in different organs
To preliminarily investigate the function of TaRBL genes, the transcriptomic data acquired from the WheatOmics 1.0 database were used to analyze the expression level of TaRBL genes in various organs (root, stem, leaf, spike, and grain).The results showed that 41 TaRBL genes exhibited different expression patterns in different organs (Figure S7).Of these, 25 TaRBL genes were highly expressed in the spike or grain, while eight TaRBL genes were highly expressed in the leaves.The TaRBL genes of the same subfamily generally showed similar expression patterns.For instance, both RhoD and PARL subfamily genes were highly expressed in spikes, while RhoA subfamily genes displayed a wide expression pattern in roots, stems, leaves, and spike/grain, except for TaRBL4 specifically expressed in spike.These results indicated that TaRBL genes in the same subfamily could be functionally conserved.
To verify the results acquired from RNA-Seq data, we selected six TaRBL genes from different subfamilies for qRT-PCR analysis (Table S4).The expression levels of these genes were measured in seedlings, roots, stems, leaves, young spikes, and grains at 5, 10, 15, 20, 25, and 30 DPA by the qRT-PCR determination (Figure 4).The qRT-PCR results showed that all these genes were generally highly expressed in young spike, and their expression levels in grain at different stages were gradually increased after anthesis.These results indicated that TaRBL genes could participate in the regulation of spike and grain development.

Analysis of the expression level of TaRBL genes under different abiotic-stressed treatments
The results of the cis-acting elements analysis showed that TaRBL genes were involved in the response to abiotic stresses.To understand the response of TaRBL genes under abiotic stress, qRT-PCR was used to investigate the expression level of six genes treated with 20% PEG 6000, 200 mM NaCl, and 100 μM ABA (Figure 5).The transcript of most of these genes was induced under 20% PEG 6000 treatment.The expression levels of TaRBL4a, TaRBL10a, and TaRBL10c gradually increased after reaching a peak at 12 or 24 h and then decreased.The expression level of TaRBL4c was continuously increased from 0 to 48 h, while TaRBL15a did not show significant up-regulation after 20% PEG 6000 treatment.Under 200 mM NaCl treatment, all TaRBL genes showed a significant up-regulation expression pattern.The expression level of these genes was gradually increased and reached the peaks at 6, 12, or 24 h.As ABA response-related ciselements were also identified in most TaRBL genes (Figure S6), the expression levels of these genes under 100 μM ABA treatment were further analyzed via qRT-PCR.After 6 h of ABA treatment, the expression levels of all six TaRBL genes showed an up-regulation trend.Notably, TaRBL10c displayed a peak expression level approximately 100-fold higher than that of the control before rapidly declining.This suggested that TaRBL family genes could play an important role in abiotic stress response.

Association analysis of TaRBL14a and grain-related traits in wheat
Based on the qRT-PCR results, TaRBL14a exhibited specific expression in the young spike, indicating its potential role in the development of spike and grain.To uncover the naturally allelic variations of TaRBL14a in wheat, we detected the polymorphisms of the TaRBL14a promoter 2000 bp DNA fragment and coding region by using 619 varieties from the WheatUnion website.We detected one SNP in each of the promoter region and the coding region of TaRBL14a (Figure 6A).Based on two SNPs, two haplotypes of TaRBL14a were identified in these wheat collections, namely TaRBL14a-Hap1 and TaRBL14a-Hap2.A specific primer was designed to amplify the SNP sequences of 10 wheat germplasm promoter regions containing two haplotypes.The SNP sequences of these materials were consistent with the resequencing results of the WheatUnion database (Figure S8).To detect the effect of TaRBL14a allele variation on yield-related traits in wheat, the phenotypic average of grain traits between TaRBL14a allele variations was compared using 111 varieties (Table S5  and S6).TaRBL14a-Hap2 had significantly higher TGW, GL, GW, and grain thickness than TaRBL14a-Hap1 (Figure S9).A KASP marker was developed at −779 bp (C/G) in the TaRBL14a promoter region to distinguish these two haplotypes (Figure 6B).This KASP marker was further validated in 263 wheat accessions.Significant association (p < 0.05) between two TaRBL14a haplotypes with grain weight, length, and width were observed in three, one, and four environments, respectively (Figure 6C).Cis-acting elements prediction in the promoter of TaRBL14a-Hap1 and TaRBL14a-Hap2 indicated that the SNP at −779 bp site in the promoter region was one of the components of binding sites to the transcription factor TCP, which has been reported to be associated with spike/grain development (Figure 6A).This suggested The quantitative real-time-PCR (qRT-PCR) analysis of expression patterns of TaRBLs at different organs.The RNA extracted from seedlings, roots, stems, leaves, and young spike at the booting stage, grains at 5, 10, 15, 20, 25, and 30 days post anthesis (DPA) were used for inverse transcription and further analysis.The relative expression levels in control sample (CK Seedling) were normalized to 1. Data are the mean ± standard errors of three independent replicates.that TaRBL14a-Hap2 was an elite haplotype for TGW and grain yield in wheat.

3.9
TaRBL14a-Hap2 was positively selected for wheat breeding in China Artificial selection leads to the gradual accumulation of elite haplotypes during domestication history.To investigate whether the elite haplotype TaRBL14a-Hap2 had been positively selected in wheat breeding, we evaluated the geographical distribution of two haplotypes of TaRBL14a in China using 325 wheat varieties from 14 provinces in China (Figure 7A; Table S7).The results showed that the accessions with TaRBL14a-Hap2 were the dominant accessions in Hebei (91%), Henan (66%), Shandong (83%), and Shanxi (75%), which were the main wheat-producing areas in China.To further determine whether haplotype TaRBL14a-Hap2 was positively selected during wheat breeding in China, we used 321 different wheat varieties to analyze the selection of allele variants of the TaRBL14a gene in wheat during historical breeding (Figure 7B; Table S8).The results showed that TaRBL14a-Hap2 was positively selected in wheat domestication history.The spatiotemporal distribution results revealed that the elite haplotype TaRBL14a-Hap2 had been positively selected in wheat domestication history for improving wheat grain traits in China.

DISCUSSION
As the intramembranous proteases, RBL proteins are involved in various cellular functions and development processes through the intramembranous proteolysis (Koonin et al., 2003;Urban & Dickey, 2011).The RBL genes have been identified in many high plants, such as Arabidopsis, rice, maize, and poplar (Populus L.) (García-Lorenzo et al., 2006;Li et al., 2015;Roman & Kaldunski, 1991), but a comprehensive analysis of RBL genes has not yet been performed at the genome-wide level of wheat.In this study, we identified 41 members of the TaRBL family and classified them into five subfamilies: RhoA, RhoB, RhoC, RhoD, and RAPL.Most TaRBL genes were highly expressed in spike/grain and could play critical roles in grain development.
In plants, there were differences in the copy numbers of RBL genes, ranging from 4 in green alga (Ostreococcus lucimarinus) to 13 in rice, 17 in Arabidopsis, and 41 in wheat (Li et al., 2015).After gene duplication, the members  pre-1971 1971-1980 1981-1990 1991-2000  of the RBL family lost or changed their genetic information during their evolution.This results in differences in amino acid length and the functional diversity of the RBL protein.For instance, the difference in the catalytic activity between AtRBL2 and AtRBL1 in Arabidopsis resulted in AtRBL2 being able to cleave the drosophila rhomboid substrates Spitz and Keren (Kanaoka et al., 2005).The phylogenetic analysis revealed that the variations in plant copy number were primarily attributed to the RhoA subfamily.The fragment duplication gene pairs were predominantly found in the RhoA subfamily, which could account for these differences.The regulatory genes, that is, transcriptional and developmental regulators, and signaling genes were more likely to be retained after duplication events, consistent with the previous report that most RhoA-type RBL genes were primarily involved in the signaling-related regulatory processes (Lei et al., 2012;Maere et al., 2005;Van de Peer et al., 2009).The Plant Genome Subcellular prediction results showed that TaRBL proteins exhibit a wide distribution in various cellular organelles, including mitochondria, chloroplasts, Golgi apparatus, and ER.Alterations in the subcellular localization of these proteins may consequently cause functional differentiation.For example, AtRBL2 was found to localize on the Golgi apparatus and cleave the Drosophila ligands Spitz and Keren, when expressed in mammalian cells (Kanaoka et al., 2005).AtRBL10 was located in the chloroplasts and was involved in floral development and fertility (Thompson et al., 2012).A recent study reported that the ANAC013-RBL2 module localized in the ER played a role in the initial reaction phase of Arabidopsis hypoxia, rbl knockout mutants exhibited impaired low-oxygen tolerance (Eysholdt-Derzsó et al., 2023).In this study, we found that both TaRBL14a and TaRBL14b contained the zf-RanBP domain at their C-terminus, which were predicted to be localized on the plasma membrane and specifically expressed in spike.The C-terminus of TaRBL14c, TaRBL14d, and TaRBL14e contained ZnF_RBZ domains, which were predicted to be located on chloroplasts and mainly expressed in leaf, spike, and grain (Nakielny et al., 1999;Schultz et al., 1998;Yaseen & Blobel, 1999).These results explained that different subcellular localization, sequence structure, and expression patterns implied functional differentiation of proteins within the same family.
The analysis of promoter cis-acting elements and tissue localization expression patterns suggested that most of the TaRBL family were associated with the development of wheat spike/grain.In rice, inhibiting the expression of OsDER1 led to decreased GL and width via changing starch and protein body I morphology (Qian et al., 2018).In Arabidopsis, the RBL8 affected the morphological changes of pollen outer wall, and RBL10 plays a role in floral development (Kanaoka et al., 2022).
A higher grain yield is an important goal for wheat breeders.Marker-assisted selection (MAS) plays a crucial role in improving wheat crops through breeding (L.Guo et al., 2022;Khan et al., 2022).The increase in wheat yield largely depends on higher TGW (Ur Rehman et al., 2019).Our results showed that TaRBL14a-Hap2 was associated with higher TGW, GL, and GW compared to TaRBL14a-Hap1.This suggested that TaRBL14a-Hap2 was an elite haplotype for improving wheat yield.Given that the elite haplotype of TaRBL14a was actively selected in the main wheat-producing areas and breeding history, further selection of the elite haplotype might be helpful for the continuous improvement of wheat grain yield.In this study, the KASP molecular marker developed for TaRBL14a provides an important function marker for improving TGW by using the marker-assisted selection (MAS) strategy in future wheat breeding.
In this study, qRT-PCR results indicated that TaRBL genes could participate in response to drought stress, salt stress, and ABA treatment.It has been reported that the ANAC013-RBL2 module localized in the ER and played a role in the initial reaction of hypoxia in Arabidopsis (Eysholdt-Derzsó et al., 2023).RBL genes have also been shown to be involved in ABA signaling transduction (Li et al., 2015).Transgenic rice with OsDER1 overexpression or suppression showed hypersensitivity to ER stress with increased levels of ubiquitinated proteins (Qian et al., 2018).These results provided insights for further exploration of the function of the RBL genes.Therefore, TaRBL genes may serve important roles in response to diverse abiotic stress.

CONCLUSIONS
A total of 41 TaRBL genes were identified and divided into five subfamilies with potentially similar functions.Fragment duplication emerged as the primary driver of TaRBL family genes expansion.The results of qRT-PCR showed that TaRBL genes were highly expressed in young spikes and differentially expressed under PEG, NaCl, and ABA treatments, suggesting that they were involved in spike/grain development and abiotic stress response.TaRBL14a-Hap2 was an elite haplotype for TGW and grain yield in wheat.These findings will provide new insights into further exploring the biological function of RBL genes and potentially applying the elite haplotype in wheat breeding program.

C O N F L I C T O F I N T E R E S T S T A T E M E N T
The authors declare no conflicts of interest.

D A T E AVA I L A B I L I T Y S T A T E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Some of the data are shown in Supporting Information.

O R C I D
Tao Chen https://orcid.org/0000-0002-5147-9918Delong Yang https://orcid.org/0000-0001-5370-1589 kb 3 kb 4 kb 5 kb 6 kb 7 kb 8 kb 9 kb 10 kb 11 kb 12 kb kb 0 kb F I G U R E 2 Phylogenetic relationships, conserved protein motifs and gene structures of the TaRBLs.(A) The phylogenetic tree was constructed based on the full-length sequences of wheat rhomboid-like (RBL) proteins.(B) The conserved motifs and functional domain of TaRBL proteins.(C) The Exon-intron structures of TaRBL genes.UTR, untranslated region.F I G U R E 3 Collinearity analysis of RBL genes.(A) Duplicated gene pairs in the wheat genome.(B) Colinearity analysis of TaRBL genes with Arabidopsis genome.(C) Colinearity analysis of TaRBL genes with rice genome.(D) Colinearity analysis of TaRBL genes with maize genome.Duplicated gene pairs are linked by lines with the corresponding color.Gray lines indicate collinear blocks within the wheat genome and other plant genomes, and the red curve indicates RBL genes with collinearity.
qRT-PCR analysis of selected TaRBL genes under PEG6000, NaCl and ABA treatment.(A) Relative expression patterns of TaRBL genes in leaves after PEG 6000 treatment.(B) Relative expression patterns of TaRBL genes in leaves after NaCl treatment.(C) Relative expression patterns of TaRBL genes in leaves after ABA treatment.The relative expression levels in control sample (CK 0 h) were normalized to 1. Data are the mean ± standard errors of three independent replicates.ABA, abscisic acid; PEG, polyethylene glycol.
Spatial and temporal distribution of TaRBL14a haplotype.(A) Geographic distribution of varieties with TaRBL14a haplotypes in China.The map was downloaded in the Standard Map Service System (http://bzdt.ch.mnr.gov.cn/).(B) Frequencies of TaRBL14a allelic variation in Chinese wheat breeding programs in different decades.

T A B L E 1 Characteristics of the TaRBL gene members in wheat Name Gene ID Chromosome location Size (aa) pl MW (kD) Subcellular location
Abbreviation: MW, molecular weight.The Plant Genome We are grateful to Prof. Xueyong Zhang at the Chinese Academy of Agricultural Sciences for providing the 111 wheat genotypes data.We thank the scientists for providing a large amount of resequencing data in the WheatUnion website (http://wheat.cau.edu.cn/WheatUnion/).This work was financially supported by the Key Sci & Tech Special Project of Gansu Province (22ZD6NA009), the National Natural Science Foundation ofChina (32260520, 32360518,  and 32160487), the Development Fund Project of National Guiding Local Science and Technology (23ZYQA0322), the Industrial Support Plan of Colleges and Universities in Gansu Province (2022CYZC-44), the Key Research and Development Program of Gansu Province, China (21YF5NA089), and the Graduate Innovation Star Project 2023 by Gansu Provincial Education Department, China (2023CXZX-684).
Yanyan Zhang: Data curation; formal analysis; validation; visualization; writing-original draft.Xiaoya Huang: Formal analysis; investigation.Long Zhang: Formal analysis; investigation.Weidong Gao: Investigation; visualization.Jingfu Ma: Data curation; software.Tao Chen: Conceptualization; funding acquisition; resources; writing-review and editing.Delong Yang: Conceptualization; funding acquisition; resources; writing-review and editing.A C K N O W L E D G M E N T S