# afila, the origin and nature of a major innovation in the history of pea breeding

Nadim Tayeh, Julie M I Hofer, Grégoire Aubert, Françoise Jacquin, Lynda Turner, Jonathan Kreplak, Pirita Paajanen, Christine Le Signor, Marion Dalmais, Stéphanie Pflieger, et al.

► To cite this version:

Nadim Tayeh, Julie M I Hofer, Grégoire Aubert, Françoise Jacquin, Lynda Turner, et al., afila, the origin and nature of a major innovation in the history of pea breeding. New Phytologist, 2024, 243 (3), pp.1247-1261. 10.1111/nph.19800. hal-04665329

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afila, the origin and nature of a major innovation in the history of
pea breeding

Nadim Tayeh¹ ID, Julie M. I. Hofer² ID, Grégoire Aubert¹ ID, Françoise Jacquin¹, Lynda Turner², Jonathan Kreplak¹ ID, Pirita Paajanen² ID, Christine Le Signor¹ ID, Marion Dalmais³,⁴ ID, Stéphanie Pflieger³,⁴ ID, Valérie Geffroy³,⁴ ID, Noel Ellis² ID and Judith Burstin¹ ID

¹Agroécologie, INRAE, Institut Agro, Univ. Bourgogne, Univ. Bourgogne Franche-Comté, Dijon, F-21000, France; ²John Innes Centre, Norwich Research Park, Colney Lane, Norwich, NR4 7UH, UK; ³Université Paris-Saclay, CNRS, INRAE, Université Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Gif sur Yvette, 91190, France; ⁴Université Paris-Cité, CNRS, INRAE, Institute of Plant Sciences Paris-Saclay (IPS2), Gif sur Yvette, 91190, France

Author for correspondence:
Nadim Tayeh
Email: nadim.tayeh@inrae.fr

Received: 24 August 2023

Accepted: 22 March 2024

New Phytologist (2024) **243**: 1247–1261

doi: 10.1111/nph.19800

**Key words:** afila (af) mutation, breeding history, compound leaf morphology, introgressed haplotypes, legume, megabase-scale deletion, *PALMATE-LIKE* PENTAFOLIATA (PALM) genes, pea (*Pisum sativum* L.).

Summary

* The *afila* (*af*) mutation causes the replacement of leaflets by a branched mass of tendrils in the compound leaves of pea – *Pisum sativum* L. This mutation was first described in 1953, and several reports of spontaneous *af* mutations and induced mutants with a similar phenotype exist. Despite widespread introgression into breeding material, the nature of *af* and the origin of the alleles used remain unknown.

* Here, we combine comparative genomics with reverse genetic approaches to elucidate the genetic determinants of *af*. We also investigate haplotype diversity using a set of *AfAf* and *afaf* cultivars and breeding lines and molecular markers linked to seven consecutive genes.

* Our results show that deletion of two tandemly arranged genes encoding Q-type Cys(2)His (2) zinc finger transcription factors, *PsPALM1a* and *PsPALM1b*, is responsible for the *af* phenotype in pea. Eight haplotypes were identified in the *af*-harbouring genomic region on chromosome 2. These haplotypes differ in the size of the deletion, covering more or less genes.

* Diversity at the *af* locus is valuable for crop improvement and sheds light on the history of pea breeding for improved standing ability. The results will be used to understand the function of *PsPALM1a/b* and to transfer the knowledge for innovation in related crops.

Introduction

Several outstanding crop-improvement successes have been achieved by modifying plant architecture, as exemplified by the introduction of dwarfing genes in cereals (Borlaug, 1983; Lumpkin, 2015). However, in most cases, the molecular determinants underlying increased genetic gain remain unknown. Although not required for the breeding process *per se*, the identification of such determinants is important to minimize linkage drag around target loci (Collard & Mackill, 2008) and to reveal useful variant alleles. It enables smarter and more effective use of the genetic effects at the intra-specific level and also at the inter-specific level through translational approaches (Jacob *et al.*, 2018).

Pea (Pisum sativum L.) is an important crop for food and feed production due to its protein-rich dry seeds and can play a strategic role in achieving food security (McDermott & Wyatt, 2017; Poore & Nemecek, 2018). Since the mid-1970s, pea has been developed as a high-quality protein crop in Europe (Snoad, 1981), in part to reduce overdependence upon imported soy (de Visser *et al.*, 2014). It is a cool-season legume belonging to the Fabeae tribe of the inverted repeat-lacking clade of the Papilionoideae subfamily of

legumes (Azani *et al.*, 2017). It was a model organism for studying the basic principles of inheritance and independent segregation (Knight, 1799; Mendel, 1866), as well as plant physiology and morphology (Dostál, 1941; Murfet & Reid, 1985; Smýkal, 2014). Wild-type (WT) peas have pinnately compound leaves; a mature adult leaf consists of a basal pair of stipules, proximal leaflet pairs, distal tendril pairs and a terminal tendril (Fig. 1a; Hofer & Ellis, 1998; Gourlay *et al.*, 2000).

Leaves are usually flat organs, which account for the majority of a plant's photosynthetic activity and carbon supply (Schiltz *et al.*, 2005). Leaf shape and structure affect photosynthesis and transpiration and are critical for the energy efficiency and water economy. Leaf shape also contributes to the microclimate of the canopy and thus influences disease progression (Schoeny *et al.*, 2008). Genes that control leaf size and shape are therefore of primary importance to agriculture. The molecular bases of different mutations affecting leaf morphogenesis in pea have been described, including *unifoliata* (*uni*; Eriksson, 1929; Hofer *et al.*, 1997), *stamina-pistilloida* (*stp*; Monti & Devreux, 1969; Taylor *et al.*, 2001), *crispa* (*cri*; Lamm, 1949; Tattersall *et al.*, 2005), *tendril-less* (*tl*; Vilmorin, 1910; Hofer *et al.*, 2009),
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Mutants with altered compound leaf morphology in pea (*Pisum sativum* L.). (a) A leaf of cultivar Cameor representing conventional (wild-type) pea leaf morphology. It is composed of two basal stipules, three proximal leaflet pairs, two distal tendril pairs and a terminal tendril. (b) A leaf from accession line FN1210_1_42BC1 taken at a flowering node to show the combined effect of the *unifoliata* (*uni*) mutation on leaf and flower morphogenesis. The *uni* leaf has two basal stipules and a single leaflet. (c) A leaf from accession line VCC0350 carrying the *tendril-less* (*tl*) mutation. All tendrils are replaced by leaflets. (d) A leaf of cultivar Kayanne exhibiting a semi-leafless phenotype resulting from the *afila* (*af*) mutation. No leaflets are present and tendrils are located on the branches (termed rachides or rachillae) of the leaf midrib. This phenotype is common to all currently available semi-leafless pea cultivars. (e) A leaf of accession JI3129, also known as line 11/47, that shows a pair of leaflets of normal size and morphology and the ramification of the central rachis. JI3129, descending from cultivar Raman, holds a novel *af* allele induced by a combination of gamma-rays and fast neutrons (Ambrose, 2004). (f) A leaf from accession NGB101745 carrying simultaneously *af* and *tl* mutations. It is representative of the pea 'parsley leaf' type. Seeds for accession JI3129 were provided by the Germplasm Resources Unit at the John Innes Centre. Seeds for VCC0350 and NGB101745 were provided by INRAE CRB PROTEA based in Dijon, France and the Nordic Genetic Resource Centre in Alnarp, Sweden, respectively. Photographs are from this study. Bars: 1 cm.</figcaption></figure>

*cochleata* (*coch*; Wellensiek, 1959; Couzigou *et al.*, 2012), *apulvinic* (*apu*; Harvey, 1979; Chen *et al.*, 2012), *crispoid* (*crd*; Swiecicki, 1989; Berdnikov *et al.*, 2000; McAdam *et al.*, 2017) and *stipules reduced* (*st*; Pellew & Sverdrup, 1923; Moreau *et al.*, 2018). These mutations affect various aspects of leaf morphology (Fig. 1b,c): degree of pinnation (*uni* and *stp*), leaf polarity (*cri*), organ identity (*tl*, *apu*, *coch*) or organ patterning and development (*crd*, *st*).

One of the major successes in pea breeding over the last 50 yr has been the development of semi-leafless varieties carrying an *afila* (*af*) mutation (Snoad & Gent, 1976; Kielpinski &

Blixt, 1982; Mikić *et al.*, 2011a; Tayeh *et al.*, 2015b). In a mature *af* leaf, the leaflets are replaced by pinnate structures bearing lateral and terminal tendrils, while the distal part of the midrib, which already bears lateral and terminal tendrils, remains unchanged compared with a conventional leaf (Fig. 1d). Semi-leafless pea types are particularly suitable for large-scale production and mechanical harvesting; they have a significantly increased standing ability as plants with strongly intertwined leaves can better support each other. They also show a reduced severity of foliar disease and enhanced uniformity of ripening and drying due to increased air movement and homogeneous
light distribution inside the canopy (Snoad, 1974; Mikić *et al.*, 2011a). The yield superiority of semi-leafless types com-
pared with conventional leafy types under normal competitive
field conditions has been reported (Snoad, 1974; Kielpinski &
Blixt, 1982); however, this type has been shown to be associated
with reduced plant nitrogen content and seed size potential (Bur-
stin *et al.*, 2007). Contemporary dry pea breeding programmes
world-wide almost exclusively use the semi-leafless ideotype.

The *af* or semi-leafless phenotype in pea arises as a consequence of a recessive mutation at a single locus (Kujala, 1953; Solov'eva, 1958; Goldenberg, 1965; Khangildin, 1966; Swiecki, 1982) on linkage group (LG) I (Marx, 1969; Blixt, 1972; Bordat *et al.*, 2011; Tayeh *et al.*, 2015a). In WT (*AfAf*) genotypes, the proximally positioned lateral primordia, on the flanks of the leaf primordium, develop into determinate leaflet laminae, but in *af* mutants, this fails to happen; the lateral and apical primordia behave in the same way so that second-order, midrib-like, primordia in turn initiate organ primordia (Meicenheimer *et al.*, 1983; Côté *et al.*, 1992).

There are three early reports of spontaneously occurring *af*
mutants: The first was by Kujala (1953) based on a field observa-
tion in 1950, the second report was by Solov'eva (1958) describ-
ing a 'whiskered' (усатый, usatyy, sometimes transliterated as
usatyy) pea first seen in 1949 (Kielpinski & Blixt, 1982), and the
third report from Goldenberg (1965) described a phenotypically
similar spontaneous mutation that occurred in cultivar Cuaren-
tona in Argentina. Multiple induced mutants from different
genetic backgrounds with similar phenotypes have also been
identified (Fig. 1e; Kielpinski & Blixt, 1982; Ambrose, 2004)
and are available in germplasm collections. These include a
gamma-ray-induced *af* allele described by Jaranowski (1970).

The interaction of *Af* with other genes has been studied in
double or triple mutants (Marx, 1974; Gourlay *et al.*, 2000)
(Fig. 1f) in efforts to understand *Af* function. These suggested
that *Af* is a negative regulator of *Uni* transcription (Hofer &
Ellis, 1998; Gourlay *et al.*, 2000) and that *Af* is a negative regula-
tor of auxin synthesis or is involved in auxin conjugation or cata-
bolism (DeMason *et al.*, 2013). Thus, pinna initiation
and determination during leaf development in pea and the estab-
lishment of pinna identity are likely to depend on auxin gradients
together with antagonistic interaction between *Uni* and *Af* (Hofer
& Ellis, 1998; Gourlay *et al.*, 2000; DeMason *et al.*, 2013). In
*Medicago truncatula* Gaertn. and *Cicer arietinum* L., close rela-
tives of pea, it has been shown that Cys(2)His(2)-zinc finger tran-
scription factors named PALM1 and MULTI-PINNATE
LEAF1 (MPL1) bind to the promoters of *SINGLE LEAFLET1*
(*SGL1*), the orthologue of *Uni* in *M. truncatula*, and *CaLEAFY*
(*CaLFY*), the orthologue of *Uni* in *C. arietinum*, thereby repres-
sing *SGL1* and *CaLFY* expressions (Chen *et al.*, 2010; He
*et al.*, 2020; Liu *et al.*, 2023).

Besides the lack of information on the molecular identity of *af*,
it was also unclear how the pea community managed to trans-
form *af* from simple spontaneous mutations observed in the
USSR (Solov'eva, 1958), Finland (Kujala, 1953) and Argentina
(Goldenberg, 1965) to a major breeding criterion in dry pea. Did
pioneer breeders simply rely on spontaneous mutations in their

programmes? What part did mutation breeding (Blixt, 1972)
play in developing semi-leafless pea varieties? Therefore, the aims
of this study were to: elucidate the genes underlying the *af* pheno-
type, thus contributing to the identification and description of a
key element in the determination of leaf formation in pea; and
highlight the origin and prevalence of different alleles and haplo-
types associated with the semi-leafless phenotype.

**Materials and Methods**

Plant material

Four recombinant inbred line (RIL) populations, namely Pop2, Pop4, Pop5 and BxP, obtained by single seed descent from crosses between the accessions listed in Supporting Information Table S1, were used to place *af* on the consensus map of Tayeh *et al.* (2015a). The pea (*Pisum sativum* L.) cultivars Ballet, Terese and Baccara show a semi-leafless phenotype (Fig. 1d), while the cultivars/landraces Cameor, Champagne, PI180693 and VavD265 have a conventional leaf type. The 450 RILs from all four populations were visually scored for leaf type, and phenotypic data were used for the genetic analyses. Another 58 individual RIL population and a fine-mapping population of 967 F<sub>7</sub> and F<sub>8</sub> individuals were used for the same purpose in an independent experiment. Details are given in Notes S1.

Fifty-nine accessions with conventional leaf type and 107 *af* accessions, including 63 and 72% cultivars, respectively, were used as a validation panel to confirm the identity of the candidate molecular determinants for *af* and to assess the different haplotypes at this locus (Table S2; Fig. 4). The cultivars were bred in Europe, North America and Australia. For some mutants and cultivars, different seed sources were used for confirmation, bringing the total number of examined accessions to 173 (Table S2). Seeds were mainly provided by the Pulse Genetic Resources Unit at INRAE in Dijon, France (CRB PROTEA), the John Innes Centre and the Nordic Genetic Resource Centre in Sweden, except for gamma-ray-induced mutants from cultivars Auralia, Borek and Raman. These mutants were provided by the Bulgarian Academy of Sciences, Ulpia Serdica, Sofia-Capital, Bulgaria. Mutant FN2070 was identified as a homozygous recessive segregant in the M3 generation of a fast neutron mutant population and backcrossed twice. The fast neutron-mutagenized population, derived from accession JI2822, is described by Domoney *et al.* (2013).

To identify TILLING mutants in *PsPALM1a* and *PsPALM1b*, an ethylmethane sulfonate (EMS) mutant population from pea cultivar Cameor was screened. M2 mutant lines were screened for substitutions in the coding sequences of these genes, and homozygotes were selected in the M3 or M4 generation. All recovered homozygous plants, 51 for *PsPALM1a* and 37 for *PsPALM1b*, were subjected to leaf phenotyping in a glasshouse. Three lines carrying mutations provoking non-synonymous amino acid changes or premature stop codons were selected for further analyses. Two cycles of backcross to Cameor were performed. Homozygotes for the mutant alleles were then crossed to a semi-leafless accession (JI1195) where both *PsPALM1a* and
*PsPALM1b* are lacking. The F₁ plants, hemizygous for either one WT *PsPALM1b* allele and one mutant *PsPALM1a* allele or one WT *PsPALM1a* allele and one mutant *PsPALM1b* allele, were analysed for their leaf types.

DNA extraction and SNP genotyping

DNA extraction was performed on fresh young leaves using the Macherey-Nagel™ NucleoSpin™ Plant II mini kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. Elution was performed in 50 µl ultrapure water. DNA purity and concentration were assessed using a Nano-Drop™ ND-1000 spectrophotometer (Thermo Scientific, Wilmington, NC, USA).

Polymerase chain reactions (PCR) were conducted to determine the presence or absence of seven candidate genes located in the *af*-harbouring region. The products were run on agarose gel for visual analysis. Polymerase chain reaction primers (Table S3) were designed on pea transcript sequences (Alves-Carvalho *et al.*, 2015) sharing the highest homology scores with the sequences from the model legumes *Medicago truncatula* Gaertn. and *Glycine max* (L.) Merr. (see 'Orthologous gene identification and synteny analysis' in the Materials and Methods section). A PCR primer set corresponding to a *sucrose phosphate synthase* gene located outside of the *af* region and on a separate chromosome (LG IV; Aubert *et al.*, 2006) was regularly used as a control to rule out any DNA or PCR mix problems in the presence/absence detection.

Orthologous gene identification and synteny analysis

The transcript sequences corresponding to the *af*-flanking SNP markers from the pea consensus map (Tayeh *et al.*, 2015a) were used for orthologue search against *M. truncatula* Mt4.0 genome release (Tang *et al.*, 2014) and *G. max* Williams 82 assembly 2 (Grant *et al.*, 2010). In all cases, best hits from reciprocal BLAST were considered as orthologues. Gene order on *M. truncatula* and *G. max* pseudomolecules was cross-compared with the gene order on pea v.1a genome assembly version (Kreplak *et al.*, 2019) once available. To assess the extent of synteny conservation, *Arabidopsis thaliana* (L.) Heynh. genome assembly available from The Arabidopsis Information Resource (Berardini *et al.*, 2015) was further included for orthologue search. *Lens culinaris* Medik. CDC Redberry-v.2.0 genome assembly accessible on the Know-Pulse web portal (http://knowpulse.usask.ca) and *Cicer arietinum* L. Desi uwa-v.3.0 genome assembly (Edwards, 2016) were also used for comparative genomics.

Fast neutron mutant analysis

Raw fastq reads of the FN2070 palmate mutant, deposited in the European Nucleotide Archive at EMBL-EBI under accession no. PRJEB60035, were mapped using bwa mem (v.7.0.12; Li & Durbin, 2010), and the bam file was processed using SAMTOOLS (v.1.9; Danecek *et al.*, 2021). The pea ZW6 genome (Yang *et al.*, 2022) was split into 100-kb windows using BEDTOOLS

(v.2.2.28; Quinlan & Hall, 2010), and SAMTOOLS cov was used to find the coverage in these windows. The deletion on LG I, at Chr2:473,791,227–474,324,688, was curated manually from the raw data.

Virus-induced gene silencing

A 349-bp DNA fragment from the *PsPALM1a* gene of pea cultivar Champagne, a forage pea with conventional leaf type, was PCR-amplified using the primer pair 11 in Table S3. The 349-bp fragment of *PsPALM1a* shares 95.4% nucleotide identity with *PsPALM1b* and is located at the 3'-end of both genes. The PCR product was cloned into pGEM-T plasmid (Promega) and sequenced. This 349-bp fragment was then subcloned into the BamHI cloning site of the pBPMV-IA-V1 plasmid (Zhang *et al.*, 2010). The orientation of the insert was determined by PCR using a combination of vector-specific and fragment-specific primers (Table S3; primer pair 12). The insert sequence and its reverse orientation were confirmed by Sanger sequencing (GATC, Germany).

The Bean pod mottle virus (BPMV)-*PsPALM1* virus-induced gene silencing (VIGS) vector was introduced into *Phaseolus vulgaris* L. cultivar Black Valentine plants by mechanical inoculation of the two BPMV-derived infectious plasmids: pBPMV-IA-R1M (carrying RNA1 of BPMV) and pBPMV-*PsPALM1* (carrying RNA2 of BPMV with the inserted 349 bp-*PsPALM1a* fragment) (Pflieger *et al.*, 2014, 2015). After 3 wk, infected *P. vulgaris* leaves were used as viral inoculum to inoculate 15 healthy plants of cultivar Champagne as described previously (Meziadi *et al.*, 2016, 2017). Nine mock-inoculated plants and nine WT BPMV-inoculated plants were used as controls. All plants were pruned at 4 wk after inoculation to concentrate BPMV titre and increase VIGS efficiency (Meziadi *et al.*, 2016, 2017). Leaf morphology was assessed at 11.5 wk after inoculation. The chi-square statistical test was used for data analysis.

Quantitative real-time PCR

Quantitative real-time PCR was performed on the Biomark™ HD system (Fluidigm, San Francisco, CA, USA) according to the manufacturer's protocol to assess the relative gene expression of *PsPALM1a*, *PsPALM1b* and *Uni*. Total RNA extraction from different tissue samples collected from pea cultivar Cameor (D'Erfurth *et al.*, 2012; Alves-Carvalho *et al.*, 2015; Vernoud *et al.*, 2021) was performed using the same protocol as Gallardo *et al.* (2007). Tissue samples included the following: shoots, roots, nodules, developing seeds (8–29 d after pollination (DAP)) and dissected seeds at 12 DAP (embryo, endosperm and seed coat). Three biological replicates were included. Relative expression levels were evaluated using the 2^−ΔΔCt^ method, and three control genes, that is *histone H1*, *actin* and *EF1α*, were used for data normalization as described by Alves-Carvalho *et al.* (2015). For investigating gene expression in the EMS TILLING mutant 2835 and gamma-ray mutants in cultivars Auralia, Borek and Raman, 10 germinating seeds at 3 d after imbibition (DAI) were considered, each representing a separate biological replicate.
## Results

afila is located within a 1.4-cM interval on pea linkage group I with PsCam023334_13180_1532 as the closest molecular marker

Five pea RIL populations segregating for leaf morphology (Ellis et al., 1992; Tayeh et al., 2015a) (Table S1) were used in two independent studies to finely map *af* and identify candidate genes. One parent from each population has a conventional leaf type (Fig. 1a), and the other carries the *af* mutation. The approach based on the four RIL populations, namely Pop2, Pop4, Pop5 and BxP, and the synteny between closely related legume genomes is described below. The other approach, using JI1201 x JI813 RIL population and pea bacterial artificial chromosome clones, is described in Notes S1.

Phenotyping and genotyping of Pop2, Pop4, Pop5 and BxP RILs located the *af* mutation at 85.6 cM on LG I (chromosome 2) of the integrated pea consensus genetic map in Tayeh *et al.* (2015a) (Fig. 2a). Thirteen out of a total of 450 F<sub>6–9</sub> inbred lines carry recombination events near the morphological marker *af* (Fig. 2b) and were examined here. Based on their haplotypes inferred from markers exclusively polymorphic in all four populations,

including *af*, the *Af*-containing region could be narrowed down to a 1.4-cM interval located between gene-based SNP markers PsCam023334_13180_1532 and PsCam000001_1_322 (Fig. 2c). *af* is genetically closer to PsCam023334_13180_1532 than to PsCam000001_1_322 with only two recombination events between *af* and PsCam023334_13180_1532 compared with 10 recombination events between *af* and PsCam000001_1_322. Due to the recessive nature of the *af* mutation and the residual heterozygosity, the exact position of the recombination event in line Pop5-192 could not be determined (Fig. 2c). The results corroborate those obtained by the parallel approach, which identified a close linkage between *af* and PsCam002050 (Notes S1). Interestingly, a SNP marker located in PsCam002050, namely PsCam002050_1670_86, co-locates with PsCam023334_13180_1532 on the consensus map from Tayeh *et al.* (2015a).

### Co-orthologues of *Medicago truncatula* PALMATE-LIKE PENTAFOLIATA1 are candidate genes for *Afila*

Candidate genes for *Af* were mined using *af*-flanking markers from the pea consensus genetic map and the synteny relationships with close species with available genome assemblies (Fig. 3; Table S4). *Medicago truncatula* and *Glycine max* were considered.

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Fine mapping of pea (Pisum sativum L.) *afila* (*af*) locus. (a) Schematic representation of marker density on pea linkage group I on the integrated consensus map of Tayeh *et al.* (2015a); the region near *af* is shown in detail. Maps were constructed using LinkageMapView R package (Ouellette *et al.*, 2018). (b) Graphical genotype illustration for 13 recombinant inbred lines carrying recombination events near the *af* locus. Lines are from four different populations: Pop2, Pop4, Pop5 and BxP. The orange colour refers to alleles inherited from parents characterized by a conventional leaf type. The green colour refers to alleles inherited from parents showing semi-leafless phenotypes. The blue colour indicates heterozygosity. The grey colour is used when data are not available: monomorphic markers or missing data. (c) Graphical genotype illustration considering only the closest markers to *af* and polymorphic in all four mapping populations. The order of the individuals is the same as in section (b). The position of *af* in sections (a–c) is marked in magenta.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3** Collinearity between the pea (*Pisum sativum* L.) linkage map at the *afila* (*af*) locus and the physical maps of *Medicago truncatula* chromosome 5, *Glycine max* chromosome 11 and *Arabidopsis thaliana* chromosome 4. (a) Order of the gene-based SNP markers on the integrated consensus genetic map at the *af* locus. The polymorphic markers in the four recombinant inbred line populations Pop2, Pop4, Pop5 and BxP, delimiting the fine-mapped *af* interval, are in bold. (b–d) Schematic illustration of the order of genes in the syntenic regions in *M. truncatula* (b), *G. max* (c) and *A. thaliana* (d). These regions are of total length of 407, 331 and 101 kb, respectively. Colour-filled boxes refer to predicted genes that have orthologue counterparts in the species under investigation. Black, blue and orange lines are used to link putative orthologs. Grey boxes refer to predicted genes that do not have equivalents in other species. *Arabidopsis thaliana* is used as an outgroup to show the extent of collinearity conservation beyond the legume family. In total, 30 genes are contained in the illustrated genomic region from *A. thaliana* chromosome 4 but, for the sake of clarity, only those having putative orthologues in *M. truncatula* or *G. max* are illustrated. The orthologues of *PsPALM1a* and *PsPALM1b*, candidate genes for *Af*, are shown as magenta squares.</figcaption></figure>

Fifty-two and 32 genes were identified in the synteny blocks on *M. truncatula* chromosome 5 and *G. max* chromosome 11, respectively (Table S4). As shown in Fig. 3, overall collinearity was conserved between pea, *M. truncatula*, *G. max* and even *A. thaliana*. *Medtr5g014400*, named *PALMATE-LIKE PENTAFO-LIATA1 (PALM1)*, was one of the genes located in the collinear block from *M. truncatula*. It is the equivalent of MtrunA17-Chr5g0400571 in the latest r5.0 assembly version of the *M. truncatula* A17 genome and has conserved orthologues in *G. max* and *A. thaliana* (Fig. 3). Two strong lines of evidence indicated that the pea orthologue of *Medtr5g014400* is a relevant candidate gene for *Af*. *Medtr5g014400* was reported to control compound leaf morphology in *M. truncatula* (Chen et al., 2010); and *Medtr5g014400* is only 15.5- and 68.9-kb distant from *Medtr5g014410* and *Medtr5g014260*, the orthologues of the pea genes hosting PsCam023334_13180_1532 and PsCam002050_1670_86, respectively (Table S4).

Contrary to *M. truncatula*, *C. arietinum*, *G. max* and *A. thaliana*, but similar to *L. culinaris* (Figs 4, S1), not one but two sequences similar to *PALM1* were identified by homology search against pea genomic sequence data from cultivar Cameor (Aluome et al., 2016). The putative intron-less genes encode highly similar Q-type Cys(2)His(2)-zinc finger proteins (93.3% identity) with conserved functional domains (Notes S2; Fig. S2) and putative auxin response elements in their promoter regions (Fig. S3; Table S5). They were designated *PsPALM1a* (scaffold_45110) and *PsPALM1b* (scaffold_30425). Three copies of

the neighbouring gene, encoding an N2-acetylornithine deacetylase, were identified in pea, two copies in *L. culinaris* (Fig. S1), whereas *M. truncatula* and *G. max* each have a single copy (*Medtr5g014410* and *Glyma11g04650*). Pea genes predicted to encode N2-acetylornithine deacetylase according to the PsCam_LowCopy Unigene set (Alves-Carvalho et al., 2015) are hereafter referred to as: *PsNAOD1* (PsCam045967), *PsNAOD2* (PsCam027203) and *PsNAOD3* (PsCam023334).

Pea Cameor v.1a genome sequence (Kreplak et al., 2019), which became available later in the study, placed *PsPALM1a* and *PsPALM1b* 521 kb apart (Fig. 4) most likely because of a misorientation of respective scaffolds. The genomic sequence of pea cultivar ZW6 made available by Yang et al. (2022) and an Oxford Nanopore PromethION sequencing-based new assembly of Cameor genome (unpublished results) showed that *PsPALM1a* and *PsPALM1b* are separated by 259.6 kb. The two *M. truncatula* *PALM1* co-orthologues in *L. culinaris* cultivar CDC Redberry genome assembly v.2.0 are separated by 62.8 kb, with three predicted open reading frames between them, most likely related to repeat elements (Fig. 4).

*PsPALM1a*, *PsPALM1b* and *PsNAOD1* are absent in semi-leafless parent and recombinant inbred lines

Primer pairs were designed to clone *PsPALM1a* and *PsPALM1b* from RIL parental lines (Table S3). The aim was to identify mutations in the coding or regulatory regions that could
<figure><img src="image_7.png" /><figcaption>**Fig. 4** Evolution of the *afila* (*af*)-containing region in the Fabeae tribe. Two representatives of the Fabeae tribe (*Pisum sativum* L. and *Lens culinaris* Medik.) and one representative of each of the sister tribes Trifoliæae (*Medicago truncatula* Gaertn.) and Cicereae (*Cicer arietinum* L.) are included. Each rectangle represents a different gene unit. Colours are used to distinguish the orthologue sets: in grey, putative small auxin-up RNA coding genes; in magenta, putative Q-type C2H2 zinc finger coding genes; in blue and green, putative N2-acetylornithine deacetylase coding genes; in orange, putative CAAX amino-terminal protease coding genes. Phylogenetic analyses were performed to confirm orthology and the phylogenetic trees are shown in Supporting Information Fig. S1. Numbers in grey refer to the number of genes separating two depicted gene units (left) and to the physical distance between them (right; in kb). The following genome assembly versions were used to generate this figure: *P. sativum* v.1a, *M. truncatula* r5.0, *C. arietinum* Desi uwa-V3.0 and *L. culinaris* CDC Redberry Genome v.2.0. 1, both numbers are uncertain due to a possible misorientation of the corresponding scaffolds on the pseudomolecule. A new nanopore sequencing-based assembly of this region of the Cameor cultivar genome shows no genes between *PsPALM1a* and *PsPALM1b* and an intergenic distance of 259.6 kb. This is further confirmed by the genome assembly of *P. sativum* cultivar ZW6 (Yang et al., 2022). *PsPALM1a* and *PsPALM1b* do not appear in the current annotation of the cultivar ZW6 genome, but the corresponding sequences can be retrieved by BLAST search on chromosome 2 pseudomolecule, confirming the information about the distance from each other.</figcaption></figure>

potentially explain the *af* phenotype. Interestingly, no amplicons could be obtained from semi-leafless lines, whereas amplicons from accessions with conventional leaves were of the expected size (Fig. S4A). The same observation was made with primers for *PsNAOD1* (Fig. S4B). Genotyping the recombinant lines from Pop2, Pop4, Pop5 and BxP at the *af* locus (Fig. 2c), taking advantage of the presence/absence polymorphism for these three genes, confirmed the co-segregation of *PsPALM1a*, *PsPALM1b* and *PsNAOD1* with *Af*. Indeed, all semi-leafless RILs showed no amplification. Recombinant inbred lines with conventional compound leaves, used as controls, showed normal amplification. Furthermore, mapping of whole-genome sequence reads from pea accessions included in the SNP detection panel of Tayeh *et al.* (2015a) against cultivar Cameor reference genomic

scaffolds revealed an absence of read coverage for the semi-leafless genotypes for the scaffolds corresponding to *PsPALM1a*, *PsPALM1b* and *PsNAOD1*, whereas read coverage was normal for all conventional-leaved genotypes (Fig. S4C). The results thus suggest that the deletion of at least three genes, including the *M. truncatula* *PALM1* co-orthologues, is tightly linked to the *af* mutation.

Eight haplotypes define the genomic region harbouring the *afila* mutation and show that deletion of *PsPALM1a* and *PsPALM1b* confers the afila phenotype

To extend the results above, the presence/absence of seven consecutive pea genes, including *PsPALM1a* and *PsPALM1b*, was assessed in 166 cultivars, breeding lines and mutants of different geographical origins with conventional or leafless/semi-leafless phenotypes (Table S2). In total, eight haplotypes were identified, with none to seven genes being absent (Fig. 5). All genes examined were present in all accessions presenting a conventional leaf type. These latter were thus assigned to a single haplotype, referred to as Haplotype 1 (Fig. 5). *PsPALM1b* was the only gene to be systematically absent in all leafless/semi-leafless accessions (Haplotypes 2–8). *PsPALM1a* was similarly absent in all *af* cultivars but was present in three Bulgarian gamma-ray-induced mutant lines derived from cultivars Auralia (Haplotype 6), Borek and Raman (Haplotype 7). The mutation in cultivar Raman (Fig. S5A) was confirmed by Ambrose (2004) as a new, weak allele at the *af* locus. The mutants derived from cultivars Auralia and Borek were not investigated in detail, but their compound leaves occasionally produce leaflets (Fig. S5B,C).

For three haplotypes, the origin of the *af* alleles can be determined as follows:

(1) Haplotype 2 includes four accessions designated Usatuj or Usatyj; this is most likely Solov'eva's Usatyj-5 (Solov'eva, 1958). We propose to call this spontaneous mutant allele, *af*<sup>S</sup>.

(2) Haplotype 3 includes cultivars Filby, Barton and Melton, which are recorded as being derived from a cross with an 'Argentinian af' obtained from G.A. Marx at Cornell (Table S2). This haplotype also includes Marx's isogenic series MISOG-1 and MISOG-2. This is consistent with Marx receiving the *af* mutants from Goldenberg (Marx, 1969); we can call this spontaneous allele, *af*<sup>G</sup>.

(3) Haplotype 4 includes the line Wasata-af; this is clearly Jaranowski's induced allele (Jaranowski, 1970) and can be named *af*<sup>J</sup>.

Haplotype 5 is represented by five cultivars and must have a different origin from Haplotypes 2, 3 or 4. The origin of this *af* allele is unknown. The *af* allele described by Kujala (1953) has no known progeny. Kujala (1953) suggests that this mutant was lost, so it would be unwise to assume that this haplotype is related to it.

Haplotypes 6, 7 and 8 occur in more recently generated mutant lines. We propose to call the respective alleles *af*<sup>A</sup>, *af*<sup>B</sup>/*af*<sup>R</sup> and *af*<sup>N</sup> (Table S2).

These results are consistent with the *af* phenotype being the consequence of the loss of both *PsPALM1a* and *PsPALM1b*, and that different sources of *af* mutations have been used in released cultivars.
<figure><img src="image_8.png" /><figcaption>**Fig. 5** Diversity of haplotypes at the *afila* (*af*) locus in pea (*Pisum sativum* L.). Black arrows represent *Af* candidate genes (*PsPALM1a* and *PsPALM1b*) and their neighbouring genes identified following a synteny-based strategy. Haplotypes are numbered 1–8. Green and grey boxes indicate whether the corresponding genes/regions are present or absent, respectively. White boxes represent intergenic regions where the deletion endpoints lie, but the positions are uncertain. It is noteworthy that the haplotypes in this figure are based on information obtained from polymerase chain reaction with specific primers for the seven examined genes. Each haplotype may have several subgroups once the deletion boundaries are precisely defined. All accessions with conventional leaf type are clustered under Haplotype 1. Haplotypes 2–8 are exclusively found in leafless or semi-leafless accessions. The number of cultivars and mutants belonging to each haplotype, as well as the total number of examined accessions, are provided at the right side of the figure (Supporting Information Table S2).</figcaption></figure>

A fast neutron mutant with a deletion of *PsPALM1a* but not *PsPALM1b* exhibits a palmate leaf phenotype in the JI2822 background

A line isolated from a fast neutron-irradiated mutant population derived from accession JI2822 (Domoney *et al.*, 2013) was identified by a forward genetic screen as exhibiting an altered leaf phenotype. This line, designated FN2070, has extra leaflets at lower nodes (Fig. S6) and normal leaves at upper nodes. Comparison of FN2070 genomic reads with the recently published genomic sequence of the conventional-leaved pea cultivar ZW6 (Yang *et al.*, 2022), having a higher assembly resolution at the *af* locus than pea Cameor v.1a genomic sequence (Kreplak *et al.*, 2019), showed a deletion encompassing multiple genes. The deletion removes the consecutive genes *Psat2g173880* (*PsPALM1a*), *Psat2g173920* (*PsCam002388*; Fig. 5) and *Psat2g173960* in Cameor v.1a genome annotation, but not *Psat2g173360* (*PsPALM1b*). This confirms that the *PsPALM1a* genomic region is involved in leaf development and shows that the deletion of *PsPALM1a* alone does not induce the *af* phenotype.

TiLLING mutants of *PsPALM1a* or *PsPALM1b* in cultivar
Cameor do not have the *afila* phenotype and no double
mutant could be obtained

To further investigate whether *PsPALM1a* and/or *PsPALM1b* are
involved in the regulation of compound leaf morphogenesis in pea,
the Cameor EMS-mutagenized population (Dalmais *et al.*, 2008)
was screened for mutants. No nucleotide substitutions introducing
premature stop codons were found for *PsPALM1a*; however, the
TiLLING screen identified 15 missense mutations including two
nonconservative amino acid changes in the C2H2 zinc finger
domain of the protein (Table S6). Line 3715 has a change of

Cysteine into Tyrosine at position 81 (C81Y) corresponding to the first conserved Cysteine residue of the zinc finger. Line 2512 has a H101Y substitution corresponding to the second conserved Histidine residue, which occurs in a region that is expected to form an α-helix in the three-dimensional structure (Wei *et al.*, 2016). Both changes are expected to disrupt Zn⁺ ion binding within the zinc finger of the PsPALM1a protein and affect its DNA-binding function. By contrast, 13 missense and two nonsense mutations were identified for PsPALM1b (Table S6). Translation termination would occur immediately before the QALGGH motif (Q91*) in line 2835 leading to a short protein without a C2H2 zinc finger and no repressor domain (EAR motif). Unlike the bipinnate af leaf phenotype exhibited by the gamma-ray mutants lacking PsPALM1b (Haplotypes 6 and 7; Figs 1e, S5) and the palmate leaf phenotype exhibited by the PsPALM1a deletion in mutant FN2070 (Fig. S6), all three EMS mutants in Cameor exhibited a conventional leaf phenotype (Fig. S7A,B). F₁ plants resulting from crosses between TiLLING lines 3715, 2512 or 2835 and JI1195, a semi-leafless accession lacking both PsPALM1a and PsPALM1b, showed palmate or bipinnate leaf phenotypes at occasional nodes (Fig. S7C–F), but most leaves were conventional. Bipinnate tendrilled leaves without leaflets, typical of *af* cultivars (Fig. 1d), were not observed consistently on any F₁ plants. This showed that homozygous or hemizygous missense or nonsense alleles at either PsPALM1a or PsPALM1b did not confer an *af* phenotype. However, in hemizygous F₁ plants, some nodes produced bipinnate or palmate leaves.

Virus-induced gene silencing did not confer the *afila*
phenotype

Since the physical proximity of *PsPALM1a* and *PsPALM1b* hinders the construction of double mutants by simple crosses
between single mutants and since pea is rather recalcitrant to
transformation, VIGS was attempted as a reverse genetics tool
to test the functions of *PsPALM1a* and *PsPALM1b*. VIGS is a
suitable strategy for knocking down both *PsPALM1a* and
*PsPALM1b* due to their high degree of sequence similarity, and
this was attempted using a BPMV VIGS vector (Zhang
et al., 2010; Meziadi et al., 2016) (Fig. S8). The
BPMV-*PsPALM1* VIGS vector was delivered to pea plants of cul-
tivar Champagne at the two-leaf stage by mechanical rubbing of
the stipules and leaflets of both leaves. No leaves with a clear af
phenotype were observed at maturity.

Pea leaves are heteroblastic, with additional pinna pairs being
added as the plant matures. This increase in complexity is seen
first as an additional pair of tendrils and then as an additional
pair of leaflets (Blixt, 1972). Occasionally, a leaflet forms oppo-
site a tendril as this increase in pinna number occurs. We
observed a slight increase in these two leaf forms (an additional
pair of tendrils or a leaflet opposite a tendril) in the VIGS-
treated compared with the control (35 out of 352 leaves in the
BPMV-*PsPALM1* compared with 2 out of 85 in the BPMV-0-
inoculated plants; χ² = 5.09; P = 0.024) (Fig. S8). These results
suggested that *PsPALM1a* and *PsPALM1b* are involved in the
control of leaf formation and that a complete knockout of both
genes is likely required to obtain an af phenotype.

*PsPALM1a* and *PsPALM1b* exhibit similar expression profiles and contribute to the regulation of leaf morphology in a complex, likely dose-dependent, manner

Microfluidic droplet-based gene expression profiling was con-
ducted to study the expression of *PsPALM1a* and *PsPALM1b* at
the spatio-temporal level. RNA was extracted from root, nodule,
shoot, flower, expanded leaf and seed tissues of cultivar Cameor
(Fig. S9). The expression of *PsPALM1a* and *PsPALM1b* was not
detected in root, nodule or flower samples. It was detected mainly
in developing seeds (12 to 29 DAP), but also in shoots. These
results are consistent with data from the pea gene atlas (Alves-
Carvalho *et al.*, 2015), visible in the pea genome browser
(https://urgi.versailles.inra.fr/Species/Pisum), for *Psat2g173880*
(*PsPALM1a*) and *Psat2g173360* (*PsPALM1b*; Kreplak *et al.*,
2019). At 12 DAP, *PsPALM1a*, *PsPALM1b* and *Uni* gene expres-
sion was detected in the embryo (when cotyledons are expanding)
and not in the endosperm or the seed coat (Fig. S9). At later
stages of seed development (16 to 29 DAP), when scale leaf pri-
mordia and compound leaf primordia are developing on the
embryo axis, *PsPALM1a* and *PsPALM1b* expression levels were
consistently higher than *Uni*. Together, these results suggest that
*PsPALM1a* and *PsPALM1b* may repress *Uni* during seed develop-
ment as they do in adult pea leaves (Gourlay *et al.*, 2000).

The expression of *PsPALM1a* and/or *PsPALM1b* was further
investigated in the context where *PsPALM1b* carries a mutation
causing a premature stop codon (EMS TiLLING mutant 2835
in the Cameor background) or is deleted (Bulgarian gamma-ray
mutants in the Auralia, Borek, and Raman cultivars). The expres-
sion level of *PsPALM1a* and *PsPALM1b* did not change signifi-
cantly in the EMS mutant compared with the WT in

germinating seeds at 3 DAI (Fig. 6a). However, the expression of *PsPALM1a* was significantly decreased in gamma-ray mutants (Fig. 6b–d). The mutant in cultivar Raman showed the least decrease (Fig. 6d). This differential regulation of *PsPALM1a* may explain the divergent phenotypes observed in these mutants, with the TiLLING mutant 2835 having a normal leaf morphology, the mutation in cultivar Raman causing an intermediate af phe-notype (Ambrose, 2004; Fig. 1e), and the mutations in cultivars Auralia and Borek resulting in bipinnate tendrilled leaves very similar to those of *af* cultivars lacking both *PsPALM1* genes (Fig. S5).

Discussion

Fifty years ago, the pea crop was different from today. Plants
were tall and had conventional leaves. This often resulted in a
heavy canopy with poor standing ability. Much of the yield was
lost at harvest due to lodging, and seed quality was often poor
due to pathogen attacks arising from damp soil conditions (Ban-
niza et al., 2005). The success story of improving lodging resis-
tance in pea owes much to the introduction of the *af* mutation
(Kujala, 1953; Solov'eva, 1958; Goldenberg, 1965; Khangil-
din, 1966; Swiecicki, 1982) together with a mutation in Men-
del's stem length gene *Le* (White, 1917; Lester et al., 1997;
Martin et al., 1997) into breeding material. The *af* mutation
results in leaves with an increased number of tendrils, whereas
the *le* mutation causes short-stem internodes (Lester et al., 1997;
Martin et al., 1997). The main scientific and breeding efforts
to achieve a short-stem semi-leafless ideotype were made in
the seventies, mainly in Europe (Duparque, 1996; Mikić
et al., 2011b). Today's dry pea cultivars almost all carry the *af*
and *le* mutations.

Here, we show that *af* mutants carry a large deletion on pea
chromosome 2. Large deletions are known to contribute to phe-
notypic variation in living organisms (Hurles *et al.*, 2008;
Springer *et al.*, 2009), and these are often associated with trans-
posable elements, DNA recombination, replication and repair-
associated processes (Morgante *et al.*, 2005; Hastings
*et al.*, 2009). In rice, a 38.3-kb region harbouring genes regulat-
ing grain number and plant height, together with the *heading
date7* gene, is completely deleted in cultivar Zhenshan 97 (Xue
*et al.*, 2008). In maize, the deletion of a 5.16-Mb segment on
chromosome 2 in the spontaneous *multi-trait weakened* mutant
results in the loss of 48 genes, which is responsible for a range of
phenotypic defects, including wilting, reduced plant height
and decreased yield (Han *et al.*, 2019). Pea has a genome of
c. 4.45 Gb, of which an estimated 83% consists of repetitive ele-
ments, mostly retrotransposons (Kreplak *et al.*, 2019). The pre-
sence or absence of these elements across the genome is known to
be highly polymorphic between pea accessions and may
be related to the presence/absence of the genomic regions
in between, thus contributing to genomic diversity (Ellis
*et al.*, 1998; Jing *et al.*, 2010).

Seven haplotypes were identified at the *af* locus in the pea
accessions showing an af phenotype, namely Haplotypes 2 to 8
(Fig. 5; Table S2). The assignment of 107 semi-leafless (*afaf StSt*)
<figure><img src="image_9.png" /><figcaption>**Fig. 6** Expression of *PsPALM1a* and *PsPALM1b* in germinating seeds of four pea (*Pisum sativum* L.) cultivars and their respective mutants. Gene expression was detected by quantitative real-time polymerase chain reaction. The pea cultivars are Cameor, Auralia, Borek and Raman. All mutants carry mutations at *PsPALM1b*. The mutant in Cameor background was identified in an ethylmethane sulfonate-mutagenized population (line 2835) and has a G:C to A:T transition leading to a premature stop codon. The mutants in the other backgrounds have full deletions of *PsPALM1b* that arose from mutagenesis with gamma-ray. Seeds were sampled at 3 d after imbibition and 10 biological replicates were used per condition. Graphics and statistics were done with the software R v.4.0.0 using the *GGPUBR* package v.0.3.0, available at https://github.com/kassambara/ggpubr. The horizontal lines in the box plots indicate the medians and the boxes indicate the first and third quartiles. The ends of the whiskers extend the boxes from each side to the next observed data points within 1.5 times the interquartile ranges. All data points outside the boundaries of the whiskers are plotted as outliers. ns, *P* > 0.05; *, *P* ≤ 0.05; **, *P* ≤ 0.01; ***, *P* ≤ 0.001; ****, *P* ≤ 0.0001 (using paired *t*-test).</figcaption></figure>

and leafless (*afaf stst*) pea accessions corresponding to cultivars, mutants and research and breeding lines, to these seven haplotype groups is largely congruent with available pedigree information, reflecting the robustness of the molecular tools developed in this study to discriminate the haplotypes.

The cultivar Ballet, selected from a cross between Filby and NRPB 60242 (Leterme *et al.*, 1992), and its derived RILs (Pop4-69, Pop5-22, Pop5-80, Pop5-81 and Pop5-142; Table S2) all clustered with Filby, as expected. Similarly, the varieties Sum (Porta × Wasata), Hamil ((Wasata × 1.6 l/78) × Porta) and Ramir ((Porta × Wasata) × Flavanda) were found to share the same haplotype as the induced *af* mutant in cultivar Wasata (Jaranowski, 1970) obtained in Poland (FAO/IAEA Mutant database; https://mvd.iaea.org/).

Inconsistencies exist (Table S2), but these most likely reflect inaccuracies in the sources of information or records, as the *af*

alleles have so far been indistinguishable. For example, contrary to the records of the FAO/IAEA Mutant database indicating that Legenda carries the *af*<sup>S</sup> allele from Usatyj-5, Legenda and Usatyj-*af* were found in two different haplotype clusters (Table S2), but this is consistent with Kielpinski & Blixt (1982) and reported by Kielpinski in 2017 (http://www.kielpinski.eu/). Legenda is similar to the cultivars Sum, Hamil, Ramir and Miko, all developed by hybridization with Wasata-af (allele *af*<sup>J</sup>). The records of the John Innes *Pisum* Collection (https://www.seedstor.ac.uk) also indicate that the first leafless cultivar in the UK, registered for Plant Variety Rights in 1978 (Ambrose, 2004), namely cultivar Filby (accession ID: JI1768), was derived from a cross between cultivar Dik Trom and a spontaneous *af* mutant from Argentina. The Filby *af* allele clusters with the *af* used by Marx in lines B268-394-3, MISOG1 and MISOG2, and this is the Argentinian *af* mutant from Goldenberg (1965) according to
handwritten breeding notes. However, the lines JI2400 and NGB101692, from a common origin, are recorded as Goldenberg's *af*, but in fact, they carry the Solov'eva (1958) *af*<sup>S</sup> allele and not *af*<sup>G</sup>: It is therefore likely that these lines have been misidentified. Future complete sequencing of pea accessions will shed more light on these inconsistencies and precisely delineate the deletion breakpoints in each line.

The different haplotypes at the *af* locus show that several pro-
genitors have been used for the development of improved pea
cultivars, since the first initiatives to breed semi-leafless and leaf-
less pea cultivars launched in Poland and England (Snoad, 1981;
Kielpinski & Blixt, 1982). Of the 77 *af* cultivars we examined
(Fig. 5; Table S2), six were derived from the spontaneous mutant
reported by Solov'eva (1958) and 51 from Goldenberg (1965).
The allele from the induced mutant in Wasata (Jaranowski, 1970)
is present in 15 cultivars. Five cultivars have an *af* allele of
unknown origin.

All cultivars carrying the *af* mutation share a deletion involving two genes, viz. *PsPALM1a* and *PsPALM1b*. After *PALM1* in *M. truncatula* (Chen *et al.*, 2010), *MPL1* in *C. arietinum* (Liu *et al.*, 2023) and *St* in pea (Moreau *et al.*, 2018), this provides further evidence for the involvement of Q-type C2H2 zinc finger transcription factors in the control of leaf morphology in legumes belonging to the inverted repeat-lacking clade. *PALM1* negatively regulates the expression of *SGL1* in *M. truncatula* lateral leaflet primordia (He *et al.*, 2020) and is in turn the direct target of *M. truncatula* AUXIN RESPONSE FACTOR3 (MtARF3), which acts as a transcriptional repressor of *PALM1* by interacting with specific AuxREs in the *PALM1* promoter (Peng *et al.*, 2017). Mutants carrying large deletions spanning up to eight annotated genes (*palm1-3*) and tobacco Tnt1 retrotransposons (*palm1-4*, *palm1-5* and *palm1-6*) within the coding region of *PALM1* have confirmed the role of this gene as a determinant of leaf morphogenesis in *M. truncatula* (Chen *et al.*, 2010). Naturally occurring mutants carrying more or less large deletions disrupting the carboxyl terminus of the mature protein (*mpl1-1*, *mpl1-2* and *mpl1-3*) led to a similar conclusion regarding the role of *MPL1* in *C. arietinum* (Liu *et al.*, 2023). In this study, we show that the absence of both orthologues of *PALM1* in pea confers a bipinnate tendrilled phenotype and thus that the *PsPALM1a* and *PsPALM1b* genes act to restrict lateral morphogenesis in pinnate WT pea leaves.

PALM1, PsPALM1a and PsPALM1b have very similar gene
and protein structures (Notes S2; Fig. S2), and all have AuxRE
cis-elements in their promoters (Fig. S3). The similarity is also
evident in their expression profiles. Like PALM1 (Chen
et al., 2010), PsPALM1a and PsPALM1b are expressed in devel-
oping seeds and shoots but are barely detectable in other tissues,
including roots, stems, flowers, pods and seed coats (Fig. S9).
Detailed molecular studies are needed to understand and com-
pare the function of pea, C. arietinum and M. truncatula ortholo-
gous genes. The replacement of a conserved serine amino acid by
a phenylalanine residue in PsPALM1a (Fig. S2), in a site immedi-
ately adjacent to the zinc finger domain, is worth considering for
possible effects on the binding of the protein to DNA. The
genetic models proposed for Af (Gourlay et al., 2000), the

physiological studies by DeMason *et al.* (2013) and the investiga-
tions of *PALM1* by Peng *et al.* (2017) predict conserved func-
tions. Current models link auxin signalling to compound leaf
patterning and highlight a central role for *PALM1* or *Af* in this
process. Now that *PsPALM1a* and *PsPALM1b* have been identi-
fied and the orthologue of *MtARF3* in pea is known through the
current genome assembly (*Psat1g202240*; Kreplak *et al.*, 2019),
the module *PsPALM1a/PsPALM1b/Uni/Psat1g202240* can be
dissected at the molecular level. The effect of *Af*, that is
*PsPALM1a* and *PsPALM1b*, on stipule size, as suggested by Kiel-
pinski & Blixt (1982), and any possible pleiotropic effects of
these genes, such as the role of *PALM1* as an inhibitor of rust
germ tube differentiation (Uppalapati *et al.*, 2012), should
also be investigated.

Furthermore, Haplotypes 2, 7 and 8 (Fig. 5) lack the three PsNAOD genes. Molesini *et al.* (2015) have shown that NAOD contributes to the regulation of polyamine and ornithine levels in A. thaliana and that a reduced NAOD expression leads to alterations at the reproductive level, causing early flowering and impaired fruit set. It would be interesting to investigate whether reduced fitness is similarly observed in pea mutants carrying a deletion of the PsNAOD genes, and whether this has affected any attempts to use the mutants for breeding. Interestingly, an experiment using *af* near-isogenic lines from cultivar ‘Terese’ (Haplo-type 3, lacking PsNAOD1) showed that carrying *af* was associated with a lower seed weight (Burstin *et al.*, 2007).

In conclusion, this study demonstrates the causal relationship
between the *af* mutation and a large deletion affecting two
*PALM1* genes in pea, and contributes to the understanding of the
control of compound leaf morphogenesis in legumes. However,
the evolutionary dynamics in the *af* region in the Fabeae tribe,
which led to a tandem duplication of the *PALM1* genes in pea
and *L. culinaris*, remain to be elucidated. The mechanisms under-
lying the *af* phenotype and the respective roles of the two genes,
the genomic region and the genetic background need to be
further investigated. For example, the lower nodes of the fast neu-
tron mutant FN2070, which has a deletion of *PsPALM1a*, have a
leaf phenotype similar to *palm1* in *M. truncatula* (Chen
et al., 2010). Additional proximal leaflets produced by the
FN2070 mutant suggest that the proximal primordia remained
undifferentiated for longer than in WT. This is likely due to the
lack of repression of *Uni* by *PsPALM1a* in the mutant, analogous
to the *PALM1/MPL1-SGL1/CaLFY* developmental module
described in related legumes (He et al., 2020; Liu et al., 2023).
On the other hand, deletion of *PsPALM1b* in gamma-ray-
induced Bulgarian mutants recapitulates the bipinnate, tendrilled
af phenotype. Each proximal leaflet is transformed into a tendril-
bearing rachis. This suggests that *PsPALM1b* has two roles; first,
repression of *Uni* and second, repression of *Tl* (Hofer
et al., 2009). In the absence of both repressor roles, proximal pri-
mordia remain undifferentiated, *Tl* is activated and primordia
eventually acquire tendril identity. At present, we cannot explain
why the conventionally leaved TiLLING mutant 2835, which
carries a mutation in *PsPALM1b* causing a premature stop codon,
does not have the same phenotype as the Bulgarian mutants.
Nevertheless, the findings pave the way for engineering new pea
ideotypes where useful traits could be conferred by new *af* alleles
or by information-based management of breeding programmes.
Translational knowledge can be used to develop improved culti-
vars in related crops such as *L. culinaris* and *Vicia sativa*, where
lodging is a major production problem.

Note added in proof: while editing the proofs of this research
article, the authors became aware of a short communication by
Yuan *et al.* (2024), which cites the bioRxiv Preprint of this manu-
script published 19 July 2023, and identifies the association
between *PsPALM1a*, *PsPALM1b* and *afila*. In their study of
Mawan1, a pea accession with an afila phenotype, Yuan *et al.*
(2024) agree with the conclusions of this work and provide com-
plementary expression data relevant to understanding the role of
*PsPALM1a* and *PsPALM1b* in leaf development.

Acknowledgements

The authors are grateful to Julie Fievet and Philippe Brabant for
their help with the statistical analyses of the VIGS experiments.
They would like to acknowledge Karen Boucherot and Emilie
Vieille for their contribution to the molecular analyses (gene
expression quantification and genotyping), Sophie Blanchet for
the construction of the BPMV-*PsPALM1* VIGS vector and
Hélène Berges, Arnaud Bellec and Nathalie Rodde from the
French Plant Genomic Resources Centre for providing the BAC
clones and evaluating their insert size. The authors would also
like to thank the Germplasm Resources Unit at the John Innes
Centre, in particular Mr. Mike Ambrose, the Nordic Genetic
Resource Centre (NordGen) and the INRAE CRB PROTEA for
providing seeds. The authors would specially like to thank
Andrew Davis and Phil Robinson for photography and to the late
Rosemary Harvey for translating Kujala's (1953) publication into
English. The authors would also like to thank Marianne
Chabert-Martinello, Céline Rond and Anthony Klein for valu-
able passport and phenotypic data on INRAE CRB PROTEA
accessions, and Sarah Wilmot for retrieving archival records of
the *afila* breeding programme at the John Innes Institute.
Funding: French National Research Agency grant ANR-09-
GENM-026, GENOPEA; French National Research Agency
grant ANR-11-BTBR-0002, PeaMUST; UKRI (BBSRC) for the
John Innes Centre Institute Strategic Programme grant
BBS/E/J/000PR9799; European Union FP6 Framework Pro-
gram FOOD-CT-2004-506223; John Innes Centre Strategic
Fellowship (NE); French National Research Agency grant ANR-
17-EUR-0007, EUR Saclay Plant Sciences-SPS.

Competing interests

None declared.

Author contributions

NT and NE were involved in the conception and drafting of the
manuscript. NT, JMIH, GA, FJ, LT, PP, MD, CLS, JK, SP and
VG were involved in the experimental design and execution. NT,
NE and JB were involved in the supervision. NT, JMIH, GA and

NE were involved in data analysis. NE and JB were involved in
the acquisition of funding. NT, JMIH, GA, SP, VG, NE and JB
were involved in reviewing and editing the manuscript.

ORCID

Grégoire Aubert https://orcid.org/0000-0002-1867-5730

Judith Burstin https://orcid.org/0000-0002-4125-3499

Marion Dalmais https://orcid.org/0000-0002-1673-3095

Noel Ellis https://orcid.org/0000-0001-6726-9950

Valérie Geffroy https://orcid.org/0000-0003-2210-3636

Julie M. I. Hofer https://orcid.org/0000-0002-0158-7574

Jonathan Kreplak https://orcid.org/0000-0001-5563-1932

Christine Le Signor https://orcid.org/0000-0002-3204-3537

Stéphanie Pflieger https://orcid.org/0009-0006-0687-4106

Pirita Paajanen https://orcid.org/0000-0002-0561-3717

Nadim Tayeh https://orcid.org/0000-0003-0098-8322

Data availability

The data that support the findings of this study are available in
the Supporting Information of this article.

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## Supporting Information

Additional Supporting Information may be found online in the Supporting Information section at the end of the article.

**Fig. S1** Phylogenetic trees constructed using the nucleotide sequences of the genes at the *afila* locus.

**Fig. S2** Alignment of predicted amino acid sequences of legume and non-legume genes orthologous to *Medicago truncatula* *PALMATE-LIKE PENTAFOLIATA1*.

**Fig. S3** Distribution of auxin response elements in the 5' upstream regions of *Pisum sativum PsPALM1a* and *PsPALM1b* and *Medicago truncatula PALM1*.

**Fig. S4** Molecular evidence for presence/absence polymorphism of *PsPALM1a*, *PsPALM1b* and *PsNAOD1* in conventional and semi-leafless pea (*Pisum sativum* L.) accessions.

**Fig. S5** Gamma-ray-induced *Pisum sativum afila (af)* mutant lines originating from Bulgaria, carrying a deletion of *PsPALM1b*, shown in comparison with their corresponding wild-type progenitor cultivars.

**Fig. S6** Leaf phenotype of *Pisum sativum* fast neutron-induced mutant FN2070 and progenitor wild-type accession JI2822.

**Fig. S7** Photographs of adult *Pisum sativum* leaves from cultivar Cameor and related ethylmethane sulfonate-induced mutant lines.

**Fig. S8** Attempted *PsPALM1a/b* virus-induced gene silencing in *Pisum sativum* cultivar Champagne, which has a conventional leaf type.

**Fig. S9** Expression of *Unifoliata* (*Uni*), *PsPALM1a* and *PsPALM1b* in different *Pisum sativum* tissues under normal growth conditions detected by quantitative real-time reverse-transcription polymerase chain reactions.

**Notes S1.** Report on the parallel approach used to identify candidate genes for the afila phenotype in pea (*Pisum sativum* L.).

**Notes S2.** Description of the protein and the promoter sequences of *PsPALM1a* and *PsPALM1b*.

**Table S1** *Pisum sativum* recombinant inbred line populations derived by single seed descent from crosses between parents contrasting for their leaf types.

**Table S2** *Pisum sativum* accessions examined for their haplotypes at the *afila* locus.

**Table S3** Primers used to test for presence/absence variation at the *afila* locus, to perform virus-induced gene silencing and to quantify gene expression.

**Table S4** List of gene-based SNP markers tightly linked to *afila* and predicted genes in collinear regions in *Medicago truncatula*, *Glycine max* and *Arabidopsis thaliana*.

**Table S5** Positions of putative auxin response elements in the 2.5-kb 5' upstream sequences of *Pisum sativum PsPALM1a* and *PsPALM1b* and *Medicago truncatula PALM1*.

**Table S6** Ethylmethane sulfonate-induced mutants for *PsPALM1a* and *PsPALM1b* in *Pisum sativum* cultivar Cameor identified using a TiLLING approach.

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