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

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.

Plant Development in the Garden Pea as Revealed by Mutations in the Crd/PsYUC1 Gene.

In common with other plant species, the garden pea (Pisum sativum) produces the auxin indole-3-acetic acid (IAA) from tryptophan via a single intermediate, indole-3-pyruvic acid (IPyA). IPyA is converted to IAA by PsYUC1, also known as Crispoid (Crd). Here, we extend our understanding of the developmental processes affected by the Crd gene by examining the phenotypic effects of crd gene mutations on leaves, flowers, and roots. We show that in pea, Crd/PsYUC1 is important for the initiation and identity of leaflets and tendrils, stamens, and lateral roots. We also report on aspects of auxin deactivation in pea.

Mendel's pea crosses: varieties, traits and statistics.

A controversy arose over Mendel's pea crossing experiments after the statistician R.A. Fisher proposed how these may have been performed and criticised Mendel's interpretation of his data. Here we re-examine Mendel's experiments and investigate Fisher's statistical criticisms of bias. We describe pea varieties available in Mendel's time and show that these could readily provide all the material Mendel needed for his experiments; the characters he chose to follow were clearly described in catalogues at the time. The combination of character states available in these varieties, together with Eichling's report of crosses Mendel performed, suggest that two of his F3 progeny test experiments may have involved the same F2 population, and therefore that these data should not be treated as independent variables in statistical analysis of Mendel's data. A comprehensive re-examination of Mendel's segregation ratios does not support previous suggestions that they differ remarkably from expectation. The χ2 values for his segregation ratios sum to a value close to the expectation and there is no deficiency of extreme segregation ratios. Overall the χ values for Mendel's segregation ratios deviate slightly from the standard normal distribution; this is probably because of the variance associated with phenotypic rather than genotypic ratios and because Mendel excluded some data sets with small numbers of progeny, where he noted the ratios "deviate not insignificantly" from expectation.

Identification of Stipules reduced, a leaf morphology gene in pea (Pisum sativum).

Pea (Pisum sativum) is one of relatively few genetically amenable plant species with compound leaves. Pea leaves have a variety of specialized organs: leaflets, tendrils, pulvini and stipules, which enable the identification of mutations that transform or affect distinct parts of the leaf. Characterization of these mutations offers insights into the development and evolution of novel leaf traits. The previously characterized morphological gene Cochleata, conferring stipule identity, was known to interact with Stipules reduced (St), which conditions stipule size in pea, but the St gene remained unknown. Here we analysed Fast Neutron irradiated pea mutants by restriction site associated DNA sequencing. We identified St as a gene encoding a C2H2 zinc finger transcription factor that is regulated by Cochleata. St regulates both cell division and cell expansion in the stipule. Our approach shows how systematic genome-wide screens can be used successfully for the analysis of traits in species for which whole genome sequences are not available.

Linking Auxin with Photosynthetic Rate via Leaf Venation.

Land plants lose vast quantities of water to the atmosphere during photosynthetic gas exchange. In angiosperms, a complex network of veins irrigates the leaf, and it is widely held that the density and placement of these veins determines maximum leaf hydraulic capacity and thus maximum photosynthetic rate. This theory is largely based on interspecific comparisons and has never been tested using vein mutants to examine the specific impact of leaf vein morphology on plant water relations. Here we characterize mutants at the Crispoid (Crd) locus in pea (Pisum sativum), which have altered auxin homeostasis and activity in developing leaves, as well as reduced leaf vein density and aberrant placement of free-ending veinlets. This altered vein phenotype in crd mutant plants results in a significant reduction in leaf hydraulic conductance and leaf gas exchange. We find Crispoid to be a member of the YUCCA family of auxin biosynthetic genes. Our results link auxin biosynthesis with maximum photosynthetic rate through leaf venation and substantiate the theory that an increase in the density of leaf veins coupled with their efficient placement can drive increases in leaf photosynthetic capacity.

Developmental specialisations in the legume family.

The legume family is astonishingly diverse; inventiveness in the form of novel organs, modified organs and additional meristems, is rife. Evolutionary changes can be inferred from the phylogenetic pattern of this diversity, but a full understanding of the origin of these 'hopeful monsters' of meristematic potential requires clear phylogenetic reconstructions and extensive, species-rich, sequence data. The task is large, but rapid progress is being made in both these areas. Here we review specialisations that have been characterised in a subset of intensively studied papilionoid legume taxa at the vanguard of developmental genetic studies.

The B gene of pea encodes a defective flavonoid 3',5'-hydroxylase, and confers pink flower color.

The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5' position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the enzyme that hydroxylates the 5' position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3'5'H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3'5'H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3'5'H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.

Mendel, 150 years on.

Mendel's paper 'Versuche über Pflanzen-Hybriden' is the best known in a series of studies published in the late 18th and 19th centuries that built our understanding of the mechanism of inheritance. Mendel investigated the segregation of seven gene characters of pea (Pisum sativum), of which four have been identified. Here, we review what is known about the molecular nature of these genes, which encode enzymes (R and Le), a biochemical regulator (I) and a transcription factor (A). The mutations are: a transposon insertion (r), an amino acid insertion (i), a splice variant (a) and a missense mutation (le-1). The nature of the three remaining uncharacterized characters (green versus yellow pods, inflated versus constricted pods, and axial versus terminal flowers) is discussed.

Identification of Mendel's white flower character.

BACKGROUND: The genetic regulation of flower color has been widely studied, notably as a character used by Mendel and his predecessors in the study of inheritance in pea. METHODOLOGY/PRINCIPAL FINDINGS: We used the genome sequence of model legumes, together with their known synteny to the pea genome to identify candidate genes for the A and A2 loci in pea. We then used a combination of genetic mapping, fast neutron mutant analysis, allelic diversity, transcript quantification and transient expression complementation studies to confirm the identity of the candidates. CONCLUSIONS/SIGNIFICANCE: We have identified the pea genes A and A2. A is the factor determining anthocyanin pigmentation in pea that was used by Gregor Mendel 150 years ago in his study of inheritance. The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon, and we have identified a second rare mutant allele. The A2 gene encodes a WD40 protein that is part of an evolutionarily conserved regulatory complex.

A crispa null mutant facilitates identification of a crispa-like pseudogene in pea.

The genomes of several legume species contain two Phantastica-like genes. Previous studies on leaf development have found that Phantastica confers leaf blade adaxial identity in plant species with simple leaves and leaflet adaxial identity in pea (Pisum sativum L.), a legume with compound leaves. Previous characterisation of the phantastica mutant of pea, crispa, showed it had radialised leaflets, but stipules were not radialised. This suggested either that mutation of a second redundant gene was required for radialisation of stipules, or, that a null mutation was required. Previously characterised crispa mutants may not have exhibited radialised stipules because they were weak alleles. In this work we show that pea has a second Phantastica-like gene, which lies on a different chromosome to Crispa. The second gene was found to be a pseudogene in several genotypes of pea, therefore it would not have a role in conferring stipule adaxial identity. A new deletion mutant, crispa-4 was identified. The mutant has radialised stipules and leaflets, showing that Crispa confers adaxial identity on both these organs in pea. The nucleotide sequence data reported here are in the EMBL and GenBank Nucleotide Databases under the accession numbers DQ486060 (JI 2822), DQ486061 (JI 15), DQ486062 (JI 281) and DQ486063 (JI 399).

The mutant crispa reveals multiple roles for PHANTASTICA in pea compound leaf development.

Pinnate compound leaves have laminae called leaflets distributed at intervals along an axis, the rachis, whereas simple leaves have a single lamina. In simple- and compound-leaved species, the PHANTASTICA (PHAN) gene is required for lamina formation. Antirrhinum majus mutants lacking a functional gene develop abaxialized, bladeless adult leaves. Transgenic downregulation of PHAN in the compound tomato (Solanum lycopersicum) leaf results in an abaxialized rachis without leaflets. The extent of PHAN gene expression was found to be correlated with leaf morphology in diverse compound-leaved species; pinnate leaves had a complete adaxial domain of PHAN gene expression, and peltate leaves had a diminished domain. These previous studies predict the form of a compound-leaved phan mutant to be either peltate or an abaxialized rachis. Here, we characterize crispa, a phan mutant in pea (Pisum sativum), and find that the compound leaf remains pinnate, with individual leaflets abaxialized, rather than the whole leaf. The mutant develops ectopic stipules on the petiole-rachis axis, which are associated with ectopic class 1 KNOTTED1-like homeobox (KNOX) gene expression, showing that the interaction between CRISPA and the KNOX gene PISUM SATIVUM KNOTTED2 specifies stipule boundaries. KNOX and CRISPA gene expression patterns indicate that the mechanism of pea leaf initiation is more like Arabidopsis thaliana than tomato.

PROLIFERATING INFLORESCENCE MERISTEM, a MADS-box gene that regulates floral meristem identity in pea.

SQUAMOSA and APETALA1 are floral meristem identity genes from snapdragon (Antirrhinum majus) and Arabidopsis, respectively. Here, we characterize the floral meristem identity mutation proliferating inflorescence meristem (pim) from pea (Pisum sativum) and show that it corresponds to a defect in the PEAM4 gene, a homolog of SQUAMOSA and APETALA1. The PEAM4 coding region was deleted in the pim-1 allele, and this deletion cosegregated with the pim-1 mutant phenotype. The pim-2 allele carried a nucleotide substitution at a predicted 5' splice site that resulted in mis-splicing of pim-2 mRNA. PCR products corresponding to unspliced and exon-skipped mRNA species were observed. The pim-1 and pim-2 mutations delayed floral meristem specification and altered floral morphology significantly but had no observable effect on vegetative development. These floral-specific mutant phenotypes and the restriction of PIM gene expression to flowers contrast with other known floral meristem genes in pea that additionally affect vegetative development. The identification of PIM provides an opportunity to compare pathways to flowering in species with different inflorescence architectures.