Genetic architecture and evolution of the S locus supergene in Primula vulgaris.

Darwin's studies on heterostyly in Primula described two floral morphs, pin and thrum, with reciprocal anther and stigma heights that promote insect-mediated cross-pollination. This key innovation evolved independently in several angiosperm families. Subsequent studies on heterostyly in Primula contributed to the foundation of modern genetic theory and the neo-Darwinian synthesis. The established genetic model for Primula heterostyly involves a diallelic S locus comprising several genes, with rare recombination events that result in self-fertile homostyle flowers with anthers and stigma at the same height. Here we reveal the S locus supergene as a tightly linked cluster of thrum-specific genes that are absent in pins. We show that thrums are hemizygous not heterozygous for the S locus, which suggests that homostyles do not arise by recombination between S locus haplotypes as previously proposed. Duplication of a floral homeotic gene 51.7 million years (Myr) ago, followed by its neofunctionalization, created the current S locus assemblage which led to floral heteromorphy in Primula. Our findings provide new insights into the structure, function and evolution of this archetypal supergene.

Integration of genetic and physical maps of the Primula vulgaris S locus and localization by chromosome in situ hybridization.

Heteromorphic flower development in Primula is controlled by the S locus. The S locus genes, which control anther position, pistil length and pollen size in pin and thrum flowers, have not yet been characterized. We have integrated S-linked genes, marker sequences and mutant phenotypes to create a map of the P. vulgaris S locus region that will facilitate the identification of key S locus genes. We have generated, sequenced and annotated BAC sequences spanning the S locus, and identified its chromosomal location. We have employed a combination of classical genetics and three-point crosses with molecular genetic analysis of recombinants to generate the map. We have characterized this region by Illumina sequencing and bioinformatic analysis, together with chromosome in situ hybridization. We present an integrated genetic and physical map across the P. vulgaris S locus flanked by phenotypic and DNA sequence markers. BAC contigs encompass a 1.5-Mb genomic region with 1 Mb of sequence containing 82 S-linked genes anchored to overlapping BACs. The S locus is located close to the centromere of the largest metacentric chromosome pair. These data will facilitate the identification of the genes that orchestrate heterostyly in Primula and enable evolutionary analyses of the S locus.

Oakleaf: an S locus-linked mutation of Primula vulgaris that affects leaf and flower development.

In Primula vulgaris outcrossing is promoted through reciprocal herkogamy with insect-mediated cross-pollination between pin and thrum form flowers. Development of heteromorphic flowers is coordinated by genes at the S locus. To underpin construction of a genetic map facilitating isolation of these S locus genes, we have characterised Oakleaf, a novel S locus-linked mutant phenotype. We combine phenotypic observation of flower and leaf development, with classical genetic analysis and next-generation sequencing to address the molecular basis of Oakleaf. Oakleaf is a dominant mutation that affects both leaf and flower development; plants produce distinctive lobed leaves, with occasional ectopic meristems on the veins. This phenotype is reminiscent of overexpression of Class I KNOX-homeodomain transcription factors. We describe the structure and expression of all eight P. vulgaris PvKNOX genes in both wild-type and Oakleaf plants, and present comparative transcriptome analysis of leaves and flowers from Oakleaf and wild-type plants. Oakleaf provides a new phenotypic marker for genetic analysis of the Primula S locus. We show that none of the Class I PvKNOX genes are strongly upregulated in Oakleaf leaves and flowers, and identify cohorts of 507 upregulated and 314 downregulated genes in the Oakleaf mutant.

Clinical Relevance of Foam Rolling on Hip Extension Angle in a Functional Lunge Position.

The objective of this study was to examine the duration of effectiveness of foam rolling on hip extension angles in a dynamic lunge position. Thirty-one subjects were assigned to control (n = 15) or intervention (n = 16) group. All the subjects followed the same testing timeline; 3 testing sessions, with 2 lunges in each session. The intervention group performed foam rolling between each lunge in sessions 1 and 2, and 5 times in 7 days between sessions 1 and 2. They did not foam roll during the week between sessions 2 and 3 or in session 3. The control group did not foam roll at all. Hip extension angles were recorded using Dartfish software and subjects filled out a global perceived effect scale rating the feeling of the second lunge and the intervention for each session. A 6 × 2 mixed-effects analysis of variance was run with post hoc t-tests revealing significant gains in hip extension within session 2 for the intervention group (p ≤ 0.05). Hip extension angles returned to baseline values after subject's ceased foam rolling for 1 week. Global perceived effect scores were higher for the intervention group and 29 of 32 words of descriptive feedback included positive words regarding foam rolling. We concluded that consistent foam rolling produced increases in hip extension during a dynamic lunge, but these effects are not seen within the first exposure. Foam rolling received positive reception and perceived improvements in hip extension. The findings indicate that repeated foam rolling is beneficial, both objectively and subjectively, for increasing range of motion immediately preceding a dynamic activity.

Floral heteromorphy in Primula vulgaris: progress towards isolation and characterization of the S locus.

BACKGROUND: The common primrose, Primula vulgaris, along with many other species of the Primulaceae, exhibits floral heteromorphy in which different individuals develop one of two possible forms of flower, known as pin and thrum. Both flower types are hermaphrodite and exhibit reciprocal positions of male and female reproductive structures, which together with a sporophytic incompatibility system, prevent self-pollination and promote out-crossing. The development of the two different forms of flower is controlled by a co-adapted linkage group of genes known as the S locus. SCOPE: Here progress towards identification and characterization of these genes is described to provide a molecular genetic explanation of the different floral characteristics that define heterostyly in Primula as observed and described by Charles Darwin. Previous work to identify and characterize developmental mutations linked to the P. vulgaris S locus, together with the isolation of S locus-linked genes and polymorphic DNA sequences markers, is summarized. The development of tools are described which will facilitate isolation and characterization of the S locus and its environs, including the creation of two expressed sequence tag libraries from pin and thrum flowers, as well as the construction and screening of two bacterial artificial chromosome (BAC) libraries containing thrum genomic DNA. Screening of these libraries with four S locus-linked sequences has enabled us to assemble four BAC contigs representing over 40 individual overlapping BAC clones which represent over 2·2 Mb of S locus-linked genomic sequence. PCR-based approaches for identification of the allelic origin of these BACs are described as well as identification of an additional 14 S locus-linked genes within BAC-end sequences. CONCLUSIONS: On-going work to assemble the four S locus-linked contigs into one contiguous sequence spanning the S locus is outlined in preparation for sequence analysis and characterization of the genes located within this region.

Hose in Hose, an S locus-linked mutant of Primula vulgaris, is caused by an unstable mutation at the Globosa locus.

Hose in Hose mutants of primrose and cowslip have been cultivated since the early 17th century and show dominant homeotic conversion of sepals to petals. The phenotype shows variable penetrance and expressivity and is linked to the S locus, which controls floral heteromorphy in Primula species. Here we demonstrate that the homeotic conversion of sepals to petals in Hose in Hose is associated with up-regulation of both Primula B-function MADS box genes PvDef and PvGlo in the first floral whorl. We have defined a restriction fragment length polymorphism associated with PvGlo that cosegregates with the Hose in Hose phenotype and have also identified and characterized a retrotransposon insertion in the PvGlo promoter which is associated with the up-regulated expression of PvGlo. Excision of this retrotransposon, associated with epigenetic changes at the locus, causes reversion toward normal calyces and restores wild-type flower development. These data define the molecular basis of the Hose in Hose mutation and provide an explanation for its long-documented phenotypic instability.

The S locus-linked Primula homeotic mutant sepaloid shows characteristics of a B-function mutant but does not result from mutation in a B-function gene.

Floral homeotic and flower development mutants of Primula, including double, Hose in Hose, Jack in the Green and Split Perianth, have been cultivated since the late 1500s as ornamental plants but until recently have attracted limited scientific attention. Here we describe the characterization of a new mutant phenotype, sepaloid, that produces flowers comprising only sepals and carpels. The sepaloid mutation is recessive, and is linked to the S locus that controls floral heteromorphy. The phenotype shows developmental variability, with flowers containing three whorls of sepals surrounding fertile carpels, two whorls of sepals with a diminished third whorl of sepals surrounding a fourth whorl of carpels, or three whorls of sepals surrounding abnormal carpels. In some respects, these phenotypes resemble the Arabidopsis and Antirrhinum homeotic B-function mutants apetala3/deficiens (ap3/def) and pistillata/globosa (pi/glo). We have isolated the Primula vulgaris B-function genes PvDEFICIENS (PvDEF) and PvGLOBOSA (PvGLO), expression of both of which is affected in the sepaloid mutant. PvGLO, like sepaloid, is linked to the S locus, whereas PvDEF is not. However, our analyses reveal that sepaloid and PvGLO represent different genes. We conclude that SEPALOID is an S-linked independent regulator of floral organ identity genes including PvDEF and PvGLO.

Identification and characterization of pin and thrum alleles of two genes that co-segregate with the Primula S locus.

The study of heteromorphy in Primula over the past 140 years has established the reproductive significance of this breeding system. Plants produce either thrum or pin flowers that demonstrate reciprocal herkogamy. Thrums have short styles and produce large pollen from anthers at the mouth of the flower; pins have long styles and produce small pollen from anthers located within the corolla tube. The control of heteromorphy is orchestrated by the S locus with dominant (S) and recessive (s) alleles that comprise a co-adapted linkage group of genes. Thrum plants are heterozygous (Ss) and pin plants are homozygous (ss). Reciprocal crosses between the two forms are required for fertilization; within-morph crosses are impeded by a sporophytic self-incompatibility system. Rare recombination events within the S locus produce self-fertile homostyles. As a first step towards identifying genes located at the S locus, we used fluorescent differential display to screen for differential gene expression in pin and thrum flowers. Rather than only detecting differentially regulated genes, we identified two S locus linked genes by virtue of allelic variation between pin and thrum transcripts. Analysis of pin and thrum plants together with homostyle recombinant reveals that one gene flanks the locus, whereas the other shows complete linkage. One gene is related to Arabidopsis flower-timing genes Col9 and Col10; the other encodes a small predicted membrane protein of unknown function. Notwithstanding the diallelic behaviour of the Primula S locus, analysis of pin and thrum plants reveal three alleles for each gene: two pin and one thrum.

Analysis of late stage flower development in Primula vulgaris reveals novel differences in cell morphology and temporal aspects of floral heteromorphy.

Heterostyly in Primula is characterized by the development of long-styled pin and short-styled thrum flowers, with anthers midway down the corolla tube in pin flowers, and at its mouth in thrum flowers. Other differences include pollen size and stigmatic papillae length. Several linked genes at the S locus control these differences. In this study we have analyzed pin and thrum flowers through the temporal development of heteromorphy.These studies indicate that the S locus linked genes that orchestrate heteromorphic flower development act in coordination, but with different temporal and spatial dynamics. Style length is differentiated by longer style cells in pin than thrum. However, our studies on cell shape and size within the corolla tube show that a different mechanism mediates the dissimilar elevation of anthers between pin and thrum types. These studies have also revealed that upper corolla tube cells in thrum flowers are wider than those in pin flowers. This results in a larger corolla tube mouth in thrum flowers and represents a new and previously undocumented heteromorphic variation between pin and thrum flowers.

A comparison of early floral ontogeny in wild-type and floral homeotic mutant phenotypes of Primula.

Primula flowers are heteromorphic with individual plants producing either pin-form or thrum-form flowers. We have used scanning electron microscopy to observe early development of wild-type flowers of primrose (Primula vulgaris), cowslip (P. veris), and the polyanthus hybrid (P. x tommasinii x P. vulgaris). Floral ontogeny in Primula is different from that observed in the well-studied models Antirrhinum majus and Arabidopsis thaliana and our studies reveal morphological landmark events that define the sequence of early floral development in Primula into specific stages. Pin-form and thrum-form flowers are indistinguishable during early development with differentiation of the two floral morphs occurring beyond the differentiation of floral organs. Early ontogeny of flowers with homeotic mutant phenotypes was also studied to determine the timing of developmental reprogramming in these mutants. Phenotypes studied included Hose in Hose and Jack in the Green that develop petaloid sepals and leafy sepals, respectively, and Jackanapes plants that carry both these dominant mutations. Recessive double and semi- double flowers that produce additional whorls of petals and/or stamens in place of carpels were also studied. We describe a previously undocumented recessive Primula mutant phenotype, sepaloid, that produces sepals in place of petals and stamens, and a new non-homeotic, dominant mutant phenotype Split Perianth, in which sepals and petals fail to fuse to form the typical calyx and corolla structures. The molecular basis of these mutant phenotypes in relation to the ABC model is discussed.