Genetic architecture underlying variation in floral meristem termination in Aquilegia

Abstract

Floral organs are produced by floral meristems (FMs), which harbor stem cells in their centers. Since each flower only has a finite number of organs, the stem cell activity of an FM will always terminate at a specific time point, a process termed floral meristem termination (FMT). Variation in the timing of FMT can give rise to floral morphological diversity, but how this process is fine-tuned at a developmental and evolutionary level is poorly understood. Flowers from the genus Aquilegia share identical floral organ arrangement except for stamen whorl number (SWN), making Aquilegia a well-suited system for investigation of this process: differences in SWN between species represent differences in the timing of FMT. By crossing A. canadensis and A. brevistyla, quantitative trait locus (QTL) mapping has revealed a complex genetic architecture with seven QTL. We explored potential candidate genes under each QTL and characterized novel expression patterns of select loci of interest using in situ hybridization. To our knowledge, this is the first attempt to dissect the genetic basis of how natural variation in the timing of FMT is regulated, and our results provide insight into how floral morphological diversity can be generated at the meristematic level.

Keywords: Aquilegia; QTL; floral meristem termination; stamen whorl.

Fig. 1.

FMT is an important and fine-tuned developmental process that occurs in all flowers. Upper panel: diagram of floral organ initiation and FMT during flower development. Organs of the same whorl share the same colors. Lower panel: example of four flowers with different whorl numbers. From left to right: Crocus vernus ‘Pickwick’, Pyrus communis, Helleborus orientalis, and Portulaca umbraticola. Numbers in parentheses indicate the number of whorls (or ranks for H. orientalis) of floral organs in each flower. Photos of C. vernus, P. communis, and P. umbraticola were taken by Ya Min, and the photo of H. orientalis was taken by Evangeline S. Ballerini.

Fig. 2.

Phenotyping SWN in the parental and F2 populations. (A) Photos of flowers and floral diagrams of A. brevistyla and A. canadensis. (B) Examples of three F2 flower buds with different SWN. (C) Flowers that were sampled per inflorescence. (D) Developmental stages for which SWN was counted. (E) Histogram and summary statistics of SWN distribution in parental species and the F2s. In (A) and (B), stamen whorls positioned above the sepals are colored in blue while stamen whorls positioned above the petals are colored in green. Scale bar in (B)=100 µm. se, sepals; pe, petals: st, stamens; sd, staminodes; car, carpels.

Fig. 3.

FM width measurements of the parental species during the early developmental stages. (A) Examples of FM morphologies in A. brevistyla and A. canadensis at early developmental stages. Numbers in parentheses indicate the number of newly initiated stamen whorls in each FM. Scale bars=100 µm. (B) Comparison of FM width of different developmental stages between A. brevistyla and A. canadensis. Each data point represents a measurement of an FM width from a section. Three to six continuous sections were measured for each FM, and at least three FMs were measured for each developmental stage of each species. Comparison of FM widths of each stage between the parental species was done using Tukey’s HSD. ns, not significant; ***P-value <0.001; ****P-value <0.0001; se, sepal; B, A. brevistyla; C, A. canadensis.

Fig 4.

Genetic architecture and candidate genes. (A) LOD scores across seven chromosomes. Red line: α=0.05 genome-wide significance cut-off based on 1000 permutations. (B) Locations of QTL interval (pink regions on the chromosomes and magenta vertical bars), candidate genes, and genetic markers. All the genetic markers were named in numeric forms (e.g. 1.67 and 2.25) and only markers with the highest LOD scores under each QTL are shown.

Fig 5.

In situ hybridization of AqWUS and candidate genes. (A–E) Expression patterns of AqWUS (A–C) and its sense probe (E). (A) An FM that has not produced any organs. (B) An FM that is in the process of initiating either a petal primordium or the outermost stamen primordium (and thus indicated by an arrow with a question mark). (C) An FM that has at least eight whorls of stamens initiated; AqWUS expression was also detected in an axillary meristem (arrowhead). (D) An FM that has at least 11 whorls of stamens produced. (E) Sense probe of AqWUS. (F–L) Expression patterns of AqZPR3a (F–K) and its negative control probe (L). (F) An FM that has not produced any organs subtended by a bract (br). (G) An FM that has just started to produce sepal primordia (asterisk). (H, Hʹ) Serial sections through the same FM that has only produced sepal primordia (asterisks). (I) An FM that has produced 1–2 whorls of stamens. (J, Jʹ, Jʹʹ) Serial sections through the same floral bud that has initiated all floral organs. (K) A young floral bud in which all floral organs are differentiating. (L) Sense probe of AqZPR3a. (M–P) Expression of AqROXYa (M–Oʹ) and its negative control (P). (M) An FM that has not produced any organs. (N) An FM that is in the process of initiating petal or utermost stamen primordia. (O, Oʹ) Serial sections through the same FM, with brackets indicating newly emerging primordia. (P) Sense probe of AqROXYa. (Q–U) Expression of AqATH1 (Q–T) and its negative control (U). (Q) An FM that has not produced any organs. (R) A young FM that is in the process of initiating petal and stamen primordia. (S) An FM initiating stamen primordia and an associated axillary meristem (arrowhead). (T) A floral bud with carpel primordia just initiated and an axillary meristem (arrowhead). (U) Sense probe of AqATH1. All scale bars=100 µm.