S-Locus Genotyping in Japanese Plum by High Throughput Sequencing Using a Synthetic S-Loci Reference Sequence.

Self-incompatibility in Prunus species is governed by a single locus consisting of two highly multi-allelic and tightly linked genes, one coding for an F-box protein-i.e., SFB in Prunus- controlling the pollen specificity and one coding for an S-RNase gene controlling the pistil specificity. Genotyping the allelic combination in a fruit tree species is an essential procedure both for cross-based breeding and for establishing pollination requirements. Gel-based PCR techniques using primer pairs designed from conserved regions and spanning polymorphic intronic regions are traditionally used for this task. However, with the great advance of massive sequencing techniques and the lowering of sequencing costs, new genotyping-by-sequencing procedures are emerging. The alignment of resequenced individuals to reference genomes, commonly used for polymorphism detection, yields little or no coverage in the S-locus region due to high polymorphism between different alleles within the same species, and cannot be used for this purpose. Using the available sequences of Japanese plum S-loci concatenated in a rosary-like structure as synthetic reference sequence, we describe a procedure to accurately genotype resequenced individuals that allowed the analysis of the S-genotype in 88 Japanese plum cultivars, 74 of them are reported for the first time. In addition to unraveling two new S-alleles from published reference genomes, we identified at least two S-alleles in 74 cultivars. According to their S-allele composition, they were assigned to 22 incompatibility groups, including nine new incompatibility groups reported here for the first time (XXVII-XXXV).

Male Meiosis as a Biomarker for Endo- to Ecodormancy Transition in Apricot.

Dormancy is an adaptive strategy in plants to survive under unfavorable climatic conditions during winter. In temperate regions, most fruit trees need exposure to a certain period of low temperatures to overcome endodormancy. After endodormancy release, exposure to warm temperatures is needed to flower (ecodormancy). Chilling and heat requirements are genetically determined and, therefore, are specific for each species and cultivar. The lack of sufficient winter chilling can cause failures in flowering and fruiting, thereby compromising yield. Thus, the knowledge of the chilling and heat requirements is essential to optimize cultivar selection for different edaphoclimatic conditions. However, the lack of phenological or biological markers linked to the dormant and forcing periods makes it difficult to establish the end of endodormancy. This has led to indirect estimates that are usually not valid in different agroclimatic conditions. The increasing number of milder winters caused by climatic change and the continuous release of new cultivars emphasize the necessity of a proper biological marker linked to the endo- to ecodormancy transition for an accurate estimation of the agroclimatic requirements (AR) of each cultivar. In this work, male meiosis is evaluated as a biomarker to determine endodormancy release and to estimate both chilling and heat requirements in apricot. For this purpose, pollen development was characterized histochemically in 20 cultivars over 8 years, and the developmental stages were related to dormancy. Results were compared to three approaches that indirectly estimate the breaking of dormancy: an experimental methodology by evaluating bud growth in shoots collected periodically throughout the winter months and transferred to forcing chambers over 3 years, and two statistical approaches that relate seasonal temperatures and blooming dates in a series of 11-20 years by correlation and partial least square regression. The results disclose that male meiosis is a possible biomarker to determine the end of endodormancy and estimate AR in apricot.

Adaptive reduction of male gamete number in the selfing plant Arabidopsis thaliana.

The number of male gametes is critical for reproductive success and varies between and within species. The evolutionary reduction of the number of pollen grains encompassing the male gametes is widespread in selfing plants. Here, we employ genome-wide association study (GWAS) to identify underlying loci and to assess the molecular signatures of selection on pollen number-associated loci in the predominantly selfing plant Arabidopsis thaliana. Regions of strong association with pollen number are enriched for signatures of selection, indicating polygenic selection. We isolate the gene REDUCED POLLEN NUMBER1 (RDP1) at the locus with the strongest association. We validate its effect using a quantitative complementation test with CRISPR/Cas9-generated null mutants in nonstandard wild accessions. In contrast to pleiotropic null mutants, only pollen numbers are significantly affected by natural allelic variants. These data support theoretical predictions that reduced investment in male gametes is advantageous in predominantly selfing species.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

An Introduction to Male Germline Development.

In this introductory chapter, we describe male germline development in plants taking Arabidopsis thaliana as a reference species. We first describe the transition from sporophytic to germline development, then microsporogenesis including meiosis, followed by male gametophyte development prior to pollination, and finally the progamic phase culminating in double fertilization, which leads to the formation of the embryo and the endosperm. For detailed information on some of these processes or on the molecular underpinning of certain fate transitions, we refer the reader to recent reviews. An important but often neglected aspect of male gametophyte development is the formation of the unique pollen cell wall. In contrast to that of other plant cells, the pollen cell wall is composed of two principal layers, the intine and exine. While the intine, the inner pecto-cellulosic cell wall layer, is biochemically and structurally similar to a "classical" plant cell wall, the exine is a unique composite with sporopollenin as its main component. Biosynthesis of the cell wall is remarkably similar between the spores of mosses and ferns, and pollen of seed plants, although slight differences exist, even between closely related species (reviewed in Wallace et al., AoB Plants 2011:plr027, 2011). In the latter sections of this chapter, we will present a brief overview of cell wall development in Arabidopsis pollen, where this aspect has been intensively studied.

Starch Turnover and Metabolism during Flower and Early Embryo Development.

The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed.

Global warming and sexual plant reproduction.

The sexual reproductive phase in plants might be particularly vulnerable to the effects of global warming. The direct effect of temperature changes on the reproductive process has been documented previously, and recent data from other physiological processes that are affected by rising temperatures seem to reinforce the susceptibility of the reproductive process to a changing climate. But the reproductive phase also provides the plant with an opportunity to adapt to environmental changes. Understanding phenotypic plasticity and gametophyte selection for prevailing temperatures, along with possible epigenetic changes during this process, could provide new insights into plant evolution under a global-warming scenario.