Distinguishing 2N gamete nuclei from doublets in pollen using flow cytometry and pulse analysis

Abstract

The value of flow cytometry for quantifying unreduced (2N) pollen production in plants is well recognized; however, the approach has been limited by technical obstacles to obtaining high quality nuclei fluorescence histograms and the difficulty in distinguishing 2N nuclei from 1N doublets. Here, we use mathematical arguments and observations of fluorescence properties of angiosperm pollen nuclei to generate guidelines for applying pulse analysis to correct for doublets in pollen nuclei data. We show that the theoretical requirements for applying pulse analysis for doublet correction are met when nucleus fluorescence height and/or width measures in the unreduced gamete (2C DNA content) region exhibit bimodality (reflecting singlets and doublets) in combination with unimodal distributions of the same parameters in the reduced gamete (1C) region. These conditions are regularly met in the family Brassicaceae but not in the Asteraceae and Poaceae. We further show that when these requirements are met, pulse analysis estimates of doublet proportions are well correlated with estimates obtained with microscopy. We propose guidelines for doublet correction when estimating frequencies of unreduced male gametes. V C 2015 International Society for Advancement of Cytometry Key terms unreduced gametes; flow cytometry; pulse analysis; doublet discrimination; pollen POLYPLOIDY has played an important role in angiosperm evolution (1,2) and is believed to arise through the union of unreduced gametes (3-7). Despite the important role unreduced gamete production may play in plant evolution, there is much that we still do not know about it, including its frequency and distribution among individuals in natural populations and the factors that affect its occurrence (5,7,8). The limits on our knowledge are due in part to the time-consuming nature of measuring unreduced gamete production, which has traditionally involved microscopic examination of individual gametes or gametophytes or crossing experiments (6,7). More recently, high throughput methods have been developed that promise greater sampling intensity, at least for male gametes, including pollen volumetric measures (9) and flow cytometry.Since the first flow cytometric measures of DNA content of pollen nuclei, researchers have recognized the method could be used to estimate unreduced male gamete production (10,11). In fact, one of the main uses of pollen flow cytometry has been to qualitatively identify individuals with high unreduced gamete production (12). Attempts to quantify unreduced gamete production in pollen more precisely and to test statistically for small differences among individuals are less common (but see Refs. 10 and 13). Studies of this kind have been limited by the difficulties in obtaining good quality histograms with nuclei from large numbers of pollen grains, but recent methodological improvements have removed some of these

Genetic and environmental determinants of unreduced gamete production in Brassica napus, Sinapis arvensis and their hybrids

“…In this study, combined correction for doublets and somatic tissue reduced overall unreduced gamete estimates (from 0.84 to 0.59%), but did not qualitatively affect the results and conclusions with respect to the effects of our treatments. This result is encouraging if it proves to be the rule in future studies, as these kinds of corrections may not be possible in all cases (for example, doublet correction in species with elongate nuclei, Kron and Husband, 2015; somatic correction in species without somatic endopolyploidy).…”

“…As we have demonstrated elsewhere (Kron and Husband, 2015), aggregate correction is important when estimating unreduced gametes with flow cytometry. Although we have stated previously Husband, 2012, 2015) that somatic tissue contamination should not be a problem if clean pollen is collected, completely avoiding such contamination may be difficult, especially in species with small flowers where whole flower collection is the most practical approach.…”

“…The use of FCM to estimate unreduced gamete production is not always feasible, because high-quality histograms may be difficult to obtain in some species (Kron and Husband, 2012) and because doublet correction may not be practical in species with elongate nuclei (Kron and Husband, 2015). However, using FCM in a system for which these limits either do not apply or can be corrected for, we were able to rapidly determine unreduced male gamete production from a large number of plants, and we were able to estimate frequencies for relatively rare events that may have been missed with methods employing smaller sample sizes.…”

“…The fact that we had no 0% estimates is an interesting observation about unreduced gamete production in general, and speaks to the sensitivity of the method in detecting low frequencies of 2C events. Some caution should be used in interpreting very low (near-zero) values, because the presence of low levels of background noise might inflate such estimates to an extent that can be ignored with higher unreduced gamete estimates (Kron and Husband, 2015). In addition Pan et al, 2004 argue that some 2N nuclei in mature pollen could be the product of post-meiotic, mitotic failures.…”

“…Second, B. napus and other Brassica species have been previously used in studies that identify rates of unreduced gametes using pollen mother cells analysis (Mason et al, 2011), providing a frame of reference for our results. Third, species in the Brassicaceae have pollen nuclei morphology conducive to the correction for aggregates when analyzing flow cytometric data, a crucial step when quantifying unreduced gametes cytometrically (Kron and Husband, 2015).…”