Regular Article
Estimation of the Genome Sizes of Males and Females in the Marine Green Alga Monostroma angicava Using Flow Cytometry
2020 年 85 巻 2 号 p. 169-175
詳細
2020 年 85 巻 2 号 p. 169-175
Genome size provides important information in ecology and evolution as well as genomics. Genome size may be different between the sexes within a species. However, little information on the genome size of both sexes is available, particularly in ulvophycean marine green algae, because few methods of genome size estimation are suitable for these algae. We developed a method to examine the genome sizes of males and females in the dioicous ulvophycean marine green alga Monostroma angicava. We examined three methods to isolate haploid nuclei: 1) chopping of a haploid gametophyte; 2) homogenization of protoplasts made from haploid gametophyte cells; and 3) homogenization of gametes and found homogenization of gametes to be the most suitable method for isolation of nuclei in M. angicava. Isolated nuclei were stained with propidium iodide. We measured the fluorescence intensity of nuclei using flow cytometry and successfully estimated the genome sizes of males and females as 178.8 Mbp and 185.4 Mbp, respectively, using Arabidopsis thaliana and Brassica rapa as standard plants with an internal standard method. The genome size of males was slightly smaller than that of females. This may be due to the difference in the length of sex-specific genome regions.
Genome size is basic and important information in various study areas (e.g., genomics, ecology, genome sequencing, biodiversity, and evolution) (Gregory and Hebert 1999, Bennett et al. 2000, Bennett and Leitch 2005, Pellicer et al. 2018). Genome size has been estimated in many organisms (e.g., more than 7000 angiosperm species, see Plant DNA C-values database Release 7.1). In ulvophycean marine green algae, genome size has been estimated using various methods. Le Gall et al. (1993) estimated the genome sizes of some ulvophycean marine green algae using flow cytometry as follows: Blidingia minima (360 Mbp); Enteromorpha (Ulva) clathrata (270 Mbp); E. (U.) linza (270 Mbp); E. (U.) prolifera (460 Mbp); Ulva curvata (360 Mbp); U. fasciata (270 Mbp); and U. rigida (150 Mbp). Kagami et al. (2005) estimated the genome size of U. partita (treated as U. compressa in Kagami et al. 2005) to be 135 Mbp using laser scanning cytometry. De Clerck et al. (2018) estimated the genome size of U. mutabilis to be 98.5 Mbp by counting the base pairs of assembled long reads obtained by next-generation sequencing. However, these researchers did not estimate the genome sizes of both sexes separately. In haplontic or haplo-diplontic green algae, sex is expressed at the haploid phase (Coelho et al. 2018). If sex is genetically determined, there will be genomic differences between the two sexes. As a result, the genome size is different between the sexes in some species (Yamazaki et al. 2017, Hamaji et al. 2018). Therefore, genome size should be separately measured in each sex in green algae. The genome sizes of both sexes have been estimated in the isogamous ulvophycean green alga, U. partita, by next generation sequence as follows: mating type minus (mt−); 110.2 Mbp, mating type plus (mt+); 116.7 Mbp (Yamazaki et al. 2017). In many other ulvophycean marine green algae, particularly anisogamous species, little is known about the genome size of both sexes.
In this study, we focus on the anisogamous ulvophycean marine green alga M. angicava, in which genome sizes have not been reported for both sexes. Male gametes are smaller than female gametes (Tatewaki 1969, Togashi et al. 1997, 2015, Horinouchi and Togashi 2018). Their haploid gametophytes have nine chromosomes (n=9) (Horinouchi and Togashi 2019). M. angicava has some useful traits for estimating the genome sizes of males and females. First, this alga has a heteromorphic haplo-diplontic life history with a macroscopic haploid multicellular gametophyte and a microscopic diploid unicellular sporophyte (Tatewaki 1969, Horinouchi et al. 2019). Therefore, we can clearly discriminate the nuclear phase of specimens based on the morphological differences between gametophytes and sporophytes. Second, because the gametophytes are dioicous (Tatewaki 1969), we can separately isolate the nuclei of each sex. Finally, using positive phototaxis of gametes in both sexes (Tatewaki 1969, Togashi et al. 1999), we can effectively collect a sufficient number of gametes. We expect that it might be easy for us to isolate gametic nuclei because gametes have no cell walls (Tatewaki 1969).
Flow cytometry is a useful technique for estimating genome size in plants (e.g., Bennett et al. 2000, Doležel et al. 2007, Trávníček et al. 2015), because sample preparation is easy and many samples can be measured in a short time (Doležel and Bartoš 2005). Genome size estimation using flow cytometry requires the measurement and comparison of the fluorescence intensities of stained nuclei of the target plant and a standard plant for which the genome size is known. The following conditions are necessary to estimate the genome size correctly: 1) a sufficient quantity of nuclei are isolated; and 2) suitable standard plants are used (Johnston et al. 1999, Doležel and Bartoš 2005).
To isolate nuclei, plant tissues are generally chopped with a razor blade in an isolation buffer (Galbraith et al. 1983). This method might be easier than other methods (e.g., isolating nuclei from protoplasts) (Doležel and Bartoš 2005). The chopping method was unsuitable for an ulvophycean green alga U. partita because a sufficient number of nuclei was not obtained by chopping gametophytes (Kagami et al. 2005). Thus, gametes were used to obtain isolated nuclei. The chopping method might not be useful for M. angicava as well.
To estimate genome size using flow cytometry, standard plants are needed: their genome size should be close to that of the target plant and the peak of fluorescence of standard plants should not overlap with that of the target plant (Johnston et al. 1999, Doležel and Bartoš 2005). Flow cytometry compares the fluorescence intensity of nuclei between target and standard plants by the external standard method or the internal standard method. In the internal standard method, the nuclei of target and standard plants are mixed before treatment, and their fluorescence intensity is analyzed simultaneously. The internal standard method can generally estimate genome size more accurately than the external standard method because the internal standard method avoids random instrumental drifts and variations in sample preparation (Doležel and Bartoš 2005).
In this study, we first, compared three methods to isolate haploid nuclei of M. angicava: 1) chopping of a gametophyte with a razor blade; 2) homogenization of protoplasts made from haploid gametophyte cells; and 3) homogenization of gametes. Second, we stained the nuclei of M. angicava and standard plants, A. thaliana (1C=157 Mbp; Bennett et al. 2003) and B. rapa (1C=507 Mbp; Arumuganathan and Earle 1991), with PI. Finally, we estimated the genome size of M. angicava in both sexes using flow cytometry with the internal standard method.
Gametophytes of M. angicava release gametes simultaneously at spring tides between February and June (Togashi and Cox 2001). We collected matured female and male gametophytes of M. angicava at Botofurinai, Muroran, Hokkaido, Japan (42°31′N, 140°98′E) in April 2014 and May 2018, respectively. In this species, sex is distinguishable by the color of the mature parts; males are yellowish-brown and females are yellowish-green (Tatewaki 1969). We then confirmed the sex by crossing test. In the laboratory, gametes were released from the gametophytes and collected using positive phototaxis to avoid contamination (Togashi et al. 1999).
Seeds of A. thaliana and B. rapa were obtained from Inplanta Innovations Inc. (Kanagawa, Japan) and Atariya Noen Co. Ltd. (Chiba, Japan), respectively. A. thaliana and B. rapa were cultured under long-day condition (14 h : 10 h=light : dark) and ca. 50 µmol photons·m−2 s−1 with a day/night temperature cycle of 24°C/20°C.
Protoplast preparationWe used a protoplast isolation method (Saga and Kudo 1989) for M. angicava. Approximately 50–100 mg of a female gametophyte was cut into 1-mm squares in 1.0 M mannitol solution, pH 6.0. They were incubated in 1 mL enzyme solution containing 5% Cellulase Onozuka R-10 (Yakult Pharmaceutical Industry Co., Ltd., Tokyo, Japan) for 4 h at 25°C. Isolated protoplasts were filtered to remove large debris with 35-µm nylon mesh. To avoid the regeneration of cell walls, nuclei were isolated soon after protoplast preparation.
Nuclei isolation and stainingWe examined three different nuclei isolation methods for M. angicava in females using 1) gametophytes, 2) protoplasts of gametophyte cells and 3) gametes. Nuclei isolation and staining were performed on ice. Approximately 100 mg of the gametophyte was placed in a plastic Petri dish on a pre-chilled ceramic tile. We chopped the plant tissues with a razor blade to isolate nuclei in 1 mL Galbraith buffer [45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS, 0.1% (v/v) Triton X-100, pH=7.0] (Galbraith et al. 1983) and 5 µL 2-mercaptoethanol (Wako, Osaka, Japan). Nuclei of the protoplasts and the gametes were isolated in an isolation buffer (Galbraith buffer with 2-mercaptoethanol) using a homogenizer (Polytron-Kinematica GmbH, Kriens-Luzern, Sweden). The nuclei of the standard plants (A. thaliana and B. rapa) were also isolated by the chopping method (Arumuganathan and Earle 1991, Bennett et al. 2003).
Nuclei suspensions were filtered to remove debris with a 35-µm nylon mesh. To avoid the staining of double-stranded RNA by PI, 1 mL filtered nuclei suspensions were treated with 10 µL DNase-free RNase (NIPPON GENE, Tokyo, Japan) at room temperature for 10 min. The final concentration of PI (Invitrogen, California, USA) was 50 µg mL−1 and then the suspension was placed in the dark for 30 min before observation. The specimens prepared by the above three methods were observed and imaged using a CCD camera DP20-5 (Olympus, Tokyo, Japan) on an inverted microscope IX81 (Olympus, Tokyo, Japan).
Flow cytometry and genome size estimationThe fluorescence intensities of the nuclei of M. angicava and the standard plants were compared using the internal standard method. The nuclei suspensions of M. angicava and a standard plant were mixed prior to the addition of RNase and PI. Fluorescence intensity was measured by Attune Acoustic Focusing Cytometer (Thermo Fisher Scientific, Massachusetts, USA) and analyzed by Attune Cytometric Software v2.1 (Thermo Fisher Scientific). The genome size of M. angicava was estimated as follows: (genome size of M. angicava)=(mean fluorescence intensity of M. angicava)×(DNA content of the standard plant)/(mean fluorescence intensity of the standard plant) (Doležel and Bartoš 2005). For statistical analysis, we performed a Welch’s t-test using R version 3.5.2 (R Core Team 2018).
Gametophytes, protoplasts, and gametes of M. angicava are shown in Fig. 1. The gametophytes are dioicous (Fig. 1A, B). The protoplasts were almost spherical in shape and approximately 10 µm in diameter (Fig. 1C, D). The male gametes (Fig. 1E) were smaller than the female gametes (Fig. 1F).
Figure 2 shows the microscopic images of specimens prepared by the three methods: chopping a gametophyte (Fig. 2A, B), homogenization of protoplasts (Fig. 2C, D) and homogenization of gametes (Fig. 2E, F). PI successfully stained the nuclei of M. angicava in these methods. In the specimens prepared by chopping a gametophyte, we frequently observed large tissue fragments (Fig. 2A, B). Such large fragments often contained a few nuclei. In the specimens prepared by homogenization of protoplasts, we predominantly observed small fragments (Fig. 2C, D). Some of these small fragments contained nuclei. In the specimens prepared by homogenization of gametes, we observed isolated nuclei with little contamination (Fig. 2E, F). Each nucleus was uniformly stained, and little tissue contamination was observed.
Figure 3 shows the histograms of the fluorescence intensity of nuclei isolated with the three methods. The histogram of the fluorescence intensity of nuclei isolated by chopping a gametophyte had the lowest peak with the largest noise and the widest range (Fig. 3A). In the histogram of the fluorescence intensity of nuclei isolated by homogenization of protoplasts, the peak was slightly heightened but still with some noise and the range became narrower (Fig. 3B). The histogram of the fluorescence intensity of nuclei isolated by homogenization of gametes had the highest peak with the smallest noise and the narrowest range (Fig. 3C).
Figure 4 shows the results of the flow cytometry of the nuclei of M. angicava isolated by homogenization of gametes with the internal standard method. We observed clear peaks for M. angicava and standard plants. A. thaliana and B. rapa had endopolyploidy, which was represented as multiple peaks of 2C, 4C, and 8C. The ratio of cells with different levels of endopolyploidy differed among measurements. The fluorescence intensity at the peak of the histogram of the male of M. angicava was 56.95% of A. thaliana 2C, 28.86% of A. thaliana 4C, 20.00% of B. rapa 2C and 10.18% of B. rapa 4C (Fig. 4A, B). The fluorescence intensity at the peak of the histogram of the female of M. angicava was 59.05% of A. thaliana 2C, 30.14% of A. thaliana 4C, 20.58% of B. rapa 2C and 10.34% of B. rapa 4C (Fig. 4C, D). The fluorescence intensity at the peak of the histogram of A. thaliana 2C was the closest to the fluorescence intensity at the peak of the histogram of M. angicava 1C in both sexes.
The estimated genome sizes of both sexes of M. angicava using isolated gametic nuclei according to a standard plant with a different endopolyploidy level are shown in Fig. 5. The estimated genome size of M. angicava was 178.8 Mbp in males and 185.4 Mbp in females when A. thaliana 2C was used as the standard plant. The genome size of female was 3.7% larger than the genome size of males. When A. thaliana 4C was used as the standard plant, the genome size of M. angicava was 181.2 Mbp in males and 189.3 Mbp in females. The genome size was 4.5% larger in females than in males. When B. rapa 2C was used as the standard plant, the genome size of M. angicava was 202.8 Mbp in males and 208.6 Mbp in females. The genome size was 2.9% larger in females than males. When B. rapa 4C was used as the standard plant, the genome size of M. angicava was 206.4 Mbp in males and 209.8 Mbp in females. The genome size was 1.6% larger in females than in males. The genome sizes of males and females were significantly different in both standard plants with different endopolyploidy levels (A. thaliana 2C: t=−3.9668, df=24.82, p=5.4×10−4; A. thaliana 4C: t=−5.3178, df=24.509, p=1.7×10−5; B. rapa 2C: t=−3.3202, df=22.042, p=0.003; B. rapa 4C: t=−2.2475, df=26.459, p=0.03).
The specimens prepared by chopping a gametophyte contained fluoresced large tissue fragments (Fig. 2A). These fragments appeared to have caused strong noise in the fluorescence intensity histogram (Fig. 3A). The fluorescence of large tissue fragments may have been due to PI fluorescence of chloroplast and mitochondrial DNA and autofluorescence of chloroplasts. This chopping method has also proved ineffective for some other species (e.g., strawberries: Akiyama et al. 2001, U. partita: Kagami et al. 2005), in which complex macromolecules, such as the polysaccharides in strawberries (Akiyama et al. 2001) and the large amounts of anionic polysaccharide wall materials in U. partita that differ from wall materials in land plants (Kagami et al. 2005), prevent nuclei isolation. Therefore, we suggest that the gametophyte chopping method is not suitable for M. angicava, which also has polysaccharide wall materials (Mao et al. 2005).
With the protoplast homogenization method, in which cell walls were removed by cellulase before the isolation of nuclei, the contamination by tissue fragments was reduced but was not eliminated. Some tissue fragments remained (Fig. 2B) and caused some noise in the fluorescence intensity histogram (Fig. 3B). Therefore, this method appears to also be unsuitable for isolating the haploid nuclei of M. angicava.
We found that gamete homogenization was the most effective for the isolating haploid nuclei of M. angicava. In a case where we isolated nuclei by homogenizing gametes, the peak of the histogram of the fluorescence intensity of the nuclei was the clearest and showed the least noise (Fig. 3C). There was also a small second peak. A small number of isolated nuclei might not have been separated in the sample flow and might have been measured as a single nucleus. This peak was so small that it did not affect the genome size estimation. Le Gall et al. (1993) also observed flow cytometry fluorescence with little noise in marine green algae. Their nuclei isolation method requires protoplast preparation and surfactant treatment. Our gamete homogenization method is simpler and more effective than their method.
Although the estimated genome size of M. angicava slightly depended on the standard plants (A. thaliana, B. rapa) (Fig. 5), A. thaliana seems to be a more appropriate standard plant than B. rapa because the genome size of M. angicava is closer to that of A. thaliana (Doležel and Bartoš 2005). Therefore, we suggest that genome sizes of male and female of M. angicava are 178.8 Mbp and 185.4 Mbp, respectively, using the 2C of A. thaliana as the standard (Fig. 4A, C).
Female genome size of M. angicava is slightly larger than male genome size in both standard plants (3.7% with 2C of A. thaliana as the standard). This might reflect the difference in the length of sex-specific genome regions. In an isogamous ulvophycean marine green alga U. partita, sex is genetically determined, and the sex-specific region of mt+ is longer than that of mt− (Yamazaki et al. 2017). Consequently, the mt+ genome size is ca. 5% larger than mt− genome size (Yamazaki et al. 2017). Additionally, in some fresh-water green algae, the length of sex-specific genome regions is different between the sexes (e.g., anisogamous Volvox carteri: Ferris et al. 2010, isogamous Chlamydomonas reinhardtii: De Hoff et al. 2013, anisogamous Eudorina sp. and isogamous Yamagishiella unicocca: Hamaji et al. 2018). Therefore, sex in M. angicava might also be determined genetically, and the sex-specific genome region of females might be longer than that of males.
We thank the staff of the Muroran Marine Station of the Field Science Center for Northern Biosphere, Hokkaido University, for their support. This study was funded by Grants-in-Aid from the Japan Society for Promotion of Science (no. 16H04839 to TT).
