Published June 15, 2016

RESEARCH Identification and Analysis of Zbs1, a Dominant Male-Sterile Mutant of Naked ( nuda L.)

Cai Yang,* Haitao Zhou, Xiao Shi, Xinjun Zhang, Tianliang Li, Xiaohong Yang, Qian Zhang, and Tiegang Lu*

C. Yang, H.T. Zhou, X.J. Zhang, T.L. Li, and X.H. Yang, Zhangjiakou ABSTRACT Academy of Agricultural Sciences, Zhangjiakou, China 07500; A dominant male-sterile mutant of naked X. Shi, Q. Zhang, and T.G. Lu, Biotechnology Research Institute, National oat (Avena nuda L. ‘Zbs1’) was identified in a Key Facility for Gene Resources and Gene Improvement, Chinese Academy breeding program for accession A. nuda of Agricultural Sciences, Beijing, China 100081. Received 29 July 2014. ‘Pin5’, a widely cultivated naked oat variety in Accepted 19 Oct. 2015. Assigned to Associate Editor Shawn Kaeppler. northwest China. No significant differences were *Corresponding authors ([email protected]; [email protected]). detected between Zbs1 and wild-type Abbreviations: E, epidermis; EM, earlier meiosis; En, endothecium; during the vegetative growth stage. However, FDA, fluorescein diacetate; ML, middle layer; MMC, microspore at the reproductive stage Zbs1 was significantly mother cell; MP, mature pollen; Msp, miscrospore; T, tapetum; Te, different from wild-type in terms of spikelet tetrad; YM, young microspore. morphology and anther development. Iodine- potassium iodide staining, fluorescein diacetate eterosis is a phenomenon in which the progeny of diverse staining, and germination tests showed that Hvarieties of a species or crosses between species exhibit a more than 95% of the Zbs1 pollen grains were greater biomass, higher developmental rate, and greater fertility not viable. Aniline blue staining showed that than both parents (Birchler et al., 2010). Male-sterile lines do not callose was absent or degraded during the require emasculation; thus, they are ideal female lines for hybrid dyad and tetrad stages of microsporogenesis. production. Male sterility has been found in many autogamous Furthermore, genetic and histological analyses revealed that Zbs1 is a dominant male-sterile crops and is widely used in the breeding of wheat (Triticum aestivum mutant with a stable, heritable phenotype that L.) (Lucken, 1986; Whitford et al., 2013), maize (Shull, 1908), is not affected by cytoplasmic or environmental cotton (Weider et al., 2009), (Huang et al., 2012), and other factors (e.g., location and photoperiod). These vegetables (da Silva Dias, 2010). In plants, male sterility refers to a results indicate that Zbs1 is a novel, stable failure to produce dehiscent anthers, functional pollen, and viable male-sterile mutant of naked oat that can be male gametes. It includes cytoplasmic male sterility, which is caused used in breeding programs to facilitate recurrent by mitochondrial genes together with nuclear genes, and genic selection and multi-crossing schemes. male sterility, which is caused by nuclear genes alone (Vedel et al., 1994). Male-sterile mutants may show abnormal development of either sporophytic or gametophytic anther tissues. Most sporophytic male-sterile mutants exhibit defects in the tapeta and meiocytes (cells undergoing meiosis), leading to pollen abortion or pollenless sterility (Guo and Liu, 2012); in contrast, gametophytic male-sterile mutants mainly show defects in the development of microspores or pollen grains (Chen and Liu, 2014). During angiosperm microsporogenesis, callose serves as a temporary wall to separate the microsporocytes and newly formed

Published in Crop Sci. 56:1423–1428 (2016). doi: 10.2135/cropsci2014.07.0525 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. crop science, vol. 56, july–august 2016 www.crops.org 1423 microspores in tetrads, and it facilitates pollen wall formation ~700 m) and Zhangbei (with an altitude of ~1500 m) in Hebei

(Ariizumi and Toriyama, 2011; Lu et al., 2014). Abnormal Province, located in northwest China. of F1 were also callose deposition and dissolution can lead to the degeneration grown in a greenhouse under long day (14 h) or short day (11 h) of microspores (Lu et al., 2014). In transgenic tobacco, photoperiods, at 75000 LUX light intensity. Sterile plants in the F population were bagged and allowed to self or reciprocally premature dissolution of the microsporocyte callose wall 1 has been shown to cause male sterility (Worrall et al., 1992). cross with Pin5 over four successive years (1998 to 2001). The fertility rates were recorded and the data were analyzed by the In Arabidopsis, three callose synthase genes (CALLOSE chi square test. SYNTHASE 5, 11, and 12) have been shown to be involved in To determine whether the stability of the male-sterile callose synthesis during microsporogenesis (Dong et al., 2005; phenotype of Zbs1 was affected by the genetic background of the Enns et al., 2005; Nishikawa et al., 2005). A tandem CCH- paternal plant, Zbs1 plants were pollinated by 42 oat breeding type zinc finger protein, callose defective microspore1, plays lines (i.e., varieties) over five successive years (1997 to 2002). The an important role in the regulation of callose metabolism in F1 seeds were planted and the fertility rate of each F1 plant was male meiocytes and the integrity of newly formed microspores recorded. The data were analyzed by the chi square test. in Arabidopsis (Lu et al., 2014). Oat (Avena sativa L.) is an important crop both for Evaluation of Pollen Viability animal feed and human consumption. Heterosis in oat can To investigate pollen viability in Zbs1, ten plants each of field- be dated back to the 1960s (Lorencetti et al., 2006), and it grown Zbs1 and wild-type Pin5 from the field were used in each has been reported several times in recent years (Johansen- trial. The plants were planted in several different locations of Zhangbei (with an altitude of ~1500 m, located in northwest China, Morris and Latta, 2006; Lorencetti et al., 2006; Latta et al., average temperature 25°C from May to July) in Hebei Province at 2007; Prajapati et al., 2009; Crestani et al., 2012; Kapoor and the end of May. All field management practices including watering Bajaj, 2013). However, exploiting heterosis in oat breeding is and weed management were performed based on standard oat not practical due to unavailable of male-sterile oat. In 1994, production in this region of China. For each individual, five a male-sterile hulled-oat (A. sativa L.) plant was identified anthers of five florets from different branches were handled with in a breeding program, which was later confirmed to be tweezers to expel the pollen grains for 1% m/v iodine-potassium controlled by a single recessive nuclear gene (Cui et al., 1999). iodide (I2–KI) staining and light microscopic analysis, or for In 1996, we identified a spontaneous male-sterile mutant 10 mg L–1 fluorescein diacetate (FDA; Sigma-Aldrich, St. Louis, Zbs1 characterized by complete male sterility derived from MO) staining and observation by fluorescence microscopy. For A. nuda L. accession Pin5 (sterility line 1 of A. nuda L. in the pollen germination assay, liquid medium (20% sucrose, 10% PEG4000, 3 mmol L–1 calcium nitrate, 40 mg L–1 boric acid, and Zhangbei, China). Cross breeding of naked oat is hindered –1 by slow manual emasculation and a low seed setting rate due 10 mg L vitamin B1) was used. One or two drops of medium were placed on a clean slide, after which anthers were added to the to the long and soft glume. Thus, an efficient hybridization medium and squeezed to release the pollen. The pollen was then system for oat breeding is required. In this study, a genetic covered with a cover glass and cultured for 30 min at 28°C in the analysis revealed that the male sterility of Zbs1 is a dominant dark. The pollen grains were then observed under a microscope. trait; further, cytological and histological analyses indicated Pollen grains with a pollen tube that was longer than the grain abnormal microsporogenesis and microgametogenesis in diameter were deemed to be naturally germinated. Zbs1 with a lack of normal callose accumulation. It is difficult to produce and maintain seed of nuclear male sterile lines and Histological Analysis generate enough plants for hybrid breeding. However, this Spikelets from field-grown Zbs1 and segregated wild-type Pin5 mutation could facilitate recurrent selection. Plants with self- plants at flowering stages in the end of July (~60 d after sowing) pollination could use this sterile line to cross with selected were collected based on their length and fixed in Carnoy’s fluid lines to produce the population for the next cycle of selection, (containing a 3:1 v/v mixture of 100% ethanol and acetic acid). in which superior genotypes are isolated and more desirable After treatment with 70, 83, 95, and 100% ethanol for one hour, respectively, the samples were immersed in xylene for 2 genes are enriched. h and were then embedded in Paraplast Plus (Sigma-Aldrich). Microtome (RM2155; Leica Microsystems, Wetzlar, Germany) MATERIALS AND METHODS sections (8 mm thickness) were stained with 0.1% w/v toluidine blue O (Sigma-Aldrich) for 6 to 8 min and hydrated by a Plant Materials and Measurements graded ethanol series. The samples were then inspected with a A naked oat variety, accession Pin5, which is mainly cultivated in microscope (Carl Zeiss, Jena, Germany). northwest China, was used as the wild-type plant in this study. To monitor the deposition of callose during anther and The male-sterile mutant Zbs1 was identified in a Pin5 breeding microspore development, entire anthers were stained with 0.1% program and maintained by repeatedly crossing to Pin5. The w/v aniline blue in 0.067 mol L-1 phosphate buffer overnight. following characteristics of Zbs1 were investigated: plant height Squashed anther tissues were then observed under a fluorescence at the heading stage, tiller and floret number, and heading date. microscope with UV light (Hong et al., 2001). The Zbs1 mutant was cross-pollinated with Pin5, and F1 seeds were harvested and planted at Shanlizi (with an altitude of

1424 www.crops.org crop science, vol. 56, july–august 2016 RESULTS AND DISCUSSION Table 1. Growth index statistics of wild-type (WT) Pin5 and Zbs1 plants based on measurements in Zhangbei (northwest Phenotypic Characterization of Zbs1 China). Data are means ± SD (n = 50). According to a The Zbs1 mutant exhibited similar phenotypes to wild-type student’s t test, there was no significant difference between Pin5 during vegetative growth, including plant height (at the WT and Zbs1. Pin5 is a cultivated naked oat variety, and Zbs1 is a dominant male-sterile mutant of naked oat. heading stage) and tiller number (Table 1; Fig. 1a). In terms of reproductive growth, Zbs1 plants had a similar heading WT Zbs1 date and floret number (per spikelet) to wild-type plants Plant height, cm 114.5 ± 3.5 115.2 ± 2.6 (Table 1). However, the morphologies of the spikelets and Tiller number per plant 8.2 ± 1.1 8.5 ± 0.5 florets were different between wild-type and Zbs1 plants. Heading date, d 82.0 ± 2.5 83.0 ± 1.5 First, the Zbs1 plants had smaller florets compared with the Floret number per spikelet 90.5 ± 5.5 94.5 ± 3.5 wild-type plants and fewer anthers, but normal ovaries and stigmas (Fig. 1b–d). Second, spikelets of Zbs1 plants had one Pin5 when Zbs1 was used as the pollen donor, whereas 99% or two bristles and swallow tail-shaped florets, which were of the florets were able to set 1F seeds in crosses of Zbs1 absent from the spikelets of the wild-type plants (Fig. 1b). with Pin5 when Pin5 was used as the pollen donor (6014 seeds/6071 florets). The ratio of fertile/sterile plants among Third, compared with the wild-type plants, the glumes 2 2 remained open and the stigmas extended after pollination in 1198 F1 plants was 1:1 (c = 0.404 < c 0.05,1 = 3.84; 588 the Zbs1 plants (Fig. 1d), as in other male-sterile plants. fertile to 610 sterile plants). These data indicate that the male- sterile phenotype of Zbs1 is dominant and sporophytically Male Sterility in Zbs1 is a Dominant Trait controlled. In addition, small florets, small anthers, swallow Over a 4-yr period, from 1998 to 2001, no seeds were tail-shaped bolls, and spikelet bristles always co-segregated produced by selfing from 150 bagged Zbs1 plants (>10,000 with male sterility in Zbs1 plants. florets). No seeds were produced in crosses of Zbs1 with Lack of Viable Pollen in Zbs1 To investigate the sterility of the Zbs1 plants, pollen viability tests were conducted. The pollen grains of the mutant were large and round with a smooth surface. Approximately 90% (91.7 ± 1.96%) of the wild-type grains were stained dark

brown by an I2–KI solution. In contrast, the pollen grains from Zbs1 plants were small and irregular in shape with a shriveled surface; less than 1% of the grains were stained

by I2–KI (Fig. 2a and b). After staining with FDA, ~90%

Fig. 1. Morphologic comparison of Pin5 (a widely cultivated naked oat variety) and Zbs1 (a dominant male-sterile mutant), Fig. 2. Pollen viability assays in Pin5 (a cultivated naked oat variety shown on the left and right of each panel, respectively. (a) Plants at the heading stage. (b) Spikelets (c) Bolls; in northwest China) and Zbs1 (a dominant male-sterile mutant of note the short serial bolls in Pin5 and swallow tail- naked oat). I2–KI staining assay of pollen from Pin5 (A) and Zbs1 shaped bolls with one or two bristles in Zbs1; bar = 1 cm. (B) plants. FDA staining assay of pollen from Pin5 (C) and Zbs1 (D) (d) Female organs, showing normal stigma development in Zbs1; plants. Pollen germination assay using pollen from Pin5 (E) and bar = 2 mm. (e) Anthers; bar = 2 mm. Zbs1 (F) plants. Bars = 100 µm. crop science, vol. 56, july–august 2016 www.crops.org 1425 (91.9 ± 1.73%) of the wild-type pollen grains showed strong and middle layers were completely degraded, and round, fluorescence, whereas fewer than 1% of Zbs1 pollen grains densely stained pollen grains were observed in the wild- showed fluorescence (Fig. 2c and d). In vitro germination type plants (Fig. 3i). The mature wild-type pollen grains tests showed that none of the Zbs1 pollen grains had contained starch grains (Fig. 3i), and the mature Zbs1 germinated after 30 min of incubation in vitro, while the pollen grains contained only a few abnormally shaped average germination rate for wild-type pollen was ~90% starch grains (Fig. 3j). (90.1 ± 1.56%) (Fig. 2e and f). These results indicated that In addition, callose deposition in microspores at the Zbs1 mutant plants produce non-viable pollen. late meiosis and tetrad stages was investigated by aniline blue staining. Compared with the cell wall and cell plate Abnormal Microsporogenesis (which are mainly composed of callose) in wild-type at late and Microgametogenesis in Zbs1 meiosis (Fig. 4a and b) and tetrad stages (Fig. 4c and d), the Similar to other angiosperms, the cellular events of anther microspores of the Zbs1 mutant contained irregular dyad development in naked oat can be divided into eight stages: and tetrad cells (Fig. 4e and f) with no fluorescence after microsporocyte formation, early meiosis, middle meiosis, aniline blue staining (Fig. 4g and h). The fluorescence late meiosis, tetrad, young microspore, pollen mitosis, intensity represents callose content which is essential and mature pollen. From the surface to the interior, oat for the natural development of the male gametophyte. anthers are composed of an epidermis, endothecium, These results indicate a lack of callose accumulation in middle layer, and tapetum. Anther and pollen grain the microspore cell wall and cell plate, leading to the development in Zbs1 proceeded normally until the formation of irregular dyad and tetrad cells in Zbs1. The tetrad stage. Before meiosis, the epidermis, endothecium, abnormal accumulation of callose in Zbs1 may be due to a middle layer, tapetum, and pollen mother cells developed defect in callose synthesis or the degradation of callose in normally (Fig. 3a and b). During meiosis, both the wild- microspores during meiosis. type and Zbs1 pollen mother cells became associated with the tapetal layer, and the tapetum appeared vacuolated Sterility Phenotype of Zbs1 is Stable (Fig. 3c and d). Subsequently, the middle cell layer became under Different Environmental Conditions very thin and degenerated, and tetrads with four haploid To assess the stability of the male sterility phenotype of Zbs1, microspores were formed in the wild-type plants (Fig. 3e). F1 seeds produced from crosses of Zbs1 (female parent) with However, the shape and inclusion of the tetrads in Zbs1 Pin5 (male parent) were grown in the same region of China were abnormal (Fig. 3f). The young microspores were (Zhangbei) or in a greenhouse under long day or short day also irregular in Zbs1, and some of them degenerated photoperiods. For 1480 F1 plants grown under different (Fig. 3g and h). During the mature pollen stage, the tapetal environmental conditions, the ratio of fertile/sterile plants

Fig. 3. Histological features of anther development in Pin5 (a cultivated naked oat variety in northwest China) and Zbs1 (a dominant male- sterile mutant of naked oat). Cross-sections were stained with 0.25% toluidine blue O (Sigma-Aldrich, St. Louis, MO). Cross-section of a single locule at the microspore mother cell stage (MMC) in Pin5 (a) and Zbs1 (b). Cross-section of a single locule at the early meiosis stage (EM) in Pin5 (c) and Zbs1 (d). Cross-section of a single locule at the tetrad stage in Pin5 (e) and Zbs1 (f). Cross-section of a single locule at the young microspore stage (YM) in Pin5 (g) and Zbs1 (h). Cross-section of a single locule at the mature pollen stage (MP) in Pin5 (i) and Zbs1 (j). E, epidermis; En, endothecium; ML, middle layer; Msp, miscrospore; MP, mature pollen; T, tapetum; Te, tetrad. Red arrows indicate abnormal tetrads (f), abnormal young microspores (h), and abnormal mature pollen (j) in Zbs1. Bars = 15 mm.

1426 www.crops.org crop science, vol. 56, july–august 2016 Fig. 4. Aniline blue staining of microspore callose during late meiosis in wild-type (a–d) and Zbs1 (e–h) naked oat plants. Aniline blue staining was applied to fixed reproductive tissues. Note that callose deposition was nearly absent in the primary wall of gsl5 microspores and reduced in gsl5 dyads and tetrads. (a, b, e, and f) Microspores at the dyad stage. (c, d, g, and h) Microspores at the tetrad stage. (a, c, e, and g) Microspores observed under visible light. (b, d, f, and h) Microspores observed under UV light. Bars = 20 mm.

2 2 phenotype of Zbs1 was not affected by the genetic background was consistently 1:1 (c = 0.1324 < c 0.05,1 = 3.84; 747 fertile to 733 sterile plants) (Table 2). These results confirm of the paternal plant. that the male sterility phenotype of Zbs1 is a genetically stable trait that is not affected by location or photoperiod in CONCLUSIONS oat-growing regions. A male-sterile mutant, Zbs1, was identified in a breeding program in 1996 in Zhangbei, located in Hebei Province, Sterility of Zbs1 Is Unaffected by the Genetic China. Systematic analyses showed that the male-sterile Background of the Paternal Plant phenotype was complete, stable, and not affected by Over five successive years, from 1997 to 2001, Zbs1 plants environmental conditions such as the planting location were pollinated with grains from 42 different oat cultivars or photoperiod in oat-producing regions. Histological or breeding lines. The segregation ratio of male sterility to observations showed that the cause of male sterility in Zbs1 is inviable pollen, possibly due to the absence or fertility was nearly 1:1 for F1 plants from all of the crosses. Of 8640 plants investigated, 4375 plants were fertile and 4265 early degradation of callose at the dyad and tetrad stages plants were sterile, corresponding to a segregation ratio of of pollen development. 2 2 Male-sterile Zbs1 plants are normal in terms of their 1:1 (c = 1.4005 < c 0.05,1 = 3.84) (Supplemental Table S1). This indicates that the genetic stability of the male-sterile height, heading time, tiller number per plant, and floret

Table 2. Self-fertility of the F1 offspring of Zbs1 ´ Pin5, grown in different environments. Pin5 is a widely cultivated naked oat variety in northwest China, and Zbs1 is a dominant male-sterile mutant of naked oat.

Plants Fertile Sterile Expected 2 Year Location Seeds investigated plants plants ratio c 0.05,1 ––––––––––––––––––––––––– No. ––––––––––––––––––––––––– fertile/sterile 3.84 1997 Shanlizi 60 56 25 31 1:1 0.6429 1998 Shanlizi 360 300 161 139 1:1 1.6133 1999 Zhangbei 300 268 131 137 1:1 0.1343 Shanlizi 300 286 153 133 1:1 1.3986 Greenhouse 300 286 147 139 1:1 0.2238 2012 Greenhouse, 12 h of light 100 96 45 51 1:1 0.37500 Greenhouse, 8 h of light 100 98 44 54 1:1 1.0204 2013 Greenhouse, 12 h of light 50 42 20 22 1:1 0.0952 Greenhouse, 8 h of light 50 48 21 27 1:1 0.7500 Total 1620 1480 747 733 1:1 0.1324 crop science, vol. 56, july–august 2016 www.crops.org 1427 number per spike. However, small florets, small anthers, Hong, Z.L., A.J. Delauney, and D.P.S. Verma. 2001. A cell and swallow tail-shaped bolls were observed consistently plate specific callose synthase and its interaction with together with male sterility in more than 10,000 Zbs1 phragmoplastin. Plant Cell 13:755–768. doi:10.1105/ plants, indicating that these phenotypes are controlled tpc.13.4.755 Huang, F., X. Fu, A. Efisue, S. Zhang, G. Xie, W. He, A.Y.M. by one or closely linked genetic factor(s). Small anthers Nevame, and D. Jin. 2012. Genetically characterizing a and small florets with a swallow tail-shaped boll can be new indica cytoplasmic male sterility with oryza glaberrima regarded as reliable markers for male-sterile Zbs1 plants cytoplasm for its potential use in hybrid rice production. Crop by breeders. Taken together, our data indicate that the Sci. 54:132–140. naked oat mutant Zbs1 can be used as the female parent Johansen-Morris, A.D., and R.G. Latta. 2006. Fitness consequences in recurrent selection, multi-crossing, re-crossing, and of hybridization between ecotypes of Avena barbata: Hybrid convergent crossing for breeding purposes. However, the breakdown, hybrid vigor, and transgressive segregation. Evolution 60:1585–1595. doi:10.1111/j.0014-3820.2006. molecular mechanisms controlling male sterility in Zbs1 tb00503.x remain to be clarified. Kapoor, R., and R.K. Bajaj. 2013. Combining ability and heterosis studies in Avena sativa L. for green fodder yield and component Supplemental Materials Available traits. Vegetos 26:272–277. Supplemental material is available with the online version of Latta, R.G., K.M. Gardner, and A.D. Johansen-Morris. 2007. this article. Hybridization, recombination, and the genetic basis of fitness variation across environments in Avena barbata. Genetica Acknowledgments 129:167–177. We would like to thank Dr. Jingsan Sun (Institute of Botany, Lorencetti, C., F.I.F. de Carvalho, A.C. de Oliveira, I.P. Valerio, G. Chinese Academy of Sciences) for providing continuous Benin, P.D. Zimmer, and E.A. Vieira. 2006. Genetic distance encouragement and technical assistance during the study. 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