Chromosome Res DOI 10.1007/s10577-015-9477-5
Preferential recruitment of the maternal centromere-specific histone H3 (CENH3) in oat (Avena sativa L.) × pearl millet (Pennisetum glaucum L.) hybrid embryos Takayoshi Ishii & Naohiro Sunamura & Ayaka Matsumoto & Amin Elsadig Eltayeb & Hisashi Tsujimoto Received: 13 February 2015 / Revised: 19 May 2015 / Accepted: 24 May 2015 # Springer Science+Business Media Dordrecht 2015
Abstract Chromosome elimination occurs frequently in interspecific hybrids between distantly related species in Poaceae. However, chromosomes from both parents behave stably in a hybrid of female oat (Avena sativa L.) pollinated by pearl millet (Pennisetum glaucum L.). To analyze the chromosome behavior in this hybrid, we cloned the centromere-specific histone H3 (CENH3) genes of oat and pearl millet and produced a pearl millet-specific anti-CENH3 antibody. Application of this antibody together with a grass species common anti-CENH3 antibody revealed the dynamic CENH3 composition of the hybrid cells before and after fertilization. Despite co-expression of CENH3 genes encoded by oat and pearl millet, only an oat-type CENH3 was incorporated into the centromeres of both species in the hybrid embryo. Oat CENH3 enables a functional centromere in pearl millet chromosomes in an oat genetic background. Comparison of CENH3 genes among Poaceae species that show chromosome elimination in Responsible Editor: Jiming Jiang T. Ishii : A. Matsumoto : A. E. Eltayeb : H. Tsujimoto (*) Arid Land Research Center, Tottori University, Hamasaka, Tottori 680-0001, Japan e-mail: [email protected] N. Sunamura Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan Present Address: T. Ishii Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstraße 3, 06466 Gatersleben, Germany
interspecific hybrids revealed that the loop 1 regions of oat and pearl millet CENH3 exhibit exceptionally high similarity. Keywords oat . pearl millet . embryogenesis . centromere-specific histone H3 (CENH3) . interspecific cross . chromosome elimination Abbreviations CENH3 FISH Anti-PgCENH3 Anti-GrassCENH3
Centromere-specific histone H3 Fluorescence in situ hybridization Pearl millet CENH3-specific antibody Poaceae CENH3-specific antibody
Introduction Uniparental chromosome elimination occurs frequently in interspecific hybrids in the family Poaceae (Houben et al. 2011), allowing these crosses to generate haploid plants. In crosses between subfamily-level species in Poaceae, such as wheat-sorghum, wheat-pearl millet, wheat-maize, or oat-maize crosses, complete or incomplete elimination of the chromosomes from the male parent takes place (Laurie and Bennett 1986, 1988; Laurie 1989; Rines and Dahleen 1990). There are several reports regarding the mechanisms of chromosome
T. Ishii et al.
elimination. In wheat-maize hybrids, Mochida et al. (2004) found incomplete attachment of the spindles to the centromeres of maize. In a wheat-pearl millet hybrid, Gernand et al. (2005) observed lagging pearl millet chromosomes in anaphase cells and nuclear extrusion pearl millet chromatin from interphase cells. In the same wheat-pearl millet hybrid, Ishii et al. (2010) found chromosome breakage and non-disjunction of the pearl millet chromosome. Sanei et al. (2011) proposed that impaired loading of the centromere-specific histone H3 (CENH3) to the centromere causes uniparental chromosome elimination in barley-Hordeum bulbosum hybrids. In contrast to canonical histone H3, CENH3 is highly species-specific. Incompatibly between the centromere and CENH3 is a plausible mechanism for the process of chromosome elimination in some interspecific hybrids. Jin et al. (2004) found that the centromeres of stabilized maize chromosomes in oat-maize chromosome addition lines incorporated CENH3 of oat. CENH3 homologs in other species include CENP-A in humans (Earnshaw and Rothfield 1985) and HTR12 in Arabidopsis thaliana (Talbert et al. 2002). This protein consists of an N-terminal variable region and a conserved histone-fold domain with an important role in centromere targeting in humans (Sullivan et al. 1994). In the fruit fly (Drosophila melanogaster), the loop 1 region of the CENH3 homolog Cid is essential for centromere localization (Vermaak et al. 2002). In Arabidopsis, a chimeric protein comprised of the Nterminal tail of histone H3.3 and the CENH3 histonefold domain can compensate for the function of CENH3. This result suggests the importance of the histone-fold domain for centromere targeting (Ravi et al. 2010). Ravi and Chan (2010) demonstrated that Arabidopsis encoding a modified CENH3 produced haploids after fertilization with pollen from wild-type plants due to uniparental chromosome elimination. Maheshwari et al. (2015) demonstrated that a natural variant of CENH3 generates chromosome elimination in Arabidopsis. These results underline the significant role of CENH3 in the process of chromosome elimination in intra- and interspecific hybrids. Ishii et al. (2013) previously crossed oat with pearl millet and observed normal behavior of both parental chromosome sets, although these species are highly differentiated at the subfamily level. Hybrid plants grew until the seedling stage but were not viable because of severe necrosis. To understand why both oat and pearl millet maintain normal chromosome function, here, we
investigate the dynamics of parental CENH3 proteins during embryogenesis in hybrid pearl millet × oat.
Materials and methods Plant materials and crossing Oat (Avena sativa; 2n=6x=42) ‘Best Enbaku’ was cultivated in the experimental field under natural conditions at Tottori, Japan. Pearl millet (Pennisetum glaucum; 2n = 2x = 14) ‘Ugandi’ was grown in a greenhouse (≥20 °C) in Tottori, Japan. Crossing procedures and spike culture were performed as previously described in Ishii et al. (2010). Isolation of CENH3 genes and antibody production Total RNA was extracted from the roots of pearl millet and oat plants using the TriPure Isolation Reagent (Roche). To isolate the CENH3 genes by PCR, the following degenerate primers were designed based on the consensus sequences of CENH3 genes reported for species in Panicoideae or Pooideae: 5′-ATGGCTCGAA CCAAGCACCAGGCCGTGAGG-3′ and 5′TCAMGMCCAACGCCTTCCTCCGATACGCC-3′ or 5′-ATGGCCCGCACSAAGCRCCCSGCCRTSAGG3′ and 5′-TYASYAMMSCCTSYKCCCRCYGATACG CC-3′. The following PCR conditions were used: 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, with a final extension for 7 min at 72 °C. Electrophoresis of PCR products was performed in a 3 % (w/v) agarose gel, and bands of expected size were excised from the gel, purified, and cloned into the pGEM-T Easy vector (Promega). Purified PCR-amplified fragments were then labeled by BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystem) and sequenced by ABI 3100 Avant DNA sequencer. Wheat, maize, and sorghum CENH3 amino acid sequences from DDBJ (protein IDs: AEH95350, AEH95351, AEH95352, AAM74226, and EES19720) were used as reference for Table 1. ClustalX 2.0.12 (http://www.clustal.org/clustal2/) software was used to perform amino acid alignments among CENH3 proteins from Larkin et al. (2007). The N-terminal tail (GGAS TSGTSGRGAPARGG) of pearl millet CENH3 was used as a species-specific epitope for production of anti-CENH3 polyclonal antibodies. Operon (Japan)
25 61 78 53 75 38 77
25 49 SbCENH3
50 75
25
52 75
25
48
75
38
47 75
Preparation of specimens for cytological analysis
*No significant difference at P
Preferential recruitment of the maternal centromere-specific histone H3 (CENH3) in oat (Avena sativa L.) × pearl millet (Pennisetum glaucum L.) hybrid embryos Takayoshi Ishii & Naohiro Sunamura & Ayaka Matsumoto & Amin Elsadig Eltayeb & Hisashi Tsujimoto Received: 13 February 2015 / Revised: 19 May 2015 / Accepted: 24 May 2015 # Springer Science+Business Media Dordrecht 2015
Abstract Chromosome elimination occurs frequently in interspecific hybrids between distantly related species in Poaceae. However, chromosomes from both parents behave stably in a hybrid of female oat (Avena sativa L.) pollinated by pearl millet (Pennisetum glaucum L.). To analyze the chromosome behavior in this hybrid, we cloned the centromere-specific histone H3 (CENH3) genes of oat and pearl millet and produced a pearl millet-specific anti-CENH3 antibody. Application of this antibody together with a grass species common anti-CENH3 antibody revealed the dynamic CENH3 composition of the hybrid cells before and after fertilization. Despite co-expression of CENH3 genes encoded by oat and pearl millet, only an oat-type CENH3 was incorporated into the centromeres of both species in the hybrid embryo. Oat CENH3 enables a functional centromere in pearl millet chromosomes in an oat genetic background. Comparison of CENH3 genes among Poaceae species that show chromosome elimination in Responsible Editor: Jiming Jiang T. Ishii : A. Matsumoto : A. E. Eltayeb : H. Tsujimoto (*) Arid Land Research Center, Tottori University, Hamasaka, Tottori 680-0001, Japan e-mail: [email protected] N. Sunamura Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan Present Address: T. Ishii Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstraße 3, 06466 Gatersleben, Germany
interspecific hybrids revealed that the loop 1 regions of oat and pearl millet CENH3 exhibit exceptionally high similarity. Keywords oat . pearl millet . embryogenesis . centromere-specific histone H3 (CENH3) . interspecific cross . chromosome elimination Abbreviations CENH3 FISH Anti-PgCENH3 Anti-GrassCENH3
Centromere-specific histone H3 Fluorescence in situ hybridization Pearl millet CENH3-specific antibody Poaceae CENH3-specific antibody
Introduction Uniparental chromosome elimination occurs frequently in interspecific hybrids in the family Poaceae (Houben et al. 2011), allowing these crosses to generate haploid plants. In crosses between subfamily-level species in Poaceae, such as wheat-sorghum, wheat-pearl millet, wheat-maize, or oat-maize crosses, complete or incomplete elimination of the chromosomes from the male parent takes place (Laurie and Bennett 1986, 1988; Laurie 1989; Rines and Dahleen 1990). There are several reports regarding the mechanisms of chromosome
T. Ishii et al.
elimination. In wheat-maize hybrids, Mochida et al. (2004) found incomplete attachment of the spindles to the centromeres of maize. In a wheat-pearl millet hybrid, Gernand et al. (2005) observed lagging pearl millet chromosomes in anaphase cells and nuclear extrusion pearl millet chromatin from interphase cells. In the same wheat-pearl millet hybrid, Ishii et al. (2010) found chromosome breakage and non-disjunction of the pearl millet chromosome. Sanei et al. (2011) proposed that impaired loading of the centromere-specific histone H3 (CENH3) to the centromere causes uniparental chromosome elimination in barley-Hordeum bulbosum hybrids. In contrast to canonical histone H3, CENH3 is highly species-specific. Incompatibly between the centromere and CENH3 is a plausible mechanism for the process of chromosome elimination in some interspecific hybrids. Jin et al. (2004) found that the centromeres of stabilized maize chromosomes in oat-maize chromosome addition lines incorporated CENH3 of oat. CENH3 homologs in other species include CENP-A in humans (Earnshaw and Rothfield 1985) and HTR12 in Arabidopsis thaliana (Talbert et al. 2002). This protein consists of an N-terminal variable region and a conserved histone-fold domain with an important role in centromere targeting in humans (Sullivan et al. 1994). In the fruit fly (Drosophila melanogaster), the loop 1 region of the CENH3 homolog Cid is essential for centromere localization (Vermaak et al. 2002). In Arabidopsis, a chimeric protein comprised of the Nterminal tail of histone H3.3 and the CENH3 histonefold domain can compensate for the function of CENH3. This result suggests the importance of the histone-fold domain for centromere targeting (Ravi et al. 2010). Ravi and Chan (2010) demonstrated that Arabidopsis encoding a modified CENH3 produced haploids after fertilization with pollen from wild-type plants due to uniparental chromosome elimination. Maheshwari et al. (2015) demonstrated that a natural variant of CENH3 generates chromosome elimination in Arabidopsis. These results underline the significant role of CENH3 in the process of chromosome elimination in intra- and interspecific hybrids. Ishii et al. (2013) previously crossed oat with pearl millet and observed normal behavior of both parental chromosome sets, although these species are highly differentiated at the subfamily level. Hybrid plants grew until the seedling stage but were not viable because of severe necrosis. To understand why both oat and pearl millet maintain normal chromosome function, here, we
investigate the dynamics of parental CENH3 proteins during embryogenesis in hybrid pearl millet × oat.
Materials and methods Plant materials and crossing Oat (Avena sativa; 2n=6x=42) ‘Best Enbaku’ was cultivated in the experimental field under natural conditions at Tottori, Japan. Pearl millet (Pennisetum glaucum; 2n = 2x = 14) ‘Ugandi’ was grown in a greenhouse (≥20 °C) in Tottori, Japan. Crossing procedures and spike culture were performed as previously described in Ishii et al. (2010). Isolation of CENH3 genes and antibody production Total RNA was extracted from the roots of pearl millet and oat plants using the TriPure Isolation Reagent (Roche). To isolate the CENH3 genes by PCR, the following degenerate primers were designed based on the consensus sequences of CENH3 genes reported for species in Panicoideae or Pooideae: 5′-ATGGCTCGAA CCAAGCACCAGGCCGTGAGG-3′ and 5′TCAMGMCCAACGCCTTCCTCCGATACGCC-3′ or 5′-ATGGCCCGCACSAAGCRCCCSGCCRTSAGG3′ and 5′-TYASYAMMSCCTSYKCCCRCYGATACG CC-3′. The following PCR conditions were used: 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 30 s, with a final extension for 7 min at 72 °C. Electrophoresis of PCR products was performed in a 3 % (w/v) agarose gel, and bands of expected size were excised from the gel, purified, and cloned into the pGEM-T Easy vector (Promega). Purified PCR-amplified fragments were then labeled by BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystem) and sequenced by ABI 3100 Avant DNA sequencer. Wheat, maize, and sorghum CENH3 amino acid sequences from DDBJ (protein IDs: AEH95350, AEH95351, AEH95352, AAM74226, and EES19720) were used as reference for Table 1. ClustalX 2.0.12 (http://www.clustal.org/clustal2/) software was used to perform amino acid alignments among CENH3 proteins from Larkin et al. (2007). The N-terminal tail (GGAS TSGTSGRGAPARGG) of pearl millet CENH3 was used as a species-specific epitope for production of anti-CENH3 polyclonal antibodies. Operon (Japan)
25 61 78 53 75 38 77
25 49 SbCENH3
50 75
25
52 75
25
48
75
38
47 75
Preparation of specimens for cytological analysis
*No significant difference at P
