5 genetics and biology of cytoplasmic male sterility and its applications in forage and turf grass breeding

Plant Breeding, 133, 299–312 (2014) © 2014 Blackwell Verlag GmbH doi:10.1111/pbr.12155 Review Genetics and biology of cytoplasmic male sterility and its applications in forage and turf grass breeding M D S H O F I Q U L I S L A M 1, B R U N O S T U D E R 2, I A N M A X M Ø L L E R and T O R B E N A S P 1,3 Department of Molecular Biology and Genetics, Science and Technology, Aarhus University, Forsøgsvej 1, DK-4200, Slagelse, Denmark; 2Forage Crop Genetics, Institute of Agricultural Sciences, ETH Zurich, CH-8092, Zurich, Switzerland; 3Corresponding author, E-mail: Torben.Asp@agrsci.dk With tables Received May 23, 2013/Accepted December 7, 2013 Communicated by J Staub Abstract Hybrid breeding can exploit heterosis and thus offers opportunities to maximize yield, quality and resistance traits in crop species Cytoplasmic male sterility (CMS) is a widely applied tool for efficient hybrid seed production Encoded in the mitochondrial genome, CMS is maternally inherited, and thus, it can be challenging to apply in breeding schemes of allogamous self-incompatible plant species, such as perennial ryegrass Starting with a general introduction to the origin and the function of mitochondria in plants, this review focuses on the genetics and biology of CMS systems in plants to identify conserved and system-specific mechanisms We examine prospects of comparative mitochondrial genomics to identify candidate genes and causative polymorphisms associated with CMS across species and discuss specificities, obstacles and potentials of CMS as a breeding tool for maximizing heterosis in forage grasses The purpose of the review is to highlight the importance of CMS and hybrid breeding in grasses, with the aim of facilitating research and development of novel breeding strategies to address the future needs for food, feed and biomass production Key words: comparative mitochondrial genomics — cytoplasmic male sterility — forage grasses — mitochondrial genome — perennial ryegrass (Lolium perenne L.) Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants that prevents the production of functional pollen but maintains female fertility (Levings 1993) It has evoked major interest as a means for containment of transgenic plants in crop species (Chase et al 2010) and, more importantly, for controlling pollination during hybrid seed production Hybrid breeding aims to fully exploit heterosis, a fundamental genetic phenomenon, leading to better performance of F1 hybrid progeny by combining complementary genetic materials from both of its inbred parents (East 1908, Shull 1908) Heterosis can be manifested as increased size, growth rate and yield, as well as improved resistance and tolerance towards biotic and abiotic stress, respectively (Melchinger and Gumber 1998, Tollenaar et al 2004) The consequent increased productivity of hybrid crops contributes to global feed and food security (Godfray et al 2010) Hybrid breeding requires an efficient tool to control pollination during seed production (Horn and Friedt 1999) The use of CMS to produce hybrid seed has proven cost-effective and is widely used in some major crops such as maize (Zea mays L.), sorghum (Sorghum bicolor L.) (reviewed by Kaul 1988), rice (Oryza sativa L.) (Barclay 2010), rapeseed (Brassica napus L.) (Zhao and Gai 2006), rye (Secale cereale L.) (Geiger and Schnell 1970), wheat (Triticum aestivum L.) and pearl millet (Pennisetum glaucum L.) (Rajeshwari et al 1994, Havey 2004) In maize, the most prominent example of a hybrid breeding crop, many of the commercially used varieties are hybrids produced by CMS (Acquaah 2012) Cytoplasmic male sterility is determined outside the nuclear genome and is caused by sequence alterations in the mitochondrial genome affecting availability and/or functionality of anthers, pollen or male gametes (Hanson and Bentolila 2004, Ivanov and Dymshits 2007, Carlsson et al 2008) These alterations involve single-nucleotide polymorphisms (SNPs), large insertions or deletions (InDels), variation in the content of repetitive DNA sequence or major genome rearrangements caused by recombination events (Kubo and Newton 2008) The resulting CMS phenotype can manifest itself as varying reproductive abnormalities (Laser and Lersten 1972, Schnable and Wise 1998, Hanson and Bentolila 2004, Carlsson et al 2008) In some cases, male reproductive organs (e.g stamens) are transformed into petals or female reproductive organs (e.g carpels) (Zubko 2004, Linke and B€ orner 2005) Other CMS mutations lead to the degeneration of anthers or developing pollen that fails to develop fully, and if they develop completely, they are often not functional (Chase 2007) In some instances [e.g sunflower (Helianthus annuus L.), petunia (Petunia parodii L.) and maize], anthers are often completely missing (Rieseberg and Blackman 2010) The genetic mechanisms underlying CMS systems are as varied as the CMS phenotypes themselves Broad structural and functional variation of genes causing both CMS and the restoration of fertility makes it difficult to find a consensus mechanism in the genetics and biology of CMS for applying CMS and fertility restoration genes to other CMS sources and plant species (Hanson and Bentolila 2004) Unlike the above-mentioned major crop species, the use of hybrid breeding has had limited impact in forage and turf grass species (Kobabe 1978, 1983, Kiang and Kavanagh 1996b) This may be due to the fact that many of the most important forage grass species are characterized by highly effective self-incompatibility (SI) systems (Cornish et al 1979), which promote crosspollination Such allogamous species are usually improved as populations or synthetic varieties, thereby maintaining a high level of heterozygosity As a consequence, deleterious alleles are maintained in these populations, which makes them vulnerable 300 to inbreeding depression The effectiveness of SI systems also complicates the establishment of inbred lines, a prerequisite for maximizing heterosis in hybrid breeding schemes Such SI systems also make it difficult to maintain the CMS phenotype while improving its genetic background Cytoplasmic male sterility systems in forage and turf grass species that are consistently sterile under various environmental conditions are not available yet, or their commercial use is protected by property rights (Gaue and Baudis 2007) Therefore, new CMS sources, either naturally available or introduced by wide hybridization or mutagenesis, are of major interest to plant breeders In the past, several attempts have been made to induce male sterility in perennial ryegrass using selective gametocides (Wit 1974) However, none of these have been successful (Wit 1960, Foster 1969) Consequently, CMS was induced to perennial ryegrass by interspecific (Wit 1974) and intergeneric matings (Connolly and Wright-Turner 1984), as well as by chemical mutagenesis (Gaue and Baudis 2007) Although CMS sources in perennial ryegrass have been identified (Kiang et al 1993, Kiang and Kavanagh 1996a), mechanisms to restore fertility have not yet been established In forage crops, however, there is no need to restore fertility because biomass production constitutes a major breeding goal (Ruge et al 2003) On the other hand, the maintenance of the CMS phenotype in the highly heterozygous genetic background of allogamous species is difficult, and partial or complete restoration of fertility complicates the breeding process (Geiger and Miedaner 2009) With the significant recent progress in the understanding of the genetics and genomics of grass reproductive traits such as CMS (McDermott et al 2008), SI (Klaas et al 2011) and the use of SI in inbred line development (Thorogood et al 2005), breeding efforts towards the implementation of hybrid breeding systems in forage and turf grasses have intensified Starting with a general introduction to the origin and the function of mitochondria in plants, the main aims of this review are to (i) describe the genetics and biology of different CMS systems in flowering plants; and (ii) evaluate prospects of comparative mitochondrial genomics for the identification of candidate genes and causative polymorphisms of CMS in forage grass species Thereafter, the specificities, obstacles and potentials of CMS as a breeding tool for hybrid breeding in forage grass species will be discussed Origin of Mitochondria in Plants Cytoplasmic genomes, such as mitochondrial and chloroplast genomes, are probably the remnants of bacteria that were engulfed by another bacterium approximately billion years ago (Gray et al 1999, Lang et al 1999) The ‘endosymbiotic theory’ postulates that the original mitochondrion was an aerobic bacterium that was ingested by anaerobic bacteria (Margulis 1970) Over time, this original endosymbiont theoretically evolved into an organelle that was no longer able to survive without oxygen Later, the host cells and the endosymbiont performed mutual functions which likely had survival advantages as long as they continued their synergetic partnership These cells, in turn, eventually gave rise to all eukaryotic cells Plant cells, according to this theory, arose when a second endosymbiotic event took place This time, a mitochondrion-containing cell engulfed a photosynthetic cyanobacterium, which over time evolved inside the cell into the chloroplast The two layers, the outer and inner membrane of both mitochondria and chloroplasts, are the initial evidence of this endosymbiotic event (Margulis 1970) The inner M S ISLAM, B STUDER, I M MØLLER et al membrane is derived from the bacterial cell plasma membrane, and the outer membrane evolved through invagination of the plasma membrane of the host cell that hypothetically engulfed the bacterial cell (Taiz and Zeiger 2010) Function of Mitochondria in Plants Plant cells contain genetic information in the nucleus, chloroplasts and mitochondria (Unseld et al 1997) The chloroplasts, which convert solar radiation into chemical energy, and mitochondria, which convert chemically stored energy into adenosine triphosphate (ATP), are known as energy-producing organelles (Yurina and Odintsova 2010) Thus, the primary role of plant mitochondria is the respiratory oxidation of sugars or breakdown products from proteins and/or lipids and the transfer of electrons to oxygen through the respiratory electron transport chain coupled to the synthesis of ATP (Millar et al 2005) During flowering, tissues involved in reproduction require high rates of metabolism from the mitochondrial respiratory chain (Budar and Pelletier 2001) Under abiotic stress conditions, such as high temperatures, long photoperiods, drought and extreme cold, plants may be unable to maintain their normal level of energy production Plant mitochondria are involved in the tolerance to oxidative stress induced by abiotic stresses (Møller 2001b, Millar et al 2003) and may change their functionality in response to stress as a ‘stress sensor organelle’ (Jones 2000) Further evidence of this comes from studies of the Arabidopsis fro1 mutant (Lee et al 2002, Chinnusamy et al 2006) In this case, the FROSTBITE1 (fro1) gene encodes a defective 18-kDa subunit of NADH dehydrogenase Complex I, which displays a constitutively higher accumulation of reactive oxygen species (ROS) during cold acclimation Another example in Arabidopsis is the male gametophyte defective (MGP1), a gene essential for pollen formation, which encodes the FAd subunit of mitochondrial F0F1-ATP synthase and is expressed in the pollen grain at the dehydration stage (Li et al 2010) Mutation of the MGP1 gene leads to pollen death via destruction of the mitochondria (Li et al 2010) Mitochondrial Genomes A plant cell typically contains around 200 mitochondria, each carrying one or more copies of the mitochondrial genome (Logan 2006) In contrast to the more conserved and compact animal mitochondrial genomes that range in size from 14 to 19 kbp (Gray et al 1999), plants have the largest reported mitochondrial genomes The size of sequenced plant mitochondrial genomes ranges from 187 kbp in liverwort (Marchantia polymorpha L.) (Oda et al 1992) to 11 318 kbp in natural populations of Silene conica L., a dicotyledonous seed plant exhibiting a high mitochondrial mutation rate and abundant non-coding DNA content (Sloan et al 2012b) Characteristic for angiosperm mitochondrial genomes is the high degree of variation in terms of size and genome organization, which occurs both between and within species (Fauron et al 1995) This is due to frequent genomic recombinational rearrangements that occur in all angiosperm lineages (Wolstenholme and Fauron 1995, Handa 2003, Kubo and Newton 2008) The size variation of mitochondrial genomes depends on the content of repetitive sequences and large duplications that can range from 0.2 to 120 kbp (Kubo and Newton 2008) As shown by Clifton et al (2004), six pairs of large repeat sequences account for 17.4% of the maize NB (normal mitochondrial gen- Cytoplasmic male sterility in grass breeding ome in the B37 nuclear background) genome Similarly, six major repeated sequences cover 26.0% of the rice mitochondrial genome (Notsu et al 2002) Large units of repetitive sequences are also found in the mitochondrial genomes of Arabidopsis, sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) (Unseld et al 1997, Kubo et al 2000, Sugiyama et al 2005) Maize cytotype CMS-C (Charrua) has, in addition, three sets of repeats, which together account for the 30% larger size of the C relative to the NB genome (Kubo and Newton 2008) As repeated sequences in different plant species show no homology to each other, it has been hypothesized that they were independently acquired by each species during angiosperm evolution (Sugiyama et al 2005) As of February 2013, mitochondrial genomes of 72 plants and a large number of metazoa, fungi and other organisms have been published in the NCBI genome database (http://www.ncbi.nlm.nih gov/genomes/GenomesHome.cgi?taxid=2759&hopt=html) These plant mitochondrial genomes contain 50–71 non-redundant genes (Table 1), whereas the number of identified genes is comparatively less in animal mitochondrial genomes (Marienfeld et al 1999) These genes encode a set of approximately 24–41 non-redundant proteins of respiratory complexes I–V, subunits of cytochrome c biogenesis, ribosomal proteins and maturase proteins (Table 1) In addition, the plant mitochondrial genome contains 14–27 transfer RNA (tRNA) and three ribosomal RNA (rRNA) genes (Table 1) The greatest variation in gene number is found in ribosomal protein and tRNA gene contents (Kubo and Newton 2008) While the size of mitochondrial genomes during evolution of angiosperms has generally increased, the gene content has decreased due to gene loss and gene transfer to the nucleus (Adams et al 2002) Interestingly, ribosomal protein genes have been lost more often than respiratory genes (Gray et al 1998, Lang et al 1999) For example, the ribosomal protein gene rps14 is a pseudo-gene in Arabidopsis and rice, but is functional in rapeseed (Brandt et al 1993, Handa 2003) Comparative Mitochondrial Genome Analysis Comparative analysis of mitochondrial genomes has been used to identify similarities and dissimilarities of mitochondrial genomes both within and between species (Kubo et al 2011) These comparisons include genome size, the proportion of repetitive sequences, as well as the content of genes encoding rRNAs, tRNAs and proteins of respiratory complexes (Table 1) Due to extensive recombination of mitochondrial genomes in noncoding regions of higher plants (Fauron et al 1995, Allen et al 2007), these comparisons are rather difficult and mainly focus on genes that are highly conserved For example, although liverwort lacks a functional nad7 gene, this gene is present in the mitochondrial genomes of Arabidopsis, rice and sugar beet (Oda et al 1992, Unseld et al 1997, Notsu et al 2002, Satoh et al 2004) Similarly, the maize NB genome lacks rpl5 and rpl2, but both are present in the Arabidopsis and rice mitochondrial genomes (Unseld et al 1997, Notsu et al 2002, Clifton et al 2004) A slightly higher degree of mitochondrial genome conservation is found within species (Allen et al 2007) For example in maize, NB and CMS-C share a DNA duplication of 11 and 17 kbp (Kubo and Newton 2008) Comparative genome analysis between a CMS line and its fertile maintainer line has been used to identify candidate genes responsible for the CMS phenotype in maize (Allen et al 2007) To identify causative CMS genes, fertile revertants of well-known CMS sources were compared 301 with the CMS lines, which then led to the identification of CMS-associated regions in maize cytotypes, CMS-T (Texas) (Dewey et al 1987) and CMS-S (USDA, United States Department of Agriculture) (Zabala et al 1997), and sugar beet (Satoh et al 2004) These comparative genomic approaches can be complemented with transcriptomic and proteomic data to provide further evidence for the causative CMS sequence polymorphism (Rui-Hong et al 2010) With the aim to characterize differentially expressed genes and proteins involved in CMS, RNA-seq and two-dimensional differential gel electrophoresis (2D-DIGE) followed by mass spectrometry (MALDI-TOF/TOF) have successfully been used in various species (e.g wolfberry, Lycium barbarum L.; pummelo, Citrus grandis L.) (Zheng et al 2012a,b) Interaction Between Nuclear and Mitochondrial Genomes As mitochondria are semi-autonomous, they need to exchange genetic information with the nuclear genome to maintain their role in metabolic processes and ATP-based energy production (Yurina and Odintsova 2010) The mitochondrial genome encodes 95%) of the genetic information required for their biogenesis and function is found in the nuclear genome (Millar et al 2005, 2006, Cui et al 2011) Special mechanisms are required for coordinating gene expression in the nucleus, chloroplast and mitochondria These organelles are engaged in organelle-tonucleus regulation, known as retrograde regulation (Yang et al 2008a) Anterograde regulation, which controls the flow of information from the nucleus and cytoplasm to organelles, also plays a key role in the biogenesis and function of cell organelles (Yurina and Odintsova 2010) Retrograde regulation controls the fine-tuning of the nuclear gene expression involved in growth, development and environmental stress management (Gadjev et al 2006) The chloroplast retrograde signalling pathway has been studied extensively in plants (Nott et al 2006, Koussevitzky et al 2007) Chloroplastic retrograde regulation has been identified that is involved with signalling in response to photomorphogenesis (Surpin et al 2002) and also altered metabolic function during chloroplast biogenesis and redox regulation (Rodermel 2001) Mitochondrial retrograde signalling might be a useful tool for studying CMS (Carlsson et al 2008, Yang et al 2008a, Møller and Sweetlove 2010) Alterations in the mitochondrial genome or in mitochondrial gene expression result in changed expression of certain nuclear genes, which in turn leads to modified phenotypes (Linke and B€ orner 2005) This can occur naturally or can be induced through wide hybridization or somatic cell fusion (Chase 2007, Dalvi et al 2010) In all cases, CMS might be caused by a disrupted interaction of the nuclear and mitochondrial genomes, facilitated by changes in the mitochondrial genome Genetics of CMS Systems in Plants The genetic basis of CMS was first described by Bateson and Gairdner (1921) and Chittenden and Pellew (1927) who stated that male sterility was due to an interaction of a sterility-inducing cytoplasm and a homozygous recessive nuclear gene causing pollen sterility, where the nuclear gene was ineffective in ‘normal (male-fertile)’ cytoplasm Shinjyo (1969) reported, for instance, that a male sterility-inducing cytoplasm of rice 678 186 430 490 490 491 490 434 559 F1-30, male-fertile NA Bright yellow 4, male-fertile Nipponbare-N, male-fertile Nipponbare-S, male-fertile 93–11, Male-fertile PA64S, male-sterile Lead rice, CMS-LD Chinese wild rice strain W1, CMS-CW Khalas (male and female), Fahal (male) and Sukry (female) NA Uchiki-Gensuke male-fertile MS-Gensuke, CMS-Ogura Voucher specimens (L Bergner 003) NA Voucher specimens (D Sloan 003) Voucher specimens (L Bergner 007) KOV MTV S9L Chinese Spring, male-fertile Chinese Winter Yumai (Km3), male-fertile Yumai (Ks3), CMS-K Berken Pinot noir clone ENTAV115 conica latifolia noctiflora vulgaris Vigna radiata Vitis vinifera Triticum aestivum Silene Silene Silene Silene Physcomitrella patents Raphanus sativus Phoenix dactylifera Oryza rufipogon 40.6 45.2 45.2 43.1 42.6 40.8 41.8 42 41.8 42 NA 42 43.3 45.1 NA 647 559 401 262 773 279 45.1 44.1 42.4 45 43.8 43.8 43.8 43.8 43.8 43.8 NA 46.9 44.1 44.8 43.9 43.9 43.27 45.33 NA 45.2 NA NA 40.9 NA 44.5 G+C content (%) 340 036 426 000 413 000 000 000 000 000 528 526 105 244 258 11 318 253 728 427 361 429 422 452 452 715 001 580 608 597 520 669 515 673 735 045 982 833 414 903 432 839 Dark Green Zucchini NA NA 924 799 020 519 241 766 853 271 747 737 236 402 Cucurbita pepo Cycas taitungensis Ferrocalamus rimosivaginus Lolium perenne Marchantia polymorpha Nicotiana tabacum Oryza sativa Boea hygrometrica Brassica carinata Brassica juncea Brassica napus Brassica oleracea Brassica rapa Chara vulgaris Citrullus lanatus Cucumis melo 366 368 501 510 232 219 221 360 219 67 379 738 Columbia TK81-O, male-fertile TK81-MS, CMS-Owen NA W29, Car (male-fertile) Jiangpu-yejiecai, Jun (male-fertile) Wester, CMS-nap 08C717, Ole (male-fertile) Suzhouqing, Cam (male-fertile) Voucher number QFA468020 Florida Giant PIT92 Genome size (nt) Arabidopsis thaliana Beta vulgaris Species name Cultivar/accession name and cytoplasm type 3 (3)1 3 NA NA NA 3 3 3 (3)1 (3)1 (3)1 (3)1 (3)1 (3)1 (3)1 (1)3 10 (3)1 3 39 (34)1 (1)2 41 36 37 (2)2 NA NA NA 34 (3)2 34 (3)2 38 (1)2 42 (39)1 33 34 (33)1 25 24 26 (25)1 (25)1 (24)1 (26)1 37 (35)1 37 (33)1 (2)2 46 (34)1 (5)2 32 (31)1 38 (37)1 (3)1 (3)1 (3)1 3 (3)1 3 (3)1 rRNAs 37 39 36 (34) (2)2 33 (32)1 (2)2 29 36 (29)1 33 33 (32)1 34 (32)1 34 (33)1 56 (32)1 34 (32)1 39 37 51 (36)1 Proteins 35 (16)1 16 32 (18)1 24 17 18 11 (2)3 (6)1 (6)1 (4)1 (4)1 25 (17)1 25 (17)1 30 (15)1 248 NA NA NA NA NA NA NA NA 3 3 179 149 NA 149 29 110 NA NA NA NA NA NA NA NA NA 37 (13)1 (9)3 26 (22)1 19 28 (14)1 (1)2 29 (27)1 21 23 (17)1 (5)3 NA NA NA 22 (3)3 22 (3)3 85 93 78 (>120) NA 36 44 45 44 44 NA NA NA ORFs (>100 codons) 22 (17)1 25 (18)1 (3)2 31 (19)1 28 (19) 17 (14)1 18 (14)1 17 (14)1 35 (14)1 18 (14)1 26 27 (18)1 (2)3 39 (24)1 (2)3 tRNAs Total mitochondrial genes encoding 6.22 8.8 5.89 37 NA NA 0.5 13.6 0.9 7.2 9.9 7.5 7.6 NA NA 6.5 7.18 20.3 9.9 NA NA NA NA Na NA 3.9 10.1 8.92 10.6 11.3 10.1 7.89 15.82 17.35 17.4 17.68 17.35 90.7 10.3 1.68 Coding sequence (%)4 NA NA NA NA NA NA 182 287 189 271 NA NA NA NA NA 600 NA NA 491 NA NA NA NA NA 444 1084 NA 441 370 NA NA NA NA 427 NA NA 463 NA RNA editing site (C?U) (continued) (Liu et al 2011) (Alverson et al 2011) (Goremykin et al 2009) (Terasawa et al 2007) (Tanaka et al 2012) (Tanaka et al 2012) (Sloan et al 2012b) (Sloan et al 2010) (Sloan et al 2012b) (Sloan et al 2012b) (Sloan et al 2012a) (Sloan et al 2012a) (Sloan et al 2012a) (Ogihara et al 2005) (Cui et al 2009) (Fang et al 2012) (Islam et al 2013) (Oda et al 1992) (Sugiyama et al 2005) (Notsu et al 2002) (Tian et al 2006) (Tian et al 2006) (Tian et al 2006) (Fujii et al 2010) (Fujii et al 2010) (Unseld et al 1997) (Kubo et al 2000) (Satoh et al 2004) (Zhang et al 2012) (Chang et al 2011) (Chang et al 2011) (Handa 2003) (Chang et al 2011) (Chang et al 2011) (Turmel et al 2003) (Alverson et al 2010) (Rodriguez-Moreno et al 2011) (Alverson et al 2010) (Chaw et al 2008) (Ma et al 2012) References Table 1: List of sequenced plant mitochondrial genomes Species and cultivars name, cytoplasm type, genome size, nucleotide composition and genes encoded by mitochondria Species are presented in alphabetical order based on their Latin name 302 M S ISLAM, B STUDER, I M MØLLER et al (Allen et al 2007) (Clifton et al 2004) (Allen et al 2007) (Allen et al 2007) (Allen et al 2007) NA NA NA NA NA 7.01 7.51 7.42 7.69 7.44 NA 121 NA NA NA (15)1 (10)3 (15)1 (15)1 (15)1 23 32 (15)1 23 22 17 (3)1 (3)1 3 (33)1 (33)1 (33)1 (33)1 34 37 34 43 34 NA: Data not available in the original paper Duplicated and triplicated genes were counted once Pseudo-genes encoding proteins Pseudo-tRNAs Coding sequences of proteins, rRNAs and tRNAs; ORFs, open reading frames 43.8 43.9 43.97 43.9 44.1 046 630 719 162 825 701 569 739 557 535 A-188, NA type (male-fertile) B37N, NB type (male-fertile) B37, CMS-C B37, CMS-S B37, CMS-T Zea mays tRNAs rRNAs Proteins Species name Table (continued) Cultivar/accession name and cytoplasm type Genome size (nt) G+C content (%) Total mitochondrial genes encoding ORFs (>100 codons) Coding sequence (%)4 RNA editing site (C?U) References Cytoplasmic male sterility in grass breeding 303 (ms Boro from an Indica rice cultivar ‘Chinsurah Boro II’) interacting with a recessive nuclear gene ‘rf rf’ (derived from the Japonica rice cultivar ‘Taichung 65’ bearing wild-type cytoplasm) resulted in male sterility Both the maintainer line (pollen fertile) and the restorer line of the sterile line were genotypically ‘Rf Rf’ Mitochondrial genes are inherited maternally and thus not adhere to Mendelian inheritance laws (Hanson and Bentolila 2004) As described above, the genetic basis of CMS is complex and varies between natural sources (Frank 1989, Taylor et al 2001) Cytoplasmic male sterility resulting from nuclear– mitochondrial genome incompatibility can be created by repeated backcrossing to introduce a nuclear genome into another genotype cytoplasm within the same or closely related species, even though both parents are fully fertile (Kaul 1988, Vedel et al 1994) When sterility-inducing cytoplasm of one genotype is crossed with fertility-inducing cytoplasm having non-restorer nuclear genes of another genotype, the offspring will be male sterile A male-sterile line of any donor inbred, which does not restore fertility, can be developed by repeated backcrossing to the recurrent parent followed by selection for the male-sterile phenotype (Rogers and Edwardson 1952) Any male-sterile line developed in this manner will contain the nuclear genome of the recurrent parent and the cytoplasmic genome of the non-recurrent parent The first cytoplasmic male sterile ryegrass was reported by Nitzsche (1971) in Italian ryegrass (Lolium multiflorum Lam.) and by Wit (1974) in perennial ryegrass Although the origin of CMS in Italian ryegrass is obscure, CMS in perennial ryegrass was initially collected from a population in a Dutch pasture (Wit 1974) The CMS system was developed by crossing a diploid interspecific F4 hybrid (perennial x Italian ryegrass) with an autotetraploid meadow fescue (Festuca pratensis L.) as female and male parent, respectively, after which CMS was maintained by repeated backcrosses to Lolium (Wit 1974) The male sterility was detected after intergeneric hybridization because the chromosome counts from the interspecific F4 hybrid appeared to be tetraploid In a similar way, CMS has been introgressed into a number of crop species which was thereafter made available for commercial usage (Leclercq 1969, Shinjyo 1969, Wit 1974, Connolly and Wright-Turner 1984, Horn and Friedt 1999) The genetic changes responsible for novel plant CMS systems (e.g Ogura cytoplasm in radish, Bo cytoplasm in rice, Owen cytoplasm in sugar beet and T-, C- and S-type cytoplasm in maize) have all arisen spontaneously and are not yet fully understood Likewise, the genetic basis of CMS systems in natural populations remains to be discovered (Ivanov and Dymshits 2007) Physiological Aspects of CMS The physiological understanding of CMS in plants is based on two theories First is the inability of mitochondria to meet the energy demand during pollen development (Levings 1993), and second is the premature programmed cell death (PCD) of tapetum cells in anthers (Balk and Leaver 2001) There is a 40-fold increase in mitochondria per cell in the tapetal cell layer of maize CMS-T anthers and a 20-fold increase in the sporogenous cells (Warmke and Lee 1977, 1978, Lee and Warmke 1979) Rapid increases in the number of mitochondria per cell such as those observed in tapetum have not been seen in any other maize cell types, including cells of developing ears The increase in the number of mitochondria in the tapetal cell layer suggests an increased energy demand during pollen development It was thought that a mutated mitochondrial gene product could be a serious impairment to pollen development in CMS-T maize 304 under conditions in which heavy demand for energy exists (Levings 1993) This is supported by the fact that sterility of some CMS sources (e.g barley, Hordeum vulgare L.) is induced by adverse growth conditions, especially at high temperatures (Abiko et al 2005) The Texas cytoplasm of maize can also be used to exemplify the second theory (Flavell 1974) A mutated mitochondrial gene, designated T-urf13, causes the T-type CMS in maize (Dewey et al 1987) The T-urf13 gene encodes a 13-kDa polypeptide (URF13) that is a component of the inner mitochondrial membrane (Dewey et al 1987, Wise et al 1987) and is uniquely associated with CMS (Forde et al 1978) An anther-specific molecule interacts with the URF13 protein to permeabilize the inner mitochondrial membrane, resulting in mitochondrial dysfunction and cell death (Levings 1993) Cell death in anther cells, particularly the tapetal cell layer which plays an essential role in pollen development (Goldberg et al 1993), is hypothesized to interfere with normal pollen development and lead to sterility (Levings 1993) The tapetum is the innermost cell layer that lines the anther locule, the pollen-containing chamber within the anther (Parish and Li 2010), and has a secretory function providing essential nutrients required for microspore and pollen grain development (Papini et al 1999, Gonzalez-Melendi et al 2008) It secretes enzymes that are used to release the young haploid microspores from the callose wall surrounded by the meiotic tetrad and provides precursors for the biosynthesis of the pollen outer wall (Bedinger 1992, Wu and Cheung 2000) The deterioration of the tapetum cell is highly regulated, irreversible and is associated with biochemical and physical changes in the cytoplasm, nucleus and plasma membranes (Nguyen et al 2009) As with other biological contexts, such as senescence and defence responses to a wide variety of pathogen and environmental stresses (Greenberg 1996), the tapetal PCD is a highly regulated process (Rogers 2006) and is perhaps triggered by mitochondrial signals (Hirsch et al 1997, Vianello et al 2007) Cellular triggers for PCD might induce H2O2 (Li et al 2012) or the release of cytochrome c from the mitochondria (Liu et al 1996) Balk and Leaver (2001) showed that mutated PET1-CMS sunflower mitochondria release cytochrome c into the cytosol of tapetal cells In the cytosol, cytochrome c activates a proteolytic cascade mediated by caspases (cysteinylaspartate proteases), leading to nuclear DNA degradation as part of tapetal PCD (Enari et al 1998) Thus, PCD leads to death of the microspores, which, in turn, results in the male-sterile phenotype Another physiological process associated with CMS is RNA editing (Gott 2003) Such RNA editing is a post-transcriptional modification process that changes the nucleotide sequence of primary transcripts (Gott 2003) Organelles of flowering plants mainly show cytidine-to-uridine mRNA editing (Gray 2003), leading to modification of the coded information in some amino acids or the generation of new initiation and/or termination codons Unedited mRNA molecules associated with genes that are crucial for the production of ATP (e.g atp9) have been reported to induce CMS in tobacco (Araya et al 1998) and wheat (Hernould et al 1993), which is consistent with the idea that RNA editing is essential for RNA maturation and optimal functionality of mitochondrial genes (Zabaleta et al 1996) Therefore, RNA editing may constitute an interesting tool for production of artificial male-sterile plants via expression of unedited mitochondrial gene transcripts An inducible antisense approach to restore fertility would make such a system highly useful for plant breeding (Takenaka et al 2008) M S ISLAM, B STUDER, I M MØLLER et al Genes Involved in CMS Systems Even though progress has been made in genetically and physiologically characterizing CMS systems, the mitochondrial genes causing CMS are still largely unknown (Chase 2007) Most of the well-characterized CMS systems show insertion/deletion or recombination events in the mitochondrial genome that lead to the formation of chimeric open reading frames (ORFs) (Ivanov and Dymshits 2007, Rieseberg and Blackman 2010) Chimeric ORFs involve mitochondrial gene-coding and gene-flanking sequences or sequences of unknown origin (Chase and GabayLaughnan 2004, Hanson and Bentolila 2004, Ivanov and Dymshits 2007) Indeed, at least 14 mitochondrial genes determining CMS of different species have been characterized as new ORFs (Chase and Gabay-Laughnan 2004, Hanson and Bentolila 2004), which are often associated with the ATP synthase subunit of the mitochondrial respiratory chain (Table 2) For some species, the mechanism how chimeric ORFs induce CMS has been hypothesized For the Ogura CMS of radish (Raphanus sativa L.), several flavonoid biosynthetic genes that repress the expression level of chalcone synthase (CHS) have been identified (Yang et al 2008b) The expression of CHS was specifically inhibited by orf138 prior to bud formation, indicating that the CMS phenotype in Ogura CMS of radish is related by the suppression of biosynthesis of flavonoid compounds Mitochondrial dysfunction causing CMS can also be associated with alterations in the expression of mitochondrial genes encoding subunits of the respiratory chain complexes (Ducos et al 2001) For example, in sorghum, altered gene expression of subunit of the mitochondrial ATP synthase (atp6) seems to be involved with CMS (Howard and Kempken 1997) Expression profiling of CMS candidate genes in the tapetum cells might also be critically important for understanding their involvement in CMS Generally, most mitochondrial genes that have been observed to be associated with or responsible for CMS are unrelated in sequence (Table 2) An exception might be orf222 from the nap-CMS and orf224 from the polima-CMS of rapeseed that exhibit 79% sequence identity (L’homme et al 1997) Genes Involved in Fertility Restoration Restorers of fertility (Rf) are nuclear genes that are able to suppress the mitochondrial genes causing CMS (Chase 2007) The genetic mechanisms involved in the restoration of pollen fertility are as diverse as the mutations in mitochondrial genes that cause CMS in plants (Table 2) In some systems, more than one major gene confers the fertility restoration In maize, a single gene (Rf3), two genes (Rf1 and Rf 2) and three genes (Rf4, Rf and Rf6) regulate fertility restoration in CMS-S (Hanson and Bentolila 2004), CMS-T (Levings and Dewey 1988) and CMS-C cytoplasm (reviewed by Sotchenko et al 2007), respectively A major gene (Rfg1) and two genes (Rfp1 and Rfp2) have been identified that regulate male fertility restoration in alternative CMS-inducing G€ ulzow (G) cytoplasm (B€ orner et al 1998) and Pampa (P) cytoplasm (Stracke et al 2003) of rye, respectively For both the PET1-cytoplasm in sunflower and T-cytoplasm in onion (Allium cepa L.), two unlinked restorer genes, designated Rf1 and Rf 2, are required for full restoration of male fertility (Levings 1993, Schnable and Wise 1998) For some other CMS systems, multiple independent genes have small cumulative effects that condition sterility (Mackenzie and Bassett 1987) This has been shown to be the case in natural populations of common bean (Phaseolus vulgaris L.) (Mackenzie and Bassett 1987) Cytoplasmic male sterility in grass breeding 305 Table 2: Description of the major cytoplasmic male sterility (CMS) systems in crop species Species and their cytoplasm name, origin of the CMS systems and its corresponding restorer gene(s), genes responsible for CMS and their differential expression between male-sterile and fertile mitochondrial genomes are listed Species name Designation of the CMS systems Origin of the CMS systems Beta vulgaris G Spontaneous RfG1 nad9 and coxII Owen Spontaneous Rf1 coxII, Norf246 Brassica juncea Hau Spontaneous NA atp6 Brassica napus Nap Intraspecific Rfn Ogura Rfo Polima (pol) Tour CMS3 Spontaneous (cybrids, a) Intraspecific Unknown Interspecific orf222/nad5c/ orf139 orf138 Rfp Rft NA pol-orf (orf224) orf263 coxIII and atp6 CMS89 Interspecific NA orfH522, orfC PET1 MSL Unknown Unknown Bo Rf1 NA NA NA Rf1 Helianthus annuus Petunia parodii CMS3688 Phaseolus vulgaris Raphanus sativa Sorghum bicolor Sprite Ogura A3 (IS1112C) Interspecific Mutagenesis Interspecific Intergeneric Spontaneous (cybrids, c) Interspecific Interspecific or inter-racial Spontaneous (cybrids, b) Intraspecific Spontaneous NA Milo (A1) Intraspecific 9E Intraspecific Secale cereale Pampa (P) Spontaneous Zea mays G€ulzow (G) C Spontaneous Spontaneous S T Spontaneous Spontaneous Lolium perenne Oryza sativa BT WA Fertility restorer gene Gene(s) responsible for respective CMS system Differential expression between male-sterile and fertile mitochondrial genomes References1 34.5-kDa protein encoded by coxII Transcript variation, 35kDa protein atp6/HindIII band pattern variation Transcript variation (Ducos et al 2001) (Bonhomme et al 1992) orfH522 NA atp6 and coxI atp9 B-atp6 1.4-kbp CMS-specific transcript Transcript variation 32-kDa protein 14, 18 and 38-kDa protein 16-kDa protein, 15-kDa protein 16-kDa protein NA Transcript variation 45-kDa protein Transcript variation Rf1 or Ifr1 Rf4 atp6-orf79 orf156, orfB 2.0-kbp transcripts Transcript variation Rf S-pcf 25-kDa protein Fr or Fr2 Rfo Rf3 and Rf4 Rf1 and Rf2 NA pvs-atpA atp6 orf107 Transcript variation Transcript variation Transcript variation coxI 38-kDa protein coxI 42-kDa protein Rfp1 and Rfp2 Rfg1 Rf4, Rf5 and Rf6 Rf3 Rf1 and Rf2 pol-r Transcript variation NA atp6, atp9 and coxII orf355 or orf77 T-urf13 NA Transcript variation (Bailey-Serres et al 1986) (Bailey-Serres et al 1986) (Dohmen and Tudzynski 1994) NA (Dewey et al 1991) 130-kDa protein (S2) 13-kDa protein (Zabala et al 1997) (Dewey et al 1987) (Senda et al 1991, Satoh et al 2004) (Wan et al 2008) (L’homme et al 1997) (L’homme et al 1997) (Landgren et al 1996) (Spassova et al 1994) (K€ohler et al 1991, Laver et al 1991) (Horn et al 1996) NA (Rouwendal et al 1992) (McDermott et al 2008) (Iwabuchi et al 1993) (Kazama et al 2008) (Seth et al 1996, Das et al 2010) Review by (Hanson 1991) (Johns et al 1992) (Makaroff et al 1989) (Tang et al 1999) NA: Data not available References given in the list are based on the identification of gene(s) responsible for CMS In contrast, in many species exhibiting CMS, a single nuclear Rf gene is sufficient to restore male fertility Examples are the wild abortive (WA) cytoplasm of rice (Ahmadikhah and Karlov 2006), the Pol cytoplasm of rapeseed (Singh and Brown 1993), the Ogura cytoplasm of radish (Koizuka et al 2003) and Japanese wild radish (Yasumoto et al 2008), the 3688 cytoplasm of Petunia (Edwardso and Warmke 1967) and the GO8063 cytoplasm of common bean (Mackenzie and Bassett 1987) (Table 2) Many of the cloned Rf genes are members of the pentatricopeptide repeat (PPR) protein family (Saha et al 2007) The PPR motif is a 35-amino acid sequence motif predicted to form a helix-turn-helix structure (Small and Peeters 2000) Such PPR proteins are widespread in plants, and 450 and 477 of these proteins have been identified in the Arabidopsis and the rice genomes, respectively (O’Toole et al 2008) The PPR protein gene, orf687, plays a role in restoring male fertility in the Kosena CMS of radish (Koizuka et al 2003) The gene Orf687 consists of 16 repeats of the 35-amino acid PPR motif An allele of this gene expressed in the Kosena cytoplasm possesses four substituted amino acids in the second and third repeat positions of the PPR, leading to a reduction in the CMS-associated mitochondrial protein level encoded by the orf125 gene Interestingly, PPR proteins are involved in mitochondrial transcription, possibly in RNA editing (Kotera et al 2005) It has been shown that post-transcriptional RNA editing plays a role in fertility restoration (Schnable and Wise 1998) Editing of RNA might change the length of CMS-associated ORFs by creating new start (AUG) and/or stop (UAA, UAG and UGA) codons, because the most prevalent example of editing in plant mitochondrial sequences is C-to-U (Schnable and Wise 1998) For example, editing of the mitochondrial atp6 gene of CMS sorghum increases fertility restoration (Howard and Kempken 1997) An exception to PPR protein-based restoration of fertility found in many plant species is the maize Rf2 gene, which 306 encodes an aldehyde dehydrogenase (Cui et al 1996, Liu et al 2001) and may be involved in removing reactive aldehydes formed as a result of increased ROS production (Møller 2001a) Development of Novel CMS Systems in Grass Species Plant breeders are continuously looking for new CMS sources Cytoplasmic male sterility can arise either spontaneously in natural populations due to mutations or artificially by means of distant crosses, cell hybridization, induced mutagenesis or gene engineering (Ivanov and Dymshits 2007) Cytoplasmic male sterility has been described in more than 300 plant species and interspecies hybrids (Ivanov and Dymshits 2007) Among these, there are about 175 plant species in which CMS has arisen spontaneously, the majority being dicotyledonous In the remaining species, CMS originated from interspecific crosses (reviewed by Kaul 1988) A few cases of induced mutagenesis are also known in barley, sugar beet, pearl millet and petunia (Harten et al 1985) In a number of plant species, there is no evidence for the occurrence of CMS from spontaneous mutations To induce CMS artificially, two techniques have been successfully employed in this regard, namely wide hybridization and induced mutagenesis (Kiang and Kavanagh 1996b, Gaue and Baudis 2007) Wide hybridization refers to intergenera or interspecies crosses, resulting in alloplasmic male sterility (Lacadena 1968) Kaul (1988) defined alloplasmy as the phenomenon wherein cells have the cytoplasm of one and the nucleus of another species As such, CMS is caused by the interruption of the communication between the cytoplasm and the nucleus Through wide hybridization, the CMS trait has been introduced in a number of species, including perennial ryegrass (Wit 1974, Connolly and Wright-Turner 1984), sunflower (Leclercq 1969, Horn and Friedt 1999) and rice (Shinjyo 1969) However, wide hybridization by conventional breeding can be time-consuming For example, the CMS trait in perennial ryegrass was introduced after nine generations of backcrossing taking over years (Connolly and Wright-Turner 1984, Kiang et al 1993) Another way to induce CMS in plant species is chemical and/ or physical mutagenesis (Dalvi et al 2010) Streptomycin sulphate, sodium azide, ethyl methyl sulphate (EMS) and di-ethyl sulphate are examples of chemical mutagens that have been employed in the creation of CMS genotypes (Dalvi et al 2010) For instance, the N-ethyl form of urea has been successfully used in perennial ryegrass to induce male sterility (Gaue and Baudis 2007) Physical mutagenesis can also be carried out by irradiation of seed material using short- (e.g 254 nm) and longwavelength UV light (e.g 300–400 nm) in combination with radiation such as X-ray or gamma irradiation (Dalvi et al 2010) Use of mutagenic agents to induce male sterility in plants can be much less time-consuming than wide hybridization (Rowell and Miller 1971, Adugna et al 2004) Brief descriptions and associated citations of major cytoplasm types, their origin and regulating genes responsible for induction of sterility and restoration of fertility are given in Table Future Prospects in Mitochondrial Genetics and Genomics To genetically dissect CMS, sequencing of mitochondrial genomes and understanding of genome structure and organization are crucial Recent advances in next- and third-generation sequencing technologies, such as single-molecule sequencing, will provide an opportunity to optimize sequencing strategies, to M S ISLAM, B STUDER, I M MØLLER et al increase depth of sequence coverage and to achieve longer sequencing reads (Treffer and Deckert 2010, Thompson and Milos 2011) This, in turn, will likely greatly facilitate the rather complex assembly of highly repetitive mitochondrial genomes, which will in combination with improved bioinformatics pipelines for genome annotation increase the number of fully assembled mitochondrial genomes that can be compared More comprehensive comparative mitochondrial genome analyses will facilitate the identification of mitochondrial sequence polymorphisms associated with CMS and further elucidate to what extent the mitochondrial genome organization and gene content is conserved within and between plant species (Handa 2003) The integration of these comparative genomic approaches with additional transcriptomics and proteomics data may provide further evidence for interesting mitochondrial target genes and regions associated with CMS (Rui-Hong et al 2010) In a long-term perspective, epigenetic regulation (including DNA methylation, micro- and non-coding RNAs and RNA editing patterns) of annotated genes and novel ORFs will likely be the key to unravelling CMS mechanisms and mode of action in higher plants Likewise, by focusing on the non-coding genome regions, comparative mitochondrial genome analysis will allow for a clearer understanding of mitochondrial genome evolution in higher plants as large non-coding sequences of mitochondrial genomes are species-specific (Handa 2003) Current Forage and Turf Grass Breeding Schemes Because of the highly effective SI system, breeding of allogamous forage crop species is largely based on open pollination (Souza 2011) In general, two breeding strategies have been used – population breeding and synthetic breeding Population breeding is the direct result of continuous population improvement through recurrent selection, while synthetic breeding refers to crosses among a restricted number of selected parents followed by multiplication through repeated open pollination in isolation Both strategies only partially exploit the genetically available heterosis and result in panmictic populations consisting of highly heterozygous genotypes (Posselt 2010) Superior individuals are either selected directly based on their phenotype (phenotypic selection) or on the performance of their progeny (genotypic selection) Phenotypic selection is typically based on the evaluation of individual plants in spaced plant nurseries (mass selection) or the evaluation of vegetative replicates (clones) planted in rows (clonal selection) A more efficient exploitation of heterosis can be achieved at the population and single plant level In populations, heterotic groups can be utilized to identify specific pairs of heterotic groups expressing a high general combining ability (Aguirre et al 2012) and, consequently, high hybrid performance (Posselt 2010) The existence of such heterotic patterns has been attributed to the possibility that populations of divergent backgrounds might have unique allelic diversity that may have originated from founder effects, genetic drift or through the accumulation of unique diversity by mutation or selection (Acquaah 2012) However, the genetic diversity in perennial ryegrass varieties and accessions is found within rather than between varieties or accessions (K€ olliker et al 1999) A recent comprehensive analysis of the population structure of European elite perennial ryegrass varieties identified two groups representing maritime and continental varieties, respectively (Brazauskas et al 2011) These two geographically distinct groups of accessions (gene pools) represent an excellent starting point for reciprocal recurrent selection to establish continuously improved heterotic pools Cytoplasmic male sterility in grass breeding At the single plant level, classical single-cross or double-cross hybrids such as in maize can be generated (Hayward 1988, Scotti and Brummer 2010) In a strict sense, the parents of a single-cross hybrid are two diploid highly inbred lines However, inbred line development in allogamous forage and turf grass species is often impaired due to SI and inbreeding depression (Baumann et al 2000) Therefore, the production of singlecross hybrids from heterozygous parent genotypes is a more likely scenario for outbreeding grass species, resulting in segregating F1 populations, which are comparable to double-cross hybrids in maize Hybrid breeding schemes maximizing heterosis at both population and single plant levels require pollination control to achieve targeted crossing between gene pools or specific genotypes (Kempe and Gils 2011) Mechanical emasculation is not feasible in forage and turf grass species due to their small flowers Moreover, chemical sterilization of plants might raise public concerns (Duvick 1959, Mahajan and Nagarajan 1998, Kempe and Gils 2011) Thus, CMS remains the only male sterile-based option for reliable control of pollination in forage and turf grass species (Duvick 1959) 307 be used as parental lines for the production of F1 hybrids (Veilleux 1994) In perennial ryegrass, it has been possible to obtain DHs from anther cultures (Olesen et al 1988) As it is difficult to produce fertile homozygous perennial ryegrass lines by self-pollination, it is of great interest to utilize DH lines in hybrid breeding programmes However, several obstacles such as a the high percentage of albino plants and male sterility of plants regenerated from anther culture limit efficient DH production in perennial ryegrass Olesen et al (1988) reported that the formation of green plants is genotype specific, and consequently, the occurrence of albino plants and male sterility has limited the use of DH induction in plant breeding of this species (Kumari et al 2009) Nevertheless, single genotypes regenerating a high percentage of green plants after DH induction can be used in experimental crossings within heterotic groups enabling the identification of desirable DH phenotypes Alternatively, combining the current breeding strategies with genomic selection and DH application may constitute the tools of choice for the implementation of efficient hybrid forage grass breeding systems Summary and Conclusions Perspectives of CMS as a Breeding Tool for Forage and Turf Grasses For a wide range of crop species, CMS is used as an efficient tool for hybrid seed production (Duvick 1959, Adugna et al 2004, Havey 2004, Cheng et al 2007) For several reasons, CMS offers promising possibilities as a low cost method for improved exploitation of heterosis in allogamous forage and turf grass species (Duvick 1959) Firstly, CMS represents a tool that can be used to control pollination at both the population and single plant level, independent of the degree of heterozygosity Secondly, as biomass and not seed is the primary yield target, pollen fertility does not need to be restored in the F1 generation For these reasons, incorporation of a CMS-based breeding scheme can simplify forage grass improvement There are, nevertheless, challenges inherent in maintaining the CMS trait in highly heterozygous outbreeding species (Geiger and Miedaner 2009) To maintain non-restorer alleles in the nuclear genome, the maintainer line must be self-pollinated, which can be challenging due to SI and strong inbreeding depression (Baumann et al 2000) Consequently, maintaining CMS in highly heterozygous nuclear backgrounds is challenging and might result in partial restoration of fertility (Geiger and Miedaner 2009) For similar reasons, it has been difficult to genetically improve the CMS and maintainer lines by traditional breeding With the ultimate goal to produce classical single-cross hybrids, inbreeding of the parental genotypes would be required A sufficient degree of homozygosity in CMS lines can only be achieved by repeated back-crossing to a maintainer line which is laborious, time-consuming and requires the introduction of selffertility genes into the germplasm (Geiger 1972, Connolly and Wright-Turner 1984, Kiang et al 1993) Doubled haploid (DH) induction is a widely used breeding tool for inbred line development The production of haploids followed by genome duplication can greatly accelerate the development of inbred lines (Dunwell 2010) There are several methods available to obtain DH plants, of which in-vitro anther or isolated pollen cultures are the most effective (as reviewed by Germana 2011) Doubled haploid lines are developed mainly to achieve 100% homozygosity in diploid or allopolyploid species without the need for several generations of inbreeding Homozygous DH lines can then Cytoplasmic male sterility is a maternally inherited genetic mechanism in higher plants that affects the production or functionality of pollen leading to male sterility The genetic mechanisms causing CMS are very diverse in plants and vary even within the same species Therefore, it is difficult to find common characteristics between different CMS systems that would allow for the identification of candidate genes or regulatory pathways Recent technological advancements in sequencing whole organellar genomes will likely provide interesting possibilities for elucidation of the role of mitochondrial genes in CMS through comparative sequence analysis of mitochondrial genomes isolated from CMS plants and their corresponding male-fertile maintainer genotypes However, identification of the polymorphisms that control CMS still remains difficult as numerous nucleotide polymorphisms and small- and large-scale rearrangements are found during such mitochondrial genome comparative analyses Further advancements in sequencing technologies and assembly strategies will lead to an increased number of CMS and non-CMS mitochondrial genomes that can be compared, and the integration of additional transcriptomic and proteomic data will likely be useful for unravelling the underlying genetics of CMS systems The mechanisms and genes involved in fertility restoration seem to be more conserved, and thus, in-depth characterization of the PPR gene family might lead to cloning of fertility restorer genes in novel CMS systems Likewise, characterization of the genetic mechanisms of CMS systems and the corresponding fertility restoration may also provide new insights for the dissection of complex mitochondrial and nuclear genome interactions Cytoplasmic male sterility is a promising tool for the implementation of hybrid breeding schemes in forage grasses In contrast to CMS systems of other crop species, there is no need to restore fertility in forage grass species, as biomass and not seed is of economic interest However, the maintenance of CMS in a breeding programme will remain a major challenge in allogamous, highly heterozygous forage grass species The development of molecular marker systems for the maintenance of CMS in heterogeneous genetic backgrounds might prove useful In this regard, the use of CMS for the implementation of a hybrid system in forage grass species raises several critical questions: 308 (i) Does one need to inbreed first, or can CMS be readily used in heterozygous populations? (ii) If inbreeding is necessary, what is the optimal degree of homozygosity for return on investment (i.e low cost and optimal performance of hybrids)? and (iii) What is the best strategy for improvement of genetic backgrounds of CMS plants? 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PLoS ONE 7, e41861 Zubko, M K., 2004: Mitochondrial tuning fork in nuclear homeotic functions Trends Plant Sci 9, 61—64  ... 2011) and the use of SI in inbred line development (Thorogood et al 20 05) , breeding efforts towards the implementation of hybrid breeding systems in forage and turf grasses have intensified Starting... NA NA 40.9 NA 44 .5 G+C content (%) 340 036 426 000 413 000 000 000 000 000 52 8 52 6 1 05 244 258 11 318 253 728 427 361 429 422 452 452 7 15 001 58 0 608 59 7 52 0 669 51 5 673 7 35 0 45 982 833 414 903... parents of a single-cross hybrid are two diploid highly inbred lines However, inbred line development in allogamous forage and turf grass species is often impaired due to SI and inbreeding depression