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

Genetics and biology of cytoplasmic male sterility and its applications in forage and turf grass breeding 1 Department of Molecular Biology and Genetics, Science and Technology, Aarhus U

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

1

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 2 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

ge-nomics to identify candidate genes and causative polymorphisms

associ-ated 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 F1hybrid 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

pollina-tion during seed producpollina-tion (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 mitochon-drial 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 altera-tions involve single-nucleotide polymorphisms (SNPs), large insertions or deletions (InDels), variation in the content of repeti-tive 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 do 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 restora-tion of fertility makes it difficult to find a consensus mechanism

in the genetics and biology of CMS for applying CMS and fer-tility 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-incompat-ibility (SI) systems (Cornish et al 1979), which promote cross-pollination 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

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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

sys-tems 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

ster-ile 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

rye-grass by interspecific (Wit 1974) and intergeneric matings

(Con-nolly 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

pro-cess (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

develop-ment (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

func-tion of mitochondria in plants, the main aims of this review are

to (i) describe the genetics and biology of different CMS

sys-tems in flowering plants; and (ii) evaluate prospects of

compara-tive mitochondrial genomics for the identification of candidate

genes and causative polymorphisms of CMS in forage grass

spe-cies Thereafter, the specificities, obstacles and potentials of

CMS as a breeding tool for hybrid breeding in forage grass

spe-cies will be discussed

Origin of Mitochondria in Plants

Cytoplasmic genomes, such as mitochondrial and chloroplast

ge-nomes, are probably the remnants of bacteria that were engulfed

by another bacterium approximately 2 billion years ago (Gray

et al 1999, Lang et al 1999) The‘endosymbiotic theory’

postu-lates 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,

even-tually 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

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, chlorop-lasts and mitochondria (Unseld et al 1997) The chloropchlorop-lasts, which convert solar radiation into chemical energy, and mito-chondria, 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 cou-pled 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 func-tionality 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 1 (MGP1), a gene essential for pollen formation, which encodes the FAd sub-unit 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 mito-chondrial genomes The size of sequenced plant mitomito-chondrial genomes ranges from 187 kbp in liverwort (Marchantia polymorpha L.) (Oda et al 1992) to 11 318 kbp in natural pop-ulations of Silene conica L., a dicotyledonous seed plant exhibit-ing a high mitochondrial mutation rate and abundant non-codexhibit-ing DNA content (Sloan et al 2012b)

Characteristic for angiosperm mitochondrial genomes is the high degree of variation in terms of size and genome organiza-tion, 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 (Wolsten-holme 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-ome in the B37 nuclear background) gengen-ome 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

homo-logy to each other, it has been hypothesized that they were

inde-pendently 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

pro-tein 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

ge-nomes both within and between species (Kubo et al 2011)

These comparisons include genome size, the proportion of

repet-itive 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

non-coding 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

liver-wort 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

ge-nomes (Unseld et al 1997, Notsu et al 2002, Clifton et al

2004)

A slightly higher degree of mitochondrial genome

conserva-tion 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

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 Depart-ment of Agriculture) (Zabala et al 1997), and sugar beet (Satoh

et al 2004)

These comparative genomic approaches can be comple-mented with transcriptomic and proteomic data to provide fur-ther evidence for the causative CMS sequence polymorphism (Rui-Hong et al 2010) With the aim to characterize differen-tially 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 suc-cessfully 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<5% of the mitochondrial proteins, and the vast major-ity (>95%) of the genetic information required for their biogene-sis 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-to-nucleus regulation, known as retrograde regulation (Yang et al 2008a) Anterograde regulation, which controls the flow of infor-mation 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 met-abolic function during chloroplast biogenesis and redox regula-tion (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 mito-chondrial 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-induc-ing cytoplasm and a homozygous recessive nuclear gene caussterility-induc-ing pollen sterility, where the nuclear gene was ineffective in ‘nor-mal (‘nor-male-fertile)’ cytoplasm Shinjyo (1969) reported, for instance, that a male sterility-inducing cytoplasm of rice

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(ms Boro from an Indica rice cultivar‘Chinsurah Boro II’) inter-acting with a recessive nuclear gene ‘rf rf’ (derived from the Japonica rice cultivar ‘Taichung 65’ bearing wild-type cyto-plasm) 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 do 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 fer-tility, can be developed by repeated backcrossing to the recurrent parent followed by selection for the male-sterile phenotype (Rog-ers 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 chro-mosome 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 cyto-plasm in sugar beet and T-, C- and S-type cytocyto-plasm in maize) have all arisen spontaneously and are not yet fully understood Likewise, the genetic basis of CMS systems in natural popula-tions 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 tape-tum 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

Genome size

Coding sequen

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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

mem-brane (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

dys-function 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

hypothe-sized 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

pro-vides 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

bio-logical 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

pri-mary 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

co-dons 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

(Her-nould 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

artifi-cial male-sterile plants via expression of unedited mitochondrial

gene transcripts An inducible antisense approach to restore

fer-tility would make such a system highly useful for plant breeding

(Takenaka et al 2008)

Genes Involved in CMS Systems

Even though progress has been made in genetically and physio-logically 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 Gabay-Laughnan 2004, Hanson and Bentolila 2004, Ivanov and Dym-shits 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 6 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 associ-ated with or responsible for CMS are unrelassoci-ated 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 sup-press 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 5 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) cyto-plasm (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)

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

Japa-nese wild radish (Yasumoto et al 2008), the 3688 cytoplasm of

Petunia (Edwardso and Warmke 1967) and the GO8063

cyto-plasm of common bean (Mackenzie and Bassett 1987) (Table 2)

Many of the cloned Rf genes are members of the

pentatrico-peptide 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

pro-teins have been identified in the Arabidopsis and the rice

ge-nomes, respectively (O’Toole et al 2008) The PPR protein

gene, orf687, plays a role in restoring male fertility in the

Ko-sena 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 mito-chondrial protein level encoded by the orf125 gene Interest-ingly, 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 res-toration (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 mito-chondrial sequences is C-to-U (Schnable and Wise 1998) For example, editing of the mitochondrial atp6 gene of CMS sor-ghum 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

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 mitochon-drial genomes are listed

Species name

Designation of the CMS systems

Origin of the CMS systems

Fertility restorer gene

Gene(s) responsible for respective CMS system

Differential expression between male-sterile and fertile mitochondrial genomes References 1

Beta vulgaris G Spontaneous RfG1 nad9 and coxII 34.5-kDa protein encoded

by coxII

(Ducos et al 2001) Owen Spontaneous Rf1 coxII, Norf246 Transcript variation,

35-kDa protein

(Senda et al 1991, Satoh

et al 2004) Brassica juncea Hau Spontaneous NA atp6 atp6/HindIII band pattern

variation

(Wan et al 2008) Brassica napus Nap Intraspecific Rfn orf222/nad5c/

orf139

Transcript variation (L ’homme et al 1997) Ogura Spontaneous

(cybrids, a)

Rfo orf138 1.4-kbp CMS-specific

transcript

(Bonhomme et al 1992) Polima (pol) Intraspecific Rfp pol-orf (orf224) Transcript variation (L ’homme et al 1997) Tour Unknown Rft orf263 32-kDa protein (Landgren et al 1996) Helianthus annuus CMS3 Interspecific NA coxIII and atp6 14, 18 and 38-kDa

protein

(Spassova et al 1994) CMS89 Interspecific NA orfH522, orfC 16-kDa protein, 15-kDa

protein

(K €ohler et al 1991, Laver et al 1991) PET1 Interspecific Rf1 orfH522 16-kDa protein (Horn et al 1996)

Unknown Interspecific NA atp6 and coxI Transcript variation (Rouwendal et al 1992) Unknown Intergeneric NA atp9 45-kDa protein (McDermott et al 2008) Oryza sativa Bo Spontaneous

(cybrids, c)

Rf1 B-atp6 Transcript variation (Iwabuchi et al 1993)

BT Interspecific Rf1 or Ifr1 atp6-orf79 2.0-kbp transcripts (Kazama et al 2008)

WA Interspecific or

inter-racial

Rf4 orf156, orfB Transcript variation (Seth et al 1996, Das

et al 2010) Petunia parodii CMS3688 Spontaneous

(cybrids, b)

Rf S-pcf 25-kDa protein Review by (Hanson

1991) Phaseolus vulgaris Sprite Intraspecific Fr or Fr2 pvs-atpA Transcript variation (Johns et al 1992) Raphanus sativa Ogura Spontaneous Rfo atp6 Transcript variation (Makaroff et al 1989) Sorghum bicolor A3 (IS1112C) NA Rf3 and

Rf4

orf107 Transcript variation (Tang et al 1999) Milo (A1) Intraspecific Rf1 and

Rf2

coxI 38-kDa protein (Bailey-Serres et al.

1986) 9E Intraspecific NA coxI 42-kDa protein (Bailey-Serres et al.

1986) Secale cereale Pampa (P) Spontaneous Rfp1 and

Rfp2

pol-r Transcript variation (Dohmen and Tudzynski

1994)

G €ulzow (G) Spontaneous Rfg1 NA NA NA Zea mays C Spontaneous Rf4, Rf5

and Rf6

atp6, atp9 and coxII

Transcript variation (Dewey et al 1991)

S Spontaneous Rf3 orf355 or orf77 130-kDa protein (S2) (Zabala et al 1997)

T Spontaneous Rf1 and

Rf2

T-urf13 13-kDa protein (Dewey et al 1987)

NA: Data not available.

1

References given in the list are based on the identification of gene(s) responsible for CMS.

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

engineer-ing (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

spe-cies As such, CMS is caused by the interruption of the

commu-nication 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

gener-ations of backcrossing taking over 9 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

sul-phate, 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

long-wavelength 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

associ-ated citations of major cytoplasm types, their origin and

regulat-ing genes responsible for induction of sterility and restoration of

fertility are given in Table 2

Future Prospects in Mitochondrial Genetics and

Genomics

To genetically dissect CMS, sequencing of mitochondrial

ge-nomes 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

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 pipe-lines for genome annotation increase the number of fully assem-bled mitochondrial genomes that can be compared More comprehensive comparative mitochondrial genome analyses will facilitate the identification of mitochondrial sequence polymor-phisms associated with CMS and further elucidate to what extent the mitochondrial genome organization and gene content is con-served within and between plant species (Handa 2003)

The integration of these comparative genomic approaches with additional transcriptomics and proteomics data may provide fur-ther 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 edit-ing 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– popu-lation breeding and synthetic breeding Popupopu-lation breeding is the direct result of continuous population improvement through recur-rent 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 geno-types (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 attrib-uted 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 recur-rent selection to establish continuously improved heterotic pools

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

How-ever, inbred line development in allogamous forage and turf

grass species is often impaired due to SI and inbreeding

depres-sion (Baumann et al 2000) Therefore, the production of

single-cross hybrids from heterozygous parent genotypes is a more

likely scenario for outbreeding grass species, resulting in

segre-gating F1 populations, which are comparable to double-cross

hybrids in maize

Hybrid breeding schemes maximizing heterosis at both

popu-lation and single plant levels require pollination control to

achieve targeted crossing between gene pools or specific

geno-types (Kempe and Gils 2011) Mechanical emasculation is not

feasible in forage and turf grass species due to their small

flow-ers 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)

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 F1generation

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

hetero-zygous 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

self-fertility 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

develop-ment The production of haploids followed by genome

duplica-tion 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%

homozygos-ity in diploid or allopolyploid species without the need for

sev-eral generations of inbreeding Homozygous DH lines can then

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 pro-grammes However, several obstacles such as a the high percent-age 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 phe-notypes 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

Cytoplasmic male sterility is a maternally inherited genetic mechanism in higher plants that affects the production or func-tionality of pollen leading to male sterility The genetic mecha-nisms 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 orga-nellar genomes will likely provide interesting possibilities for elucidation of the role of mitochondrial genes in CMS through comparative sequence analysis of mitochondrial genomes iso-lated from CMS plants and their corresponding male-fertile maintainer genotypes However, identification of the polymor-phisms that control CMS still remains difficult as numerous nucleotide polymorphisms and small- and large-scale rearrange-ments 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 char-acterization of the PPR gene family might lead to cloning of fer-tility 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 imple-mentation of hybrid breeding schemes in forage grasses In con-trast 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 alloga-mous, highly heterozygous forage grass species The develop-ment 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:

(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

back-grounds of CMS plants?

Further research is needed to address these questions and to

evaluate how CMS systems, in combination with genomic

selec-tion, can be used to improve the efficiency of CMS-based hybrid

breeding programmes to fully exploit heterosis in the F1

genera-tion In the long-run, CMS-based hybrid breeding systems of

forage and turf grasses may be used to facilitate the development

and release of new hybrid varieties with higher yield, improved

nutritional value and better tolerance towards biotic and abiotic

stresses for mitigating global needs for food, feed and biomass

Acknowledgement

This study was funded by the Danish Ministry of Food, Agriculture and

Fisheries (Project number 3304-FVFP-08).

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