Research Article

Sequence Characterized Amplified Regions Linked with Apomictic Mode of Reproduction in Four Different Apomictic Cenchrus Species  

Suresh Kumar1,2 , Sheena Saxena1
1 Division of Crop Improvement, Indian Grassland and Fodder Research Institute, Jhansi, 284003, India
2 Division of Biochemistry, Indian Agricultural Research Institute, New Delhi, 110012, India
Author    Correspondence author
Molecular Plant Breeding, 2016, Vol. 7, No. 8   doi: 10.5376/mpb.2016.07.0008
Received: 16 Oct., 2015    Accepted: 30 Nov., 2015    Published: 06 Jan., 2016
© 2016 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Suresh Kumar, and Sheena Saxena, 2016, Sequence characterized amplified regions linked with apomictic mode of reproduction in four different apomictic Cenchrus species, Molecular Plant Breeding, 7(08): 1-14 (doi: 10.5376/mpb.2016.07.0008)

Abstract

The genus Cenchrus comprises tropical and sub-tropical forage grasses, many of which reproduce by aposporous apomixis. This mode of reproduction hampers genetic improvement of the species through conventional breeding; however, it may facilitate the fixing of heterosis in a hybrid. Apomixis can be deployed in plant breeding to maintain hybrid vigour over the generations, thereby accelerating the breeding process. Markers for apospory have been identified in grasses, but we have identified conserved genomic regions and apomixis-specific AFLP markers in eight Cenchrus spp. and converted them into sequence characterised amplified regions (SCARs). Seventeen of 94 identified polymorphic AFLP markers were located in the apospory-specific genomic region and were converted into SCAR markers. Only four (23.5%) were successfully converted into SCARs linked with an apomictic mode of reproduction in Cenchrus spp. The SCAR markers were validated in an F2 population of C. ciliaris consisting of 48 apomictic and 38 sexual individuals. The SCARs were conserved across the four apomictic Cenchrus spp. with >98% sequence homology. In silico mapping of the SCARs based on sequence homology indicated synteny with segments of chromosome 12 of rice and chromosome 5 of maize. These markers would be very useful for genetic/molecular analyses of apomixis, comparative mapping studies and marker-assisted breeding of Cenchrus.

Keywords
AFLP; apomixis; Cenchrus ciliaris; molecular marker; SCAR; sexual reproduction

Introduction
The genus Cenchrus includes important forage grasses of tropical and sub-tropical regions of the world. It is a member of the tribe Paniceae, sub-family Chloridoideae of the family Poaceae. Many Cenchrus species are resilient to harsh environmental conditions including acute erosion, drought, and nutrient-depleted soils and they serve as valuable bioenergy feedstock (Ziegler et al., 2000). Cenchrus species were introduced into India from Australia and Africa, and a few have become important component of the Dichanthium-Cenchrus-Lasiurus grasslands of India. Of the eight Cenchrus species available in India, only four (C. ciliaris L. syn. Pennisetum ciliare L. Link, C. glaucus Mudaliar and Sundaraj, C. pennisetiformis Hochst and Steud. ex Steud and C. setigerus Vahl.) are grown in sown pastures. The remaining four species (C. biflorus Roxb., C. prieurii Kunth, C. echinatus L. and C. myosuroides Kunth) are either grown in limited pockets under high moisture conditions (Chandra and Dubey, 2010) or they are maintained as genetic resources for basic and applied studies. C. ciliaris, C. setigerus (Fisher et al., 1954) and C. glaucus (Shanthamma, 1982) are well known to reproduce through apomixis. Sexual mode of reproduction has been reported in C. prieurii, C. echinatus (Gupta et al., 2001) and C. myosuroides (Brown and Emery, 1958); however, no information on the mode of reproduction in C. pennisetiformis and C. biflorus could be found in the available literature.
 
Many of the Cenchrus species reproduce through apomixis (asexual reproduction through seed) wherein progenies are genetically identical to the mother plant. Apomixis is a unique, naturally-occurring mode of reproduction which produces seed without meiosis or fertilisation of the egg cell. It has been reported in 35 angiosperm families and in over 300 species, 75% of which belong to the Poaceae, Rosaceae and Compositae families. It occurs mostly in polyploid genotypes (Richards, 1986; Carman, 1997). The existence of a single dominant locus for apomeiosis and parthenogenesis has been reported in aposporous grasses including Pennisetum/Cenchrus (Sherwood et al., 1994; Ozias-Akins et al., 1998), Brachiaria (Pessino et al., 1997) and Paspalum (Martinez et al., 2001) as well as in a diplosporous grass Tripsacum (Grimanelli et al., 1998). Marker studies indicate that the apospory-specific genomic region (ASGR) in Pennisetum/Cenchrus is physically large, hemizygous and heterochromatic (Roche et al., 2002; Akiyama et al., 2004; Goel et al., 2003, 2006). Gametophytic apomixis in Pennisetum squamulatum and Cenchrus ciliaris is apparently controlled by the ASGR which is conserved and macrosyntenic between these species (Conner et al., 2008).
 
Rare obligate sexual plants of buffel grass (Cenchrus ciliaris L.) have also been identified (Bray, 1978; Kumar et al., 2010a). Buffel grass is protogynous in nature, where cross-pollination of the sexual plant with pollen from an apomictic plant leads to the production of either facultative or obligate apomictic genotypes. Therefore, over a period of time, the apomictic individuals will outnumber the sexual ones (Kumar et al., 2010a). Apomixis is a well-known limitation to genetic improvement of a species through conventional breeding practices; however, it can facilitate fixing of the recombinant genotype since no recombination/segregation takes place in subsequent generations. Hence, apomixis has been attractive to plant breeders as a tool for preserving desirable combinations of genes which otherwise are broken by genetic recombination during sexual reproduction (Savidan, 2000).
 
Maheshwari et al. (1998) elaborated different strategies for engineering apomixis in crop plants. If apomixis could be engineered in plants with a sufficient degree of flexibility, its impact on agriculture could be overwhelming by: immediately fixing of a genotype in a desired plant; revolutionising breeding procedures by moving from family-based strategies to individual plant-based strategies; facilitating the development, mass production and maintenance of elite parental lines and their derived hybrids; development of hybrid varieties for most of the crop species; providing ecologically sound protection from horizontal transfer of transgenes by the introduction of autonomous apomixis into male-sterile varieties (Bhat et al., 2005). Apomixis has potential applications in maintaining hybrid vigour over the generations (Koltunow and Tucker, 2008), but it has not yet been transferred to a crop species, mainly because the gene(s) for apomixis remain to be identified (Savidan, 2001; Spillane et al., 2004; Kandemir and Saygili, 2015).
 
Molecular markers linked with apospory have been reported in several grasses including C. ciliaris (Gustine et al., 1997; Ozias-Akins et al., 1998; Pessino et al., 1998; Martinez et al., 2003; Dwivedi et al., 2007). A sequence characterised amplified region (SCAR) linked with a sexual mode of reproduction was also reported in C. ciliaris (Kumar et al., 2010b). The SCAR marker (CcSex-260) produced a specific band in an obligate sexual C. ciliaris plant, but no band in obligate apomictic plants. The CcSex-260 marker could detect a sexual mode of reproduction in C. ciliaris, but a robust and reliable marker for the apomictic mode of reproduction was still desired (Kumar et al., 2010b). In the present study, we aimed at development of a simple, robust and reliable molecular marker that is tightly linked with the apomictic mode of reproduction in Cenchrus and that could be used for genetic and molecular analyses of apomixis. Since molecular markers are not influenced by environmental factors or the developmental stage of plant, therefore they can also be used in breeding programmes for screening of segregating populations.
 
The main objectives of the present study were to generate genomic profile of the eight Cenchrus spp. using amplified fragment length polymorphism (AFLP), to identify conserved genomic regions associated with an apomictic mode of reproduction in these species and to identify efficient molecular marker(s) linked with apomictic mode of reproduction. We successfully developed four SCAR markers linked with the apomictic mode of reproduction by genetic profiling of the eight Cenchrus spp. These SCAR markers could be very useful for genetic/molecular analyses of apomixis, for fine mapping of the apomixis locus and for marker-assisted screening of segregating populations resulting from inter-specific hybridisation in Cenchrus.
 
1 Results
1.1 Mode of reproduction in Cenchrus species
Examination of cleared pistils of the Cenchrus spp. revealed the presence of an eight-nucleated embryo sac containing 3 antipodal cells (Figure 1 a, b) in C. biflorus, C. echinatus, C. myosuroides and C. prieurii, indicating a sexual mode of reproduction in these species. Cleared pistils of C. ciliaris, C. glaucus, C. pennisetiformis and C. setigerus, by contrast, showed the typical four-nucleated (one egg cell, two synergids and one polar nucleus) embryo sacs without antipodal cells (Figure 1 c, d) indicating the existence of apospory (apomictic mode of reproduction) in these species (Table 1).

 

 

Figure 1 Representative pictures of sexual and apomictic embryo sacs in Cenchrus species. a Sexual embryo sac of Cenchrus biflorus. b Sexual embryo sac of Cenchrus echinatus. c Apomictic embryo sac of Cenchrus pennisetiformis. d Apomictic embryo sac of Cenchrus setigerus. Eg = Egg cell, Sg = synergid

  

 

Table 1 Ploidy status and mode of reproduction of the different Cenchrus spp.

Note : References: (1) Chandra and Dubey (2009), (2) Gould (1968), (3) Ahsan et al. (1994), (4) Fisher et al. (1954), (5) Gupta et al. (001), (6) DeLisle (1964), (7) Gould and Soderstrom (1974), (8) Shanthamma (1982), (9) DeLisle (1963), (10) Brown and Emery (1958). (11) Mullay and Leelamma (1956), (12) Crins (1991)

  

1.2 DNA profiles of Cenchrus species
AFLP analysis of the eight Cenchrus spp. showed an average of 114.26 bands per primer combination and a maximum of 253 bands were observed with a primer combination E-ACA+M-CTC. Of the 5713 bands produced by 50 primer combinations, 4984 (87.24%) were polymorphic in the eight species (bands were present/absent at least in one of the Cenchrus spp.). Forty-four primer combinations produced a total of 94 bands that were polymorphic between the apomictic (present in all four apomictic) and sexual (absent in all four sexual) Cenchrus spp. (Figure 2).

 

 

Figure 2 Representative AFLP profiles of eight Cenchrus species as observed with two different primer combinations. Cb= C. biflorus, Cc= C. ciliaris, Ce= C. echinatus, Cg= C. glaucus

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