The Horticulture Journal
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INVITED REVIEWS
Cytoplasmic Male Sterility in Eggplant
Md Mizanur Rahim KhanShiro Isshiki
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2016 Volume 85 Issue 1 Pages 1-7

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Abstract

Cytoplasmic male sterility (CMS) is a useful system to produce hybrid seeds in a variety of crop species. In eggplant, CMS systems were developed utilizing the cytoplasms of six wild Solanum species by repeated backcrossings. These CMS systems were classified into two types. The first one is anther indehiscent-type sterility in the CMS systems of eggplant with the cytoplasms of Solanum kurzii Brace & Prain, S. violaceum Ort., and S. virginianum L., in which anther contains normal pollen but does not open to release. The second one is pollen non-formation-type sterility in the CMS systems of eggplant with the cytoplasms of S. aethiopicum Aculeatum Group, S. anguivi Lam., and S. grandifolium C.V. Morton, in which anther of the male-sterile lines is completely devoid of pollen. Both types of sterility system were characterized by investigating pollen and seed fertility. Furthermore, in pollen non-formation CMS systems, two independent dominant fertility restorer (Rf) genes were discovered and a sequenced characterized amplified region (SCAR) marker tightly linked to these genes was developed.

Introduction

Eggplant (Solanum melongena L.) is an economically important vegetable crop of the Solanaceae family that is widely cultivated around the world. Exploitation of heterosis has become a potential tool for the improvement of eggplant (Sambandam, 1962). In Japan, Nagai and Kida (1926) were the first to report quantitative characteristics in the hybrids of eggplant and found that heterosis was manifest in total yield, number of fruit per plant, earliness of blossoming, earliness of maturity, plant height, number of branches, number of spines on the pedicel and length of the fruit. The occurrence of remarkable hybrid vigor in the crosses was reported in eggplant with regard to seed weight, stem diameter and height, earliness of production, greater vigor in growth and higher yield (Kakizaki, 1930, 1931). Gotoh (1952) recorded marked yield increase in the F1 generation of a series of crosses between Japanese eggplant cultivars. The phenomenon of heterosis or hybrid vigor in eggplant is well known today, and breeders exploit it for increased production. Hence, most commercial cultivars of eggplant are now intervarietal hybrids.

Male sterility involves the failure of plants to produce functional anthers, pollen or male gametes, although the potential for female reproduction remains. Cytoplasmic male sterility (CMS) is a kind of sterility in plants that is caused by specific nuclear and mitochondrial interactions. CMS is a maternally inherited trait that facilitates hybrid seed production of many crops and allows breeders to harness yield gains associated with hybrid vigor. CMS has now been identified in over 150 plant species (Schnable and Wise, 1998). It is well documented that an unusual open reading frame (ORF) consisting of the chimeric structure of endogenous mitochondrial genes in the CMS mitochondrial genome is responsible for CMS (Hanson and Bentolila, 2004). In many cases, it has been found that male fertility can be restored by nuclear-encoded fertility restorer (Rf) gene(s), some of which have recently been isolated (Bentolila et al., 2002). The key role in the nuclear genetic control of CMS is played by Rf genes, which are responsible for the restoration of male fertility upon interaction with CMS-inducing cytoplasm. These genes, which encode pentatricopeptide repeat proteins, affect the expression of the CMS gene post-transcriptionally. This results in the disappearance of the CMS protein, at least in the male organs. Hence, CMS/Rf systems are of value in the study of interactions between nuclear and mitochondrial genomes.

Male sterility in eggplant has been described in several reports. Genic male sterility caused by recessive nuclear genes was reported (Chauhan, 1984; Jasmin, 1954; Nuttall, 1963; Phatak and Jaworski, 1989; Phatak et al., 1991). CMS derived by utilizing the cytoplasm of wild Solanum species S. gilo Raddi (Fang et al., 1985), S. kurzii Brace & Prain (Khan and Isshiki, 2009), S. violaceum Ort. (Isshiki and Kawajiri, 2002), S. virginianum L. (Khan and Isshiki, 2008), S. aethiopicum Aculeatum Group (Khan and Isshiki, 2010), S. anguivi Lam. (Khan and Isshiki, 2011), and S. grandifolium C.V. Morton (Hasnunnahar et al., 2012a; Saito et al., 2009a) was reported. In this review, we describe cytoplasmic male sterility of eggplant found in our study, focusing on its development, characterization, fertility restoration and the development of an SCAR marker linked to Rf genes.

1.  Development of cytoplasmic male sterility systems of eggplant

1)  Development of anther indehiscent male sterility systems

Anther indehiscent male sterility systems of eggplant were developed utilizing the cytoplasms of S. violaceum (Isshiki and Kawajiri, 2002), S. virginianum (Khan and Isshiki, 2008), and S. kurzii (Khan and Isshiki, 2009). Interspecific F1 hybrids between those wild Solanum species and eggplant were made using wild Solanum species as the female parents and eggplant as the male one. Through repeated backcrossings with eggplant as the recurrent pollen parent, backcross generations were produced. All of the backcross progeny expressed anther indehiscent male sterility. Anther of these male-sterile (MS) plants did not open to release pollen (Fig. 1). Malfunction of the anthers prevented the release of pollen from the anthers, although all of the backcross progeny contained normal pollen in their anthers. The pollen non-release character became fixed in the subsequent backcross generations. In the case of CMS, the degree of male sterility is known to increase with each successive backcross generation (McVetty, 1997). Pollen fertility remained lower in all backcross progeny of these three CMS systems than in eggplant (Fig. 1). The MS lines showed normal meiosis and tetrad formation. Low pollen fertility in these MS lines could not be attributed to meiotic difficulty. The characters of anther indehiscence and low pollen fertility are kinds of CMS, induced by disharmony between the cytoplasms of wild Solanum species and the nucleus of S. melongena. Some CMS systems have been reported to be temperature-unstable and not to be commercially usable for hybrid seed production (McVetty, 1997); however, these three MS lines showed stable expression of the anther indehiscent character. No fertility restorer gene of these three CMS systems has been detected yet. Seed fertility of the MS lines was found to be very high when pollinating with eggplant ‘Uttara’ and almost equal to that of eggplant ‘Uttara’ (Table 1). These MS lines could exhibit sufficient seed production ability when they were used as the seed parents for hybrid seed production. No negative effects of the cytoplasms of S. kurzii, S. violaceum, and S. virginianum on seed fertility were recognized in eggplant.

Fig. 1

Anther tips at flowering (A, B) and acetocarmine-stained pollen grains (C, D) of S. melongena ‘Uttara’ (left) and a plant of BC4 with S. virginianum cytoplasm (right). Arrows indicate the pore portion of anthers. Scale bar = 0.5 mm (A–B), 50 μm (C–D).

Table 1

Seed fertility in the male-sterile (MS) and male-fertile (MF) lines of six cytoplasmic male-sterility systems of eggplant.

PCR-RFLP analysis of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) showed maternal inheritance in the backcross progeny with the cytoplasms of S. kurzii (Khan and Isshiki, 2009) and S. violaceum (Isshiki and Kawajiri, 2002). PCR-RFLP analysis of mtDNA showed maternal inheritance in the backcross progeny with the cytoplasm of S. virginianum (Khan and Isshiki, 2008). However, in the case of PCR-RFLP analysis of cpDNA, the examined single BC1 plant showed paternal and novel restriction patterns, in addition to the maternal restriction one. This indicated the occurrence of the biparental inheritance of cpDNA. All of the subsequent backcross progeny showed the same recombinant cpDNA pattern of the BC1 plant. These results suggested that the recombinant cpDNA might be stable and harmonize with the nuclear genome of S. melongena.

2)  Development of pollen non-formation male sterility systems

Pollen non-formation-type CMS systems were reported utilizing the cytoplasms of S. aethiopicum Aculeatum Group (Khan and Isshiki, 2010), S. anguivi (Khan and Isshiki, 2011), and S. grandifolium (Hasnunnahar et al., 2012a; Saito et al., 2009a). Interspecific F1 hybrids between wild Solanum species and eggplant were made using wild Solanum species as the female parents and eggplant as the male one (Fig. 2). The cytoplasm of eggplant was substituted with the cytoplasms of wild Solanum species through repeated backcrossing using eggplant as the recurrent pollen parent. Backcross progeny of S. aethiopicum Aculeatum Group, S. anguivi, and S. grandifolium induced CMS lines of eggplant were segregated into pollen non-formation MS and pollen formation male-fertile (MF) types (Table 2; Fig. 2). Meiosis was not detected in the MS lines, whereas it was found normally in the pollen mother cells (PMCs) of MF lines. The anthers of MS lines were smaller in size and the pollen sac tended to be blackish with a deformed structure and did not contain any pollen (Fig. 3). However, the anther and pollen sac of the MF lines were normal, which contained substantial pollen (Khan and Isshiki, 2010). The MF lines have fertility restorer (Rf) genes that are capable of counteracting the male sterility trait and restoring pollen fertility. Two independent dominant fertility restorer genes were reported to control the pollen formation in these three CMS systems. In plants containing the Rf gene at either or both of these loci, pollen can form, while recessiveness for both results in failure of pollen formation. PCR-RFLP analysis of cpDNA and mtDNA showed maternal inheritance in the backcross progeny of these three CMS systems. This confirms that the repeated backcross method is suitable to develop cytoplasm substitution lines of eggplant. Seed fertility was found to be high in these CMS lines of eggplant (Table 1). No negative effects of the cytoplasms of S. aethiopicum Aculeatum Group, S. anguivi, and S. grandifolium on seed fertility were recognized in eggplant.

Fig. 2

Procedure for substituting the cytoplasm of S. aethiopicum Aculeatum Group for that of S. melongena ‘Uttara’ by continuous backcrossing.

Table 2

Segregation for male-fertile (MF) and male-sterile (MS) plants in the F1 hybrids (S. anguivi × S. melongena ‘Senryo 2 gou’), backcross progeny and the selfed progeny with the cytoplasm of S. anguivi.

Fig. 3

Flowers (A, B) and close-up cross sections of anthers (C, D) from male-fertile (left) and male-sterile (right) lines of eggplant with the cytoplasm of S. anguivi. Scale bar = 10 mm (A–B), 0.5 mm (C–D).

2.  Characterization of cytoplasmic male sterility systems in eggplant

1)  Characterization of anther indehiscent male sterility systems

Pollen and seed fertility characteristics were investigated in the anther indehiscent male sterility systems of eggplant developed by utilizing the cytoplasms of S. kurzii, S. violaceum, and S. virginianum to find out the possibilities of propagation by selfing and the production of pure lines through anther culture (Hasnunnahar et al., 2012b). Pollen fertility was assessed at 2 days before anthesis, on the day of anthesis and 2 days after anthesis by pollen stainability in acetocarmine and in Lugol’s solution, and the in vitro germination rate of pollen (Figs. 4 and 5). Pollen stainability with acetocarmine and the in vitro germination rate of pollen of all three stages of pollen maturation were lower in the MS lines than in S. melongena ‘Uttara’. Pollen stainability with Lugol’s solution revealed that the low pollen fertility of the MS lines would be caused by incomplete degradation of starch during pollen maturation (Fig. 5). Seed fertility in both selfing and backcrossing was found generally to be high in all of these three lines (Table 1). The MS lines showed a high percentage of fruit set with a large number of seeds in the fruit obtained from selfing. These results confirmed that it is possible to propagate the MS lines by selfing and to produce pure MS lines through anther culture.

Fig. 4

Stage-dependent differences of pollen stainability in Lugol’s solution between S. melongena ‘Uttara’ (A–C) and the male-sterile lines with S. violaceum cytoplasm (D–F). Two days before anthesis (A and D), on the day of anthesis (B and E) and two days after anthesis (C and F). Scale bar = 100 μm.

Fig. 5

Pollen stainability with acetocarmine (A), pollen germination (B) and Lugol’s score in three anther indehiscent male sterility systems of eggplant and in S. melongena ‘Uttara’ observed at 2 days before anthesis (2 DBA), on the day of anthesis (DA) and 2 days after anthesis (2 DAA).

2)  Characterization of pollen non-formation male sterility systems

Pollen and seed fertility characteristics of the MF lines were investigated in the pollen non-formation male sterility systems of eggplant developed by utilizing the cytoplasms of S. aethiopicum Aculeatum Group, S. anguivi, and S. grandifolium to find out the possibilities of selfing and anther culture for the development of homozygous restorer and MS lines (Khan et al., 2013). The methods of selfing and anther culture of the MF lines were applied to confirm the utility of these methods. Pollen characters of the MF lines were assessed similarly as shown in Figure 5. Pollen stainability in acetocarmine and the in vitro germination rate of pollen in all three stages of pollen maturation were lower in the MF lines than in eggplant ‘Uttara’. At anthesis, the rate of pollen stainability in acetocarmine was found to be about 50% and that of in vitro pollen germination was less than 20% in these three MF lines. Pollen stainability in Lugol’s solution revealed that the low pollen fertility of the MF lines would be caused by incomplete accumulation and degradation of starch during pollen maturation. Seed fertility was found generally to be high in pollen non-formation-type MS lines (Table 1). The MF lines showed good fruit set percentage and produced an adequate number of seeds in selfing and backcrossing. Doubled haploid MS and MF plants were obtained from anther culture of the MF lines with the cytoplasms of S. anguivi and S. grandifolium, respectively (Table 3). Pollen and seed fertility study of the MF lines revealed that homozygous restorer and MS lines could be developed by selfing and anther culture of the MF lines of pollen non-formation male sterility systems of eggplant.

Table 3

Frequency of embryo formation from cultured anther of the male-fertile (MF) lines of three cytoplasmic male sterility systems of eggplant with the cytoplasms of S. aethiopicum, S. anguivi, and S. grandifolium.

3. Fertility restoration in the CMS systems of eggplant by the Rf genes of each other’s systems

CMS systems have traditionally been characterized by the Rf genes required to overcome the CMS and to provide MF progeny in the MS cytoplasm (McVetty, 1997). An Rf gene has not been discovered yet in anther indehiscent male sterility systems of eggplant developed by utilizing the cytoplasms of S. kurzii, S. violaceum, and S. virginianum. However, Rf genes were found in the CMS systems with the cytoplasms of S. aethiopicum Aculeatum Group, S. anguivi, and S. grandifolium (Khan et al., 2014). Fertility restoration ability by the Rf genes of each other’s systems in three pollen non-formation-type CMS systems of eggplant was evaluated. The MS line of each CMS system was pollinated with the pollen of MF lines of the other CMS systems as well as its own CMS system (Fig. 6). Among the 9 cross combinations, 6 groups were obtained from crossing MS lines with MF lines of other CMS systems and 3 groups were obtained from crossing MS lines with MF lines of their own CMS system. The segregation of MF and MS plants fitted well, with a 3:1 ratio in almost all cross combinations (Table 4). The segregation patterns revealed that two independent dominant Rf genes restore fertility in each CMS system. Pollen stainability and in vitro pollen germination rates of the progeny obtained from different cross combinations varied in the ranges of 61.0%–89.8% and 0.7%–8.0%, respectively. Each Rf gene of the three CMS systems was able to restore fertility of other CMS systems as well as its own CMS system. Moreover, the fertility recovery actions in these three kinds of CMS were found to be similar.

Fig. 6

Cross combinations between male-sterile (MS) and male-fertile (MF) lines in three pollen non-formation male sterility systems of eggplant. The arrowheads indicate the direction of pollination.

Table 4

Segregation for male-fertile (MF) and male-sterile (MS) plants in different cross combinations between MS and MF lines of three cytoplasmic male sterility systems of eggplant with the cytoplasms of S. aethiopicum, S. anguivi, and S. grandifolium.

4. Development of SCAR marker linked to Rf gene

DNA markers linked to the Rf gene were screened from three pollen non-formation-type CMS systems of eggplant with the cytoplasms of S. aethiopicum Aculeatum Group, S. anguivi, and S. grandifolium by bulked segregant analysis using random amplified polymorphic DNA (RAPD) (Khan et al., 2014). Aliquots of DNA from 10 arbitrarily selected MF and MS plants from backcross progeny were bulked separately for each CMS system. The bulked DNA samples were screened with a total of 220 kinds of Operon primers and 200 kinds of Bex Common primers from Operon Technologies (Alameda, CA, USA). A total of 420 primers were used, and more than 65% of the primers yielded a considerable number of RAPD fragments in both MF and MS bulks. Only one Operon primer, OPAB10, gave a reproducible polymorphic band of 1.9 kbp (OPAB101900) between bulks and was identified as tightly linked to the Rf genes of the MF plants. Additionally, the RAPD marker OPAB101900 was converted to a sequenced characterized amplified region (SCAR) marker designated as SCAB101900 by nucleotide sequencing (Fig. 7). SCAB101900 could be successfully used for marker-assisted selection and is expected to help breeders to distinguish MF from MS plants prior to pollen shed. This technique of selection could be useful for rapid screening of inbred lines for the Rf/rf gene without developing and evaluating testcross progeny. This marker might also be useful for the isolation of Rf genes.

Fig. 7

Segregation of SCAR (SCAB101900) marker in male-fertile and male-sterile individuals of eggplant with the cytoplasm of S. anguivi. M is a λ/Hind III, EcoR I double digest marker.

5.  Conclusion and perspective

This review summarizes CMS research in eggplant, focusing on CMS development, characterization, fertility restoration, and the development of SCAR marker linked to Rf genes. The findings from studies on CMS systems in eggplant may give us a way to explore CMS and the corresponding Rf genes from wild Solanum resources for breeding programs of eggplant (Table 5). Isolation and studies of the function of the Rf gene might allow a better understanding of the mechanisms involved. The molecular basis of CMS in alloplasmic lines of eggplant was reported where genomic structures and transcription patterns of mitochondrial ATP synthase subunit and cytochrome oxidase subunit genes were studied for wild and cultivated eggplants (Yoshimi et al., 2013). Diversification of CMS sources is needed for use in commercial hybrid seed production. Parthenocarpic eggplant lines have been developed by genetic engineering (Acciarri et al., 2002; Donzella et al., 2000; Rotino et al., 1997) and conventional cross-breeding (Kikuchi et al., 2008; Saito et al., 2009b). Incorporating these parthenocarpic characters into the present CMS systems will be very useful for producing high-quality seedless fruit and for fruiting of hybrid cultivars without discovering the Rf genes or applying treatment with phytohormones. Finally, investigations on the mechanisms involved in male sterility and fertility restoration need to be extended to improve our understanding of the nuclear cytoplasmic interactions and achieve better utilization of CMS/Rf systems for hybrid breeding in eggplant. Further efforts are to be made to find out the nature and roles of nucleus-encoded proteins in the expression of essential mitochondrial genes. With all of these resources, the future of eggplant breeding research promises to be exceptionally fruitful.

Table 5

A summary of cytoplasmic male sterility systems of eggplant found in our study.

Literature Cited
 
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