Molecular and cytological evaluation of male sterile and restorer lines in hybrids rice

Transcription

1 International Research Journal of Applied and Basic Sciences. Vol., 3 (1), , 2012 Available online at ISSN X 2012 Molecular and cytological evaluation of male sterile and restorer lines in hybrids rice Ammar Gholizadeh ghara 1 *, Ghorbanali Nematzadeh 2, Nadali Bagheri 2, Asa Ebrahimi 3 and Morteza Oladi 2 1-M. Sc student, Science and Research branch, Islamic Azad University, Tehran, Iran. 2-Department of Agronomy and Plant Breeding, Genetics and Agricultural Biotechnology Institute of Tabarestan, Sari Agricultural Science and Natural Resources University, Sari, Iran 3-Assistant Professor of Biotechnology, Science and Research branch, Islamic Azad University, Tehran, Iran. Corresponding author: Ammar Gholizadeh Ghara, ammar_gholizadeh@yahoo.com ABSTRACT: Information on the genetics of fertility restoration facilitates breeding and/or selection of restorer lines used in hybrid breeding program in a cytoplasmic male sterility (CMS) system. Cytoplasmic male sterility (CMS) is a common phenomenon that has been extensively used for production of hybrid seeds in various crops. Rf genes are needed for restoring fertility to CMS lines. Searching for molecular tagging of restorer genes is of high importance where phenotyping is very time consuming and requires the determination of spikelet sterility in testcross progeny. In order to determine maintainers and fertility restorers lines in hybrids rice, 5 cytoplasmic male sterile was crossed whit 10 pure line as tester. In other year, F1 progenies arranged in RCBD design whit three Replications. Evaluation of fertility in F1 populations showed IR-9 parent for all the crosses was identified as restorer. The cross Rome parent with CMS lines observed lowest amount fertility, also molecular analysis confirmed exist gene RF only in IR-9 parent. Keywords: Hybrid Rice, genetics, Cytoplasmic male sterility and restorer. INTRODUCTION Hybrid rice offers an opportunity to boost the yield potential of rice. It has a yield advantage of 15-20% over conventional high-yielding varieties (Virmani et al., 1993). Fertility restorer alleles (Rfs) are always tightly evolved with cytoplasmic male sterility (CMS) during plant evolution. Research of Rfs inheritance is the precondition for breeders to develop elite restorer lines. For studying the inheritance of the fertility restorers, in general, the main three indexes (percentage of fertile pollen, bagged seed setting and opening seed-setting) are often used as the criteria to evaluate fertility restoration. Of these, the percentage of fertile pollens is thought the most reliable criterion for evaluating plant fertility (Li et al., 2007). Restorer lines are the critical factors for successful development of hybrid rice. With the scalable CMS-WA hybrid rice released for commercial production, the inheritance of Rf alleles have been extensively studied using various restorer lines with different origins. A number of reports about the inheritance of the restorer alleles for CMS-WA come from the restorer lines of IR24, IR64, IR8 (from IRRI), Minghui63, Milyang23 and Milyang46 that are widely used in commercial production. Different genetic models of Rf alleles such as one gene (Shen et al., 1996; Gyan et al., 2003), two linked genes (Li and Zhu, 1986; Guha Sarkar et al., 2002), and two independent genes (Li and Yuan, 1986; Virmani et al., 1986; Teng and Shen, 1994; Bharaj et al., 1995) are proposed by different groups. Yao et al. (1997) investigated the fertility segregation of plants in F2 of Zhen-shan97A/Minghui63. They found that fertility segregation confirmed to 15:1 ratio, indicating that fertility restoration in this population is controlled by two major dominant genes.gyan et al. (2003) found that the F2 of IR58025A x PRR-78, giving a good fit to monogenic 3:1 ratio. Fu and Xue (2004) analyzed the spikelet fertility of F1, F2

2 and BCF1 derived from five different crosses between the CMS-WA line and various restorer lines. They found that indica restorer lines Milyang46 and H804 possess two dominant restorer alleles, but japonica restorer lines of 921 and T984 have one restorer allele.high yield of the F1 hybrids depends largely upon high pollen or spikelet fertility which is determined by the mode of gene action prevalent in the restorer lines of the hybrids. nowledge of the genetic control of male fertility restoration is also useful to transfer fertility restoring genes to promising breeding lines and undertake improved restorer breeding programme. Tow major fertilityrestorer genes, Rf3 and Rf4, are required for the production of viable pollen in WA-type CMS and the genes have been mapped to chromosomes 1 and 10 respectively (Yao et al., 1997; Zhang et al., 1997; Ahmadikhah et al., 2006; Ahmadikhah and Alavi, 2009). Searching for restorer genes is a good Molecular markers are particularly useful for accelerating the process of introducing a gene or Quantitative Trail Loci (QTL) into an elite cultivar or breeding line via backcrossing. When introducing a gene or a trait, markers linked to the gene can be used to select plants possessing the desired trait, and markers throughout the genome can be used to select plants that are genetically similar to the recurrent parent (Young and Tanksley, 1989; Hospital et al., 1992; Semgan et al., 2006). A significant advance in the practical utilization of molecular marker was the development of SSR markers, also referred to microsatellite markers (McCouch et al., 2002).These markers are highly polymorphic and easy to detect. The high polymorphism means that these markers can be used in germplasms that is closely related (Ni et al., 2002; Yang et al., 1994). Recently, a fairly dense SSR map of rice has been published (McCouch et al., 2002). Mapping agronomically important genes can provide useful information for plant breeders. In this line, our objective was to map an important Rf locus in rice using molecular markers. The purpose of this study was to evaluate the fertility hybrids and molecular analysis whit use of microsatellite markers to Detection the RF gene in the hybrids rice. MATERIALS AND METHODS Plant materials Genotypes of rice used in this study were 5 CMS lines (NedaA, NematA, DashtA, ChampaA and AmolA3), and 10 restorer varieties accessions (Rashti sard, Paxhohesh, Rashti, IR-9, Binam, Salari, Abjiboji Ahlami tarom and Hasan sarai). The 5 A lines all have cytoplasm derived from a WA cytoplasmic source. Crosses were carried out in all combinations between CMS lines and pollen parents. In order to study the molecular hybrid of all that was over 50 % of fertility and leaf samples for DNA extraction, leaf desired Dlapvrta performed. 3 SSR Markers RM1, RM171 and RM490 reported to be linked with fertility restorer genes in other source were also used. Estimation of pollen and spikelet fertility Pollen fertility and seed-setting rate were used as the main criteria for the evaluation of fertile and sterile plants. Natural seed setting rates of F1 hybrids were used as the main criteria for the evaluation of compatibility. At least three different plants must be tested for every hybrid combination (Tan et al., 2008). For all materials, pollen grain fertility was measured using anthers collected from spikeletes at 1 to 2 days before anthesis and fixed in acetic acid-alcohol (1:3)solution (Sarial and Singh, 2000). Three undehised spikelets were randomly selected from different positions on the panicle. The anthers from each spikelet were smeared in a drop of 1% I2-KI solution on a glass slide separately and observed under compound microscope. Plants were classified into different fertility-sterility groups based on proportion of stained-round pollen grains, as the number of fertile grains/total number observed x 100. With regard to pollen fertility of population, plants were classified into the following classes: Fertile (F): Plants showing more than 60% pollen fertility; partially fertile (PF): plants showing 30 to 60% pollen fertility, partially sterile (PS): plants showing 1 to 30% pollen fertility and completely sterile (CS): plants showing no pollen fertility (Gyan et al., 2003). The panicles that emerged from the primary tiller were bagged before anthesis and the number of filled grains and chaffs in the panicle were counted at the time of maturity. The ratio of filled grains to the total number of spikelets was expressed as seed setting rate (He et al., 2006). The goodness of fit to Mendelian segregation pattern of fertile, partially fertile, partially sterile and completely sterile plants in hybrids DNA extraction and PCR analysis Total genomic DNA was extracted according to Dellaporta et al.(1983). PCR amplification was performed using the DNAs from parental lines IR-9, Salari, Abjiboji, Ahlmy Tarom, NedaA, NematA, DashtA,

3 ChampaA and AmolA3. A 20 l mixture was prepared for the PCR assay containing 40 ng template DNA, 2 l of 10X buffer, 0.5 l of 10 mm dntps, 1 l of 50 mm MgCl2, 0.5 l of each primer, and 1 unit of Taq polymerase. The PCR reaction was performed at 94 C for 1 min (initial denaturation); then for 33 cycles of 94 C for 1 min; 55 C for 1 min; 72 C for 2 min followed by 72 C for 1 min. PCR products were resolved by melectrophoresis in 3.5% agarose gel containing 0.5 g/ml ethidium bromide. Result and Discussion The results of testing for pollen and spikelet fertility (Table 1) showed that among the hybrids, the parent IR-9 in crosses with lines cytoplasmic male sterile NadaA, NematA, DashtA, ChampaA and AmolA3 more than 80% fertility, so IR-9 line can be as restorer. But based on pollen fertility, Ahlmytarom, Salari, Abjibojiand and Hasansarai showed over than 30% fertility in most crosses, therefore each of those should be partially fertile. Furthermore Rome, Rashti and Binam parents in most crosses was partially sterile with lower fertility (30%). Babaeian et al (2011) for introduce of restorer line reported that in cross [Sefidrod [(IR25 19A] and [IR-25 R2) Tarom] observed fertility amount around up 90 %. In the test spikelet fertility (table 1) for crosses Salari AmolA3, Abjiboji NadaA, Ahlmitarom AmolA3, HassanSarai DashtA and Rashtisard AmolA3 had fertility above 30% so, these paternal were as partially fertile and in the most crosses on the basis of spikelet fertility test (Table 1), hybrids fertility exhibited between 1-30%, so each paternal should declared as partial cytoplasmic male sterile lines. Table 1. pollen and spikelet fertility in hybrids rice. crosses Fertility Pollen (%) Spikelet fertility (%) crosses Fertility Pollen (%) Spikelet fertility (%) NedaA/Rashti Sard 35% 20% NedaA/Salari 30% 10% NematA/ Rashti Sard 10% 20% NematA/ Salari 40% 10% DashtA/ Rashti Sard 30% 28% DashtA/ Salari 50% 25% ChampaA/ Rashti Sard 35% 23% ChampaA/ Salari 40% 18% Amol3A/ Rashti Sard 30% 31% Amol3A/ Salari 75% 34% NedaA/Paxhohesh 10% 9% NedaA/Rome 10% 5% NematA/ Paxhohesh 5% 6% NematA/ Rome 5% 4% DashtA/ Paxhohesh 5% 13% DashtA/ Rome 5% 4% ChampaA/ Paxhohesh 62% 58% ChampaA/ Rome 18% 5% Amol3A/ Paxhohesh 20% 23% Amol3A/ Rome 5% 3% NedaA/Rashti 35% 25% NedaA/Abjiboji 63% 55% NematA/Rashti 10% 10% NematA/ Abjiboji 40% 10% DashtA/Rashti 5% 24% DashtA/ Abjiboji 40% 30% ChampaA/Rashti 40% 30% ChampaA/ Abjiboji 25% 16% Amol3A/Rashti 30% 15% Amol3A/ Abjiboji 25% 20% NedaA/ IR-9 95% 89% NedaA/Ahlamitarom 10% 16% NematA/ IR-9 95% 89% NematA/ Ahlamitarom 15% 7% DashtA/IR-9 95% 95% DashtA/ Ahlamitarom 15% 29% ChampaA/IR-9 95% 83% ChampaA/ Ahlamitarom 30% 50% Amol3A/IR-9 90% 82% Amol3A/ Ahlamitarom 35% 39% NedaA/Binam 35% 12% NedaA/Hasansarai 35% 16% NematA/Binam 10% 13% NematA/ Hasansarai 30% 14% DashtA/Binam 5% 10% DashtA/Hasansarai 25% 37% ChampaA/Binam 24% 18% ChampaA/ Hasansarai 40% 23% Amol3A/Binam 20% 30% Amol3A/Hasansarai 40% 20% F (fertile) = > 60% Pollen fertility; PF (partially fertile) = 30-60%; PS (partially sterile) = 1-30%; CS (completely sterile) =` 0.

4 F (fertile) = >80% Spikelet fertility; PF (partially fertile) = 30-80%; PS (partially sterile) = 1-30%; CS (completely sterile) = 0. Figure 1. Frequency distribution of pollen fertility in hybrids rice. Figure 2. Frequency distribution of spikelet fertility in hybrids rice. Gueimarayz and et al (1998) were reported 36% restorer lines among lines Also restorer frequency was reported high amount among indica tropical elites. Based on the results obtained from the cross restorer line and cytoplasmic male sterility due commercial used of these lines in hybrid seed production program, in addition to traits such as plant height, date to flowering, SCA and heterosis must be noted. The results of pollen and spikelet fertility showed that in crosses no exist completely sterile. The results of Frequency lines based on pollen fertility defined most partially sterile (56%). the frequency of the fertility restorer lines and lines of the partial restoration of fertility, 16 and 28%, respectively and no completely sterile line (Figure 2). The results of spikelet fertility showed that most Frequency of partially sterile (76%). Frequency of restorer line and partial restorer was 10 and 14% respectively, and no completely sterile (Figure 3). Band fragment based on polymerase chain reaction with application of RM1 markers (on chromosome number 1 and linked with RF3 gene) showed that only IR-9 cultivar had restorer fertility gene, liked spikelet fertility test (Figure 3).Also, Jing et al (2001) indicated that this marker was linked to Rf3 gene and reported that these marker had segregation potential restorer linked of other linked.

5 Figure 3. banding patterns obtained by PCR using marker RM1 for 10 varieties of rice (respectively, from left to right of ladder, Paxhohesh, IR-9, Salari, Abjiboji, Ahlmitarom, NedaA, NematA, DashtA, ChampaA and Amol3 A) on agarose gel. Band fragment based on polymerase chain reaction with application of RM171 markers (on chromosome number 10 and linked with RF4 gene) showed that only IR-9 cultivar had restorer fertility gene, liked spikelet fertility test (Figure 4). McCouch et al (2002) showed RM171 marker had segregation potential restorer linked of other linked. Figure 4. The banding patterns obtained by PCR using markers RM171 for 10 varieties of rice (respectively from left to right of ladder, Paxhohesh, IR-9, Salari, Abjiboji, Ahlamitarom, NedaA, NematA, DashtA, ChampaA and Amol3 A) on agarose gel. Also, Band fragment based on polymerase chain reaction with application of RM490 markers (on chromosome number 10 and linked with RF4 gene) showed that only IR-9 cultivar had restorer fertility gene, liked spikelet fertility test (Figure 5). Bgheri and Babaeian Jelodar (2011) showed that RM490 marker linked with restorer fertility gene in mapping of population included 36 homozigos sterile plant and 100 selection plant Randomly of F2 population, thus this marker can be used for detection improved restorer lines in mass selection program.

6 Figure 5. The banding patterns obtained by PCR using markers RM490 for 10 varieties of rice (respectively from left to right of ladder, Paxhohesh, IR-9, Salari, Abjiboji, Ahlmitarom, NedaA, NematA, DashtA, Champa A and Amol3A) on agarose gel. References Ahmadikhah A, Alvi M (2009) A cold-inducible modifier QTL affecting fertility restoration of WA CMS in rice. Int. J. Genet. Mol. Biol., 1(5): Ahmadikhah A, Karlov GI (2006) Molecular mapping of the fertility restoration gene Rf4 for WAcytoplasmic male sterility in rice. Plant Breed., 125(4), Babaeian Jelodar, Bagheri N and Hasan nataj E (2011) Esmaeil Production of cytoplasmic male sterile and restorer lines for Iranian Rice Cultivars. Proceeding of 14th national rice conference sarifebruary,28. Bagheri N Babaeian Jelodar N (2011) Genetics and combining ability of fertility restoration of 'wild abortive' cytoplasmic male sterility in rice. African Journal of Biotechnology Vol. 10(46), pp Bharaj TS, Virmani SS, Khush GS (1995) Chromosomal location of fertility restoring genes for wild abortive cytoplasmic male sterility using primary trisomics in rice. Euphytica, 83: Dellaporta SL, Wood J and Tickes JB (1993) A plantmolecular DNA miniperation Version, Plant Mol. Bio.Rep. 1: Fu HW, Xue QZ (2004) Analysis of restoring genes of three type of cytoplasmic male sterility in rice. Mol. Plant Breed. 2: Guha Sarkar CK, Zaman FU, Singh AK (2002) Genetics of fertility restoration of WA based ytoplasmic male sterility system in rice (Oryza sativa L.) using basmati restorer line. Indian J. Genet. Plant Breed. 62: Guimaraes EP, Cutrim VD, Mendonca JA (1998) Developing hybrid rice in Brazil methodology, highlights, and prospects. In: Advances in hybrid rice technology. Edited by Virmania, S. S., Siddiq, E.A., Maralidhamn, K. p Gyan PM, Singh RK, Mohapatra M, Singh AK, Prabhu KV, Zaman FU,Sharma RK (2003) Molecular mapping of a gene for fertility restoration of wild abortive (WA) cytoplasmic male sterility using abasmati rice restorer line. J. Plant Biochem. Biotechnol. 12: He H, Peng X, Gong H, Zhu C, Ye G (2006) Fertility behavior of rice (Oryza sativa) lines with dominant male sterility gene and inheritance of sterility and fertility restoration. Field Crops Res. 98: Hospital F, Chevalet C, Mulsant P (1992) Using markers in gene introgression breeding programs. Genetics 132, Jing R, Li X, Yi P, Zhu Y (2001) Mapping fertility-restoring genes of rice WA cytoplasmic male sterility using SSLP markers. Bot. Bull. Acad. Sin., 42, Li YC, Yuan LP (1986) Genetic analysis of fertility restoration in male sterile lines of rice. In: IRRI, ed. Rice Genetics. Proc. Int. Rice Genet.Symp. IRRI, Manila, pp McCouch SR, Teytelman L, Xu Y, Lobos KB, Clare K, Walton M, Fu B,Maghirang R, Li Z, Zing Y, Zhang Q, Kono I, Yano M, Fjellstrom R,DeClerck G, Schneider D, Cartinhour S, Ware D, Stein L (2002) Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.). DNA Res., 9:

7 Ni J, Colowit PM, Mackill DJ (2002) Evaluation of genetic diversity in rice subspecies using microsatellite markers. Crop Sci., 42: Sarial AK, Sinh VP (2000) Identification of restorers and maintainers for developing basmati and non-basmati hybrids in rice, Oryza sativa.plant Breed. 119: Semgan K, Bjornstad A, Ndjiondjp MN (2006) Progress and prospects of marker assisted backcrossing as a tool in crop breeding programs.afr. J. Biotechnol., 5(25): Tan YP, Li SQ, Wang L, Liu G, Hu J, Zhu YG (2008) Genetic analysis of fertility-restorer genes in rice. Biol. Planta. 52(3): Teng LS, Shen ZT (1994) Inheritance of fertility restoration for cytoplasmic male sterility in rice. Rice Genet. Newslett. 11: Virmani SS, Prasad MN, Kumar J (1994) Breaking the yield barrier of rice through exploitation of heterosis. In: Murlidharan K, Siddiq EA, editors. New frontiers in rice research. Hyderabad (India): Directorate of Rice Research. pp Virmani SS, Raj KG, Casal C, Dalmacio RD, Aurin PA (1986) Current knowledge of and outlook on cytoplasmic-genetic male sterility and fertility restoration in rice. In: IRRI, ed. Rice Genetics Proceedings of the International Rice Genetic Symposium. IRRI, Manila, pp Yang GP, Saghai-Maroof MA, Xu CG, Zhang Q, Biyashev RM (1994) Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice. Mol. Gen. Genet., 245: Yang GP, Saghai-Maroof MA, Xu CG, Zhang Q, Biyashev RM (1994) Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice. Mol. Gen. Genet., 245: Yao FY, Xu CG, Yu SB, Li JX, Gao YJ, Li XH, Zhang QF (1997) Mapping and genetic analysis of two fertility restorer loci in the wild abortive cytoplasmic male sterility system of rice (Oryza sativa L).Euphytica, 98: Young ND, Tanksley SD (1989) Restriction fragment length polymorphism maps and the concept of graphical genotypes. Theor.Appl. Genet., 77: Zhang Q, Bharaj TS, Virmani SS, Huang H (1997) Mapping of the Rf-3 nuclear fertility restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers. Theor. Appl. Genet., 94: