UDC 575.633.11 https://doi.org/10.2298/GENSR1703809Y Original scientific paper
TAGGING OF FOUR Rf GENES WITH SELECTIVE GENOTYPING ANALYSIS IN RICE (Oryza sativa L.) Saeid YARAHMADI1,5*, Mohammad Mehdi SOHANI2, Ali Akbar EBADI3, Masumeh KHEIRGOO4 1
Department of Agronomy and Plant Breeding, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 2 Biotechnology Department, Faculty of Agriculture Science, University of Guilan, Rasht, Iran 3 Rice Research Institute of Iran (RRII), Rasht, Iran. 4 Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 5 Emam Khomeini Agricultural High School, Aliabad-e Katul, Iran Yarahmadi S., M. M. Sohani, A. A. Ebadi, M. Kheirgoo (2017): Tagging of four Rf genes with selective genotyping analysis in rice (Oryza sativa L.).- Genetika, Vol 49, No.3, 809 - 818. Wild abortive type of cytoplasmic male sterility (WA-CMS) is commercially used for hybrid rice seed production. The linked markers can be used for selection of plants with desirable traits. Tagging of Rf genes was carried out using recessive and dominant class analysis in a large F2 population from the cross IR58025A×IR42686R. Pollen fertility and seed setting were evaluated at the flowering and maturity stages, respectively. Forty-seven highly sterile and 23 fertile homozygous plants were selected from F 2 population for molecular marker assay. Four Rf genes identified in a good restorer line with high-quality derived from a random mating composite population at the International Rice Research Institute (IRRI). The genetic distance from Rf3 locus with flanking markers RM443 and RM315 on chromosome 1 was 3.7 and 21.2 cM, respectively. RM258, RM591, RM271 and RM6737 on the long arm of chromosome 10 were linked with the Rf6 gene with distance of 7.4, 22.6, 6 and 2.9 cM, respectively. Rf6 was flanked by RM6737 and RM591. The Rf4 gene located on chromosome 7 was linked with RM6344 at a genetic distance of 10.6 cM. RM519 and RM7003 were linked with other Rf gene on chromosome 12 at a genetic distance of 8.5 and 20.8 cM, respectively. Closely linked markers identified in this study could be used for marker assisted selection in a hybrid rice breeding program. A new Rf locus on chromosome 12 that designated Rf7 was linked with RM7003 and RM519. ___________________________ Corresponding author: Saeid Yarahmadi, Department of Agronomy and Plant Breeding, Faculty of Agricultural sciences, University of Guilan, Rasht, Iran, Telephone no.: +98-916-3999004, Email:
[email protected]
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Key words: fertility restoration, hybrid rice, molecular mapping, recessive class analysis, SSR marker INTRODUCTION Cytoplasmic male sterility (CMS) and fertility restoration system is the most valuable genetic tool in commercializing hybrid rice technology. Hybrid rice technology and exploitation of hetrosis has tremendously improved rice production ( LOPEZ and VIRMANI, 2000). Three types of CMS in rice are widely used in hybrid rice production. They include Wild Abortive (WA), BaoTai (BT) and HongLian (HL) (HUANG et al., 2003). Cytoplasmic male sterility of the wild abortive type (WA-CMS) has been extensively used in the production of hybrid rice seed. The inheritance of fertility restoration in WA-CMS has been extensively investigated. The majority of investigators concluded that restoration of WA type CMS is controlled by two nuclear genes (Rf3, Rf4 ) (BHARAJ et al., 1991; BHARAJ et al., 1995; ZHANG et al., 1994; ZHANG et al., 2002(. The pyramiding of genes encoding the same trait is difficult to achieve with classical methods. Molecular markers, however, facilitate this process and can be used in backcross assisted selection of a gene or QTL into an elite cultivar or breeding line. Markers linked to the gene can be used to select plants possessing the desired trait, and markers positioned throughout the genome can be used to select plants that have same genetic backgrounds to the recurrent parent (AHMADIKHAH et al., 2007; HOSPITAL et al., 1992; SATTARI et al., 2007). In the present study, Rf genes for WA type of cytoplasmic male sterility were tagged utilizing an extremely sterile and fertile segregating population by microsatellite markers. MATERIALS AND METHODS Population development and fertility scoring Tagging of fertility restoration loci were carried out using a large F2 population derived from the cross IR58025A× IR42686R. IR58025A is the most commonly used CMS line in Iranian hybrid rice breeding program having WA-CMS. The F1 hybrid of IR58025AxIR42686R yields more than 1.5 t/ha over the improved check variety Khazar. Therefore, this cross was selected for the genetic analysis of Rf trait. Nearly 10000 plants at a spacing of 25cm×25cm were planted at the Rice Research Institute of Iran (RRII), Rasht, Iran. Pollen fertility and seed setting were evaluated at the flowering and maturity stages, respectively in F2 population. For seed setting evaluation, the panicles were covered by a paper bag to prevent outcrossing. Individuals were considered as sterile when contained less than 1% stainable pollen and produced no fertile seed while, individuals were considered fertile when they contained more than 85% live pollen. Selection of plants for DNA marker studies was based on F3 generation of fertile population. Finally, 47 highly sterile and 23 fertile homozygous plants were selected from F2 population for molecular marker assay. Genomic DNA extraction and PCR conditions Total genomic DNA was extracted from young and healthy leaves using the CTAB method with some modifications (MURRAY and THOMPSON, 1980). Polymerase chain reaction (PCR) was performed in 15 µL volume containing 2 mM MgCl 2, 1x PCR buffer (10mM TRIS, pH 8.2, 50mM KCl, 0.01% gelatin) )CinnaGen, Iran), 0.2mM dNTPs )CinnaGen, Iran), 36 ng of each primer (2.4 ng/µL) )CinnaGen, Iran), 1 unit of Taq polymerase (Fermentas), 50 ng of DNA. The PCR program used was: initial denaturation at 94°C for 4 min; 39 cycles of 94°C for 45s,
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annealing temperature of each primer for 45s (Table 1), 72°C for 1 min; and a final extension by 5 min at 72°C (Perklin-Elmer 9600 thermal cycler). Products from PCR reaction were separated by electrophoresis in 6% denaturing polyacrylamide gel (Bio-Rad, USA). Table 1. The sequence information of the nearest SSR primer pairs to Rf genes with their chromosome location and annealing temperature SSR marker
chromosome
Forward primer
Reverse primer
annealing temperature
RM443
1
GGGAGTTAGGGTTTTGGAGC
TCCAGTTTCACACTGCTTCG
55
RM315
1
CGGTCAAATCATCACCTGAC
CAAGGCTTGCAAGGGAAG
55
RM3740
1
ATCCCAACTCTAAGCCACCC
CTACCCGTCACCAACTCACC
50
RM3627
1
GGCTACTCGAGCAAGCTCTG
ACCTACCCGTCATCCCTCTC
50
RM1201
1
TTACCGCGCCACATATACAC
CGTACGAGCCCTAGTTACCG
55
RM5954
1
CTCGTCCTCAAGTGCCTCTC
TCGACTCCTACTCCGACTCC
55
RM1152
1
GCCTTTGTCCTTCAGTAGGC
AGAGCGCCTGGGTATAATTG
50
RM1095
1
CCCATTCAGTTGATCCTGTC
GCAAAAGCAAGGATGGAGAC
50
RM7643
1
TCCTCCTATTCGGTCGAAAC
ACCAACGAAATACCGGCAC
61
RM237
1
CAAATCCCGACTGCTGTCC
TGGGAAGAGAGCACTACAGC
55
RM6344
7
ACACGCCATGGATGATGAC
TGGCATCATCACTTCCTCAC
50
RM4098
7
CGTTTGGATGAAGAAGAAGA
AGTGTTCGTTTCGGATTAGA
55
RM320
7
CAACGTGATCGAGGATAGATC
GGATTTGCTTACCACAGCTC
55
RM70
7
GTGGACTTCATTTCAACTCG
GATGTATAAGATAGTCCC
55
RM505
7
AGAGTTATGAGCCGGGTGTG
GATTTGGCGATCTTAGCAGC
55
RM420
7
GGACAGAATGTGAAGACAGTCG
ACTAATCCACCAACGCATCC
55
RM473
7
TATCCTCGTCTCCATCGCTC
AAGGATGTGGCGGTAGAATG
55
RM171
10
CATCCCCCTGCTGCTGCTGCTG
CGCCGGATGTGTGGGACTAGCG
67
RM258
10
GCATGGCCGATGGTAAAG
TGTATAAAACCACACGGCCA
55
RM3123
10
ATTTCCCACACATCTCGCTG
GTGTCGCCGGTCAAGAAC
50
RM5271
10
CGGTGTAGATTGTAGGTACA
GTAGTTTAGTTATTGCGCAC
55
RM591
10
CGGTTAATGTCATCTGATTGG
TTCGAGATCCAAGACTGACC
55
RM6737
10
CATTGGGGGTGGATAAAGAG
TATCCTCTACTCCCTCGGCC
50
RM271
10
TCAGATCTACAATTCCATCC
TCGGTGAGACCTAGAGAGCC
55
RM6474
10
GAAGTCCTTGCGTGGCAC
AAGGCGACCTTCAGCCTC
55
RM7376
12
TCACCGTCACCTCTTAAGTC
GGTGGTTGTGTTCTGTTTGG
50
RM491
12
CCATGGCCTGAGAGAGAGAG
AGCTAAATGGCCATACGGTG
55
RM1261
12
GTCCATGCCCAAGACACAAC
GTTACATCATGGGTGACCCC
50
RM7003
12
GGCAGACATACAGCTTATAGC
TGCAAATGAACCCCTCTAGC
50
RM6123
12
TCGCCATTGCTCCTCCTC
ACTCTCTCCTCTCCCCTTCG
61
RM179
12
CATAGTGGAGTATGCAGCTGC
CCTTCTCCCAGTCGTATCTG
55
RM519
12
AGAGAGCCCCTAAATTTCCG
AGGTACGCTCACCTGTGGAC
55
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SSR analysis SSR markers from twelve rice chromosomes with equal distance within the map constructed by McCOUCH et al. (2002) were selected. Two hundred and thirty-six microsatellite primer pairs were used in this study for parental survey from which, forty-five showed polymorphism. Map distances were based on the Kosambi function ( KOSAMBI, 1944). The recombination frequency between a positive marker and the Rf locus was calculated using maximum likelihood estimator (ALLARD et al., 1956). Single-marker analysis was done with assuming that all the extremely sterile and fertile individuals were homozygous at the targeted Rf locus (ALLARD, 1956). The linkage map was constructed with Map Maker 3.0. Linkage groups were assigned to corresponding chromosomes based on SSR markers mapped by McCOUCH et al. (2002). In case of absence of linkage between the marker loci and the Rf gene a segregation ratio (1(fertile band): 2 (heterozygous band): 1 (sterile band)) expected to be observed in sterile and fertile class separately. Chi square analysis clearly showed the marker loci were strongly linked with Rf gene (Table 2) (BAZRKAR et al., 2008). The test of frequency of one specific allele in two extreme classes was used to confirm the results of chi square test in recessive class analysis (Table 3).
Table 2. Segregation deviation of markers from 1:2:1 ratio in 47 sterile individuals indicating linkage between the Rf and marker loci on four different chromosomes separately Sterile heterozygotic individuals with bands from both restorer and male sterile parents (N2);Expected if unlinked (22.5)
Sterile Individuals with Male sterile band (N3); Expected if unlinked (11.25)
Chi square with 2 degrees of freedom
SSR Primer
Chromosome
Sterile Individuals with restorer band (N1); Expected if unlinked (11.25)
RM443
1
0(11.25)
3(22.5)
42(11.25)
112.2**
RM315
1
2(11.75)
14 (23.5)
31 (11.75)
43.47**
RM6344
7
1 (11.5)
9(23)
36 (11.5)
70.30**
RM258
10
0 (11.25)
6 (22.5)
39 (11.25)
91.80**
RM591
10
2 (11)
14 (22)
28 (11)
36.55**
RM271
10
0 (11.75)
4 (23.5)
43 (11.75)
111.04**
RM6737
10
0 (11.5)
2 (23)
44 (11.5)
122.52**
RM7003
12
2 (11)
12 (22)
30 (11)
44.73**
RM519
12
1 (11.25)
5 (22.5)
39 (11.25)
91.40**
**1% significant level
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Table 3. Test of frequency of one specific allele in two extremes classes for confirming the results of chi square test in recessive class analysis Frequency of sterile band Frequency of sterile band SSR primer Z in sterile extreme in fertile extreme RM 443 0.97 0.04 25.99** RM 315 0.81 0.22 8.08** RM 6344 0.88 0.07 15.50** RM 258 0.93 0.09 17.21** RM 591 0.80 0.23 7.43** RM 271 0.96 0.10 16.55** RM 6737 0.98 0.05 26.74** RM 519 0.92 0.10 14.90** RM 7003 0.82 0.23 7.84** **1% significant level
RESULTS In the present study, two hundred and thirty-six microsatellite primer pairs were used to screen polymorphism between parental lines. Among these primers, forty-five demonstrated polymorphism between parental lines. Polymorphic markers were first used for studying recessive class. Genotyping of fertile plants were carried out using F 3 generation and genetic distance calculated using all homozygous plants (Table 4). Rf3 was flanked by RM443 and RM315, on chromosome 1 which were 3.7 and 21.2 cM away, respectively (Table 4). RM6344 was linked at a distance of 10.6 cM to Rf4 on chromosome 7 (Table 4). Four SSR markers including RM258, RM591, RM271 and RM6737 not only showed polymorphism between parental lines but also high correlation with a fertility restorer gene in a mapping population constructed from 47 homozygous sterile and 23 homozygous fertile plants (Table 2, 3). Distances of these markers from Rf6 on chromosome 10 were 7.4, 22.6, 6 and 2.9 cM respectively (Table. 3, Fig. 1). Rf6 was flanked by RM6737 and RM591 (Fig. 1). RM519 and RM7003 on chromosome 12 had a 8.5 and 20.8 cM, distance, respectively with another Rf locus that we named it Rf7 (Table. 3, Fig. 2). Table 4. Recombination frequencies and genetic distances among the positive markers and the Rf locus
Locus
chromosome
RM 443 RM 315 RM 6344 RM 258 RM 591 RM 271 RM 6737 RM 519 RM 7003
1 1 7 10 10 10 10 12 12
Early recombination frequency (%) calculated in sterile population 3.33 19.15 11.96 6.67 20.45 4.26 2.17 7.78 18.18
Final recombination frequency (%) in all homozygous plants 3.68 20 10.45 7.35 21.21 5.97 2.94 8.46 19.70
Final genetic distance (cM)
LOD score
adjacent Rf gene
3.7 21.2 10.6 7.4 22.6 6 2.9 8.5 20.8
15.82 5.86 10.43 12.71 5.06 13.59 16.55 5.65 11.38
Rf3 Rf3 Rf4 Rf6 Rf6 Rf6 Rf6 Rf7 Rf7
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Map position
centromer
Genetic distance
cM
cM
39.3
RM258
41.7
RM271 7.4 6
RM6737 2.9
52.3- 58.9
Rf6
22. 6
78.3
RM591
telomere
Chromosome 10
Fig 1. Linkage between the fertility restorer gene (Rf6) and microsatellite markers. Map positions based on gramene marker view (www.gram-ene.org, right side). Marker names and genetic distances between markers and Rf4 estimated by Kosambi function (left side).
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Fig 2. The banding pattern of simple sequence repeat (SSR) marker RM591 for parental lines and recessive class. P1: the male sterile parent IR58025 A; P 2: the fertile parent IR42686 R; A, H and R individuals of recessive class with pattern of banding similar to male sterile parent, both parent and fertile parent respectively.
DISCUSSION Recessive class analysis is a relatively easy approach for mapping because homozygous male sterile plants with 0% pollen fertility and 0% seed setting rate can be easily found, but recognition of homozygous fertile plants at all Rf loci is difficult and progeny test approach is required with F3 generation (AHMADIKHAH et al., 2006). But two homozygous classes (rfrf and RfRf) could be combined for the genotyping analysis during molecular mapping, providing larger sample size and greater statistical power (SATTARI et al., 2007). In the present study, molecular markers linked to Rf genes in a rice WA-CMS system were identified and tagged on chromosomes 1, 7, 10 and 12 by the use of recessive and dominant class analysis. We detected four different Rf loci from a single source of restorer line IR42686R. This restorer line was developed at IRRI by random mating composite population approach. In this approach, IRRI used IR36 as a genetic male sterile line to cross with 11 R lines with desirable traits such as grain quality and floral traits. At IRRI, An alternative strategy was developed to extract high quality parental lines in hybrid rice breeding program from a random mating composite population consisted of a genetic male sterile line (IR36) and a series of selected restorer lines of 'WA' cytoplasm. Materials with desired parental line traits and wide adaptability to different climate conditions were extracted slowly. Random mating composite approach for parental line improvement was discussed in detail (BHARAJ and VIRMANI, 1997; VIRMANI, 1998). Breeding of restorers through random mating composite population approach appears to be extremely useful for favorably accumulating Rf genes and quality related traits into an individual selection such as IR42686R. The Rf genes located on chromosomes 1, 7 and 10 have been identified by several researchers. KOMORI et al. (2003) converted RFLP markers to PCR based markers and fine mapped the Rf1 gene at 0.2 cM from C1361 and 0.8 cM from S10019 on chromosome 10. ICHIKAWA et al. (1997) confirmed the location of Rf1 for BT cytoplasm on chromosome 10.
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et al. (2003) also confirmed the location of Rf5 on chromosome 10 and fine-mapped this gene at 0.63 cM from microsatellite marker HL01 and 2.7 cM from RFLP marker S10019. TAN et al. (1998) mapped two QTLs on long and short arm of chromosome 10 that explained 71.5% and 27.3% of the phenotypic variance, respectively. LIU et al. (2004) mapped two nuclear fertility restorer genes of HL type CMS on chromosome 10. AKAGI et al. (1996) mapped a nuclear fertility restorer gene of BT-type CMS on chromosome 10, 3.7 cM from SSR marker RM171. JING et al. (2001) also mapped the Rf4 gene on the long arm of chromosome 10 between two SSR markers RM171 and RM228 with distances of 3.7 and 3.4 cM respectively. HUANG et al. (2000) mapped a nuclear fertility restorer gene Rf5(t) of Hl type CMS on chromosome 10, 3.6 and 7.8 cM from OSR33 and RM258, respectively. MISHRA et al. (2003) reported that RM258 located on chromosome 10 was found linked to the Rf gene at a distance of 5.9 cM. AHMADIKHAH et al. (2006) reported that Rf4 on chromosome 10 was flanked by two SSR markers RM171 and RM6737 at distance of 3.2 and 1.6 cM respectively. BAZRKAR et al. (2008) reported that RM258 and RM591 flanked the Rf6 gene at a genetic distance of 4.4 and 23.3 cM, respectively. Our results showed that RM258, RM591, RM271 and RM6737 linked with the Rf6 gene with distance of 7.4, 22.6, 6 and 2.9 cM, respectively. This paper confirmed the results of previous studies. YAO et al. (1997) identified two Rf genes on chromosomes 1 and 10. ALAVI et al. (2009) reported that Rf3 on the short arm of chromosome 1. It flanked by two SSR markers RM1 and RM3873 with distances of 5.6 and 14 cM, respectively. BAZRKAR et al. (2008) mapped a nuclear fertility restorer gene on chromosome 1. This gene flanked with RM443 and RM315 at a genetic distance of 4.4 and 20.7 cM, respectively. BHARAJ et al., (1995) using primary trisomic identified two restorer genes for wild abortive cytoplasm on chromosomes 7 and 10. ZHANG et al. (1994) identified two chromosomal regions each containing a PSGMS locus on chromosomes 3 and 7. BAZRKAR et al. (2008) reported that RM6344 linked to Rf4 locus on chromosome 7 at a genetic distance of 13.3 cM. we used the same population as BAZRKAR et al. (2008). Four Rf genes were identified in both studies. Random mating composite population approach was used for breeding IR42686R. This approach pyramided four Rf genes in a restorer line for the first time. BAZRKAR et al. (2008) used only recessive class but we used sterile and fertile classes. Therefore we have larger population than BAZRKAR et al. (2008). With larger population, more recombination events can be sampled, thus we had more precision than BAZRKAR et al. (2008). HUANG
CONCLUSION The microsatellite markers RM443, RM6344, RM6737 and RM519 will be facilitating selection of restorers for WA cytoplasm in many sets of elite lines. MAS can be effectively employed for pyramiding of Rf genes in the new genetic backgrounds. Received, December12th, 2016 Accepted July 18th, 2017
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OZNAČAVANJE ČETIRI Rf GENA PIRINČA (Oryza sativa L.) SELEKTIVNOM GENOTIPIZACIJOM Saeid YARAHMADI1,5*, Mohammad Mehdi SOHANI2, Ali Akbar EBADI3, Masoumeh KHEIRGOO4 1
Departman za agronomiju i oplemenjivanje biljaka, Fakultet poljoprivrednih nauka, Univerzitet Guilan, Rasht, Iran 2 Departman za biotehnologiju, Fakultet za poljoprivredne nauke, Univerzitet Guilan, Rasht, Iran 3 Iranski istraživački institut za pirinač (RRII), Rasht, Iran. 4 Departman za zaštitu bilja, Fakultet za poljoprivredne nauke, Univerzitet Guilan, Rasht, Iran 5 Emam Khomeini viša poljoprivredna škola, Aliabad-e Katul, Iran Izvod Divlji abortivni tip citoplazmatične muške sterilnosti (WA-CMS) se komercijalno koristi za proizvodnju hibridnog semena pirinča. Povezani mrkeri se mogu koristiti za selekciju biljaka sa odabranim svojstvima. Označavanje Rf gena je obavljeno korišćenjem recesivne i dominantne analize u velikoj F2 populaciji, nastaloj ukrštanjem IR58025A×IR42686R. Ocenjivana je fertilnost polena i ozrnjenost u fazi cvetanja i zrenja. 47 visoko sterilnie i 23 fertilne homozigotne biljke odabrane su iz F2 populacije za analizu molekularnim markerima. Četiri Rf gena identifikovana su u dobroj restorer liniji dobijenoj iz kompozitne populacije u Međunarodnom istraživačkom institutu za pirinač (IRRI). Genetička distance za Rf3 od markera RM443 i RM315 na hromozomu 1 bile su 3.7 i 21.2 cM, dok su markeri RM258, RM591, RM271 i RM6737 na dužem kraku hromozoma 10 bili udaljeni od Rf6 gena 7.4, 22.6, 6 i 2.9 cM. Rf4 gen koji se nalazi na hromozomu 7 bio je povezan sa RM6344 genetičkom distancom od 10.6 cM, a RM519 i RM7003 su bili povezani sa drugim Rf genom na hromozomu 12 genetičkim distancama od 8.5 i 20.8 cM. Blisko povezani markeri identifikovani u ovom istraživanju mogu se koristiti za marker asistiranu selekciju u opleminjivačkim programim hibridnog pirinča. Novi Rf lokus na hromozomu 12, označen sa Rf7 bio je povezan sa RM7003 i RM519. Primljeno 12.XII.2016. Odobreno 18. VII. 2017.
