Plant Breeding 131, 42—47 (2012) 2011 Blackwell Verlag GmbH
doi:10.1111/j.1439-0523.2011.01897.x
Mapping of tms8 gene for temperature-sensitive genic male sterility (TGMS) in rice (Oryza sativa L.) APPIBHAI J. HUSSAIN1,2, JAUHAR ALI3,8, EBRAHIMALI A. SIDDIQ1,4, VIDYA S. GUPTA5, UMESH K. REDDY1,6 and P R A B H A K A R K . R A N J E K A R 5 , 7 1
Directorate of Rice Research, Hyderabad 500 030, Andhra Pradesh, India; 2Seed Works India Pvt Ltd, Banjara Hills, Hyderabad 500 034, Andhra Pradesh, India; 3International Rice Research Institute, College, Los Banos, Laguna, DAPO Box 7777, Metro Manila, Philippines; 4Institute of Biotechnology, College of Agriculture, Rajendranagar, Acharya N G Ranga Agriculture University, Hyderabad 500 030, Andhra Pradesh, India; 5Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, Maharashtra, India; 6Department of Biology, West Virginia State University Institute, PO Box 1000, WV 25112-1000, USA; 7Interactive Research School for Health Affairs, Pune 411008, Maharashtra, India; 8Corresponding author, E-mail:
[email protected] With 3 figures Received February 14, 2011/Accepted July 6, 2011 Communicated by Z.K. Li
Abstract Genetic analysis of F2 and backcross populations of an induced temperature-sensitive genic male sterility (TGMS) mutant source F 61 with normal pollen parents revealed that TGMS trait was controlled by a single recessive gene. Molecular tagging of TGMS trait was attempted using polymorphic randomly amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers through bulked segregant analysis. The RAPD primers UBC 345830, UBC 313927, microsatellites RM224 and RM21 produced putative markers, which differentiate parents and bulks from sterile parent and sterile bulks. The RAPD analysis of individual F2 plants with the primer UBC345830 showed perfect marker–phenotype cosegregation. The 830-bp RAPD fragment, which segregated with TGMS locus at a distance of 1.33 cM, was eluted and cloned, and sequence information was used for designing sequence-characterized amplified region (SCAR) primer, which cosegregated with TGMS locus at a distance of 0.8 cM. TGMS locus was mapped onto chromosome 11 using RM21 and RM224, flanking it at a distance of 4.3 and 3.0 cM, respectively. The DNA markers tightly linked to TGMS gene (tms8) in F 61 can be cost effectively used for marker-assisted selection of TGMS trait.
Key words: Bulked segregant analysis — randomly amplified polymorphic DNA — SSR — sequence-characterized amplified region — rice — temperature-sensitive genic male sterility Cytoplasmic-genetic male sterility (CGMS)-based three-line hybrid technology is a readily available technological option to raise the ceiling to genetic yield by 15-20% over the high yielding semi-dwarf varieties in rice. However, inherent limitations of this approach are non-availability of quality hybrid seed in adequate quantity at affordable price and relatively cumbersome and expensive hybrid seed production on account of the need for maintainer (B) line to produce the seed of male sterile (A) line. Time-consuming process of conversion of maintainer sources into stable A-line, excessive dependence on a single cytoplasmic male sterile (CMS) source, limited scope to use diverse R lines and problem of pollen shedding in less stable CMS lines are other weaknesses of the three-line-based hybrid technology. The two-line hybrid breeding assumed importance as an alternative to the three-line approach following the discovery by Prof. Shi Ming Shong of a photoperiod-sensitive genic male sterile (PGMS) mutant
Nongken 58S in Hubei Province, China, which remains male sterile or fertile with change in day length (Shi 1981, 1985, Shi and Deng 1986). This discovery led subsequently to the identification of temperature-sensitive genic male sterile (TGMS) sources of spontaneous or induced origin such as Annong 1S (Tan et al. 1990) in China, Norin PL 12 (Maruyama et al. 1990a, 1991) in Japan, IR 32364 at IRRI (Virmani and Voc 1991) and SM 5, F61 and SA 2 in India (Ali 1993, Ali et al. 1995). In the tropics, consistent temperature differences are usually found at different altitudes and over different seasons in the same location or region that can be exploited for the establishment of two-line system using TGMS, especially for hybrid rice development. Unlike the CMS system, the TGMS system does not require a maintainer line for seed multiplication of a male sterile line; only a TGMS line and a pollen parent are required to produce a hybrid. Hence, this system of developing rice hybrids is also known as two-line system. Hybrid rice seed production via two-line system is relatively simple as compared with three-line system, as the maintenance step is avoided involving a cross between CMS and maintainer. TGMS line can produce its own seed through selfing by growing them in locations with low-temperature (32C) would produce selectively sterile pollens and thus will be used as female (seed) parent and allowing any other fertile pollen parent to be used for producing hybrid seeds (Siddiq and Ali 1999, Virmani et al. 2003).The extent of heterosis in two-line hybrids can be higher than those of three-line hybrids, as there is a wide range of choice for pollen parents unlike in the three-line system where we need only a restorer class. There is no negative effect of a sterility-inducing cytoplasm in TGMS line as encountered in CMS lines. Thus, the two-line system reduces the risk of genetic vulnerability among the hybrids. There is no need for restorer genes in the pollen parents of two-line hybrids, and this system is ideal for developing indica/japonica hybrids, as most japonica lines do not possess restorer genes in them (Siddiq and Ali 1999, Virmani et al. 2003). However, in the development and use of TGMS lines, influence of modifier gene complex affecting full expression of
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Mapping of tms8 gene for TGMS in rice
the TGMS trait is often encountered as a serious problem (Oard and Hu 1995). Use of molecular marker(s) closely linked to the TGMS trait could be a reliable approach in such situations. In rice, molecular linkage map has been constructed using restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD) and microsatellite (SSRs) markers (Huang et al. 1997, McCouch et al. 1997). Several TGMS alleles of varied origin have been discovered and mapped onto different linkage groups (Sun et al. 1989, Virmani 1992, Li et al. 2007). Advances have been made in the identification of RAPD, SSR, amplified fragment length polymorphism (AFLP), sequence-tagged site (STS), cleaved amplified polymorphic sequence (CAPS) and expressed sequence tags (ESTs) linked to TGMS genes. For instance, using STS and CAPs marker, Wang et al. (2003) could map the TGMS gene tms5 onto chromosome 2. Wang et al. (2004) identified a RAPD marker linked to the two TGMS genes tms6 and tms6-1 in a somatic mutant 0A15-1. Lee et al. (2005) developed SSR and AFLP markers linked to the gene tms6-2 in a spontaneous mutant SoKcho-MS. However, application of either type of marker is time-consuming, so discouraging their use in large-scale screening. Conversion of the amplified fragments into stable markers such as the SCAR markers makes them more convenient for use by breeders for routine screening of large populations. This paper reports the molecular mapping and chromosomal location of the TGMS gene (tms8) in F61 using RAPD, SSR and SCAR markers.
43 were resolved by 1.5% (RAPD) and 2% agarose gels (SSR), and electrophoresis was performed in 1· TAE buffer along with molecular weight marker (/ 174/Hae III). Amplicons were detected by staining with ethidium bromide and photographed under UV light and documented using Image Master VDS (Pharmacia Biotech, Pittsburgh, PA, USA). Conversion of RAPD marker to SCAR marker: The RAPD amplicon of 830 bp was excised and eluted from the gels. The excised band was re-hydrated in 100 ll of sterile water as described by Meksem et al. (1995). For PCR amplification, 2 ll of supernatant was used as template. Re-amplified products (3–5 ll) were cloned into pGEM-T Easy vector as per manufacturerÕs instructions (Promega, Madison, WI, USA) and used for transformation of Escherichia coli strain DH5a. The RAPD clones were sequenced using automated DNA sequencer (ABI 3700; Applied Biosystems, Carlsbad, CA, USA). Based on the sequence information, forward (5¢-GATGCGTGACCCGT ATACCT-3¢) and reverse (5¢-CACACCCATGTGCGACTATT-3¢) SCAR primers were designed using the software primer 5 to amplify the genomic DNA samples from the F61/IR58025B F2 segregating population. Linkage analysis: The genetic linkage analysis of markers and TGMS trait was carried out using MAPMAKER version 3.0 (Lander et al. 1987). The linkage group was established with an LOD-score threshold of 3.0 and the maximum distance of 30 cM, using the mapping function of Kosambi 1994.
Results Characteristic features of F61 TGMS mutant
Materials and methods Plant materials and screening for TGMS trait: F61 was first developed by Ali et al. (1995) through sodium azide–induced mutation breeding in IET11990 variety. Two hundred F2 plants derived from cross F61/ IR 58025B along with the parents were screened for fertility and sterility phase at Directorate of Rice Research (DRR), Hyderabad, India. Genomic DNA from the fertile parent, the sterile parent and the F2 plants was extracted at maximum tillering stage using the protocol proposed by Dellaporta et al. (1983). The plant from which DNA was isolated was uprooted and pot-planted for phenotyping with respect to fertility–sterility transformation over the season. Bulked segregant analysis and RAPD: Two DNA bulks were made for bulked segregant analysis (BSA) by pooling equal amounts of genomic DNA from 10 individual F2 plants each of fertile and sterile phenotypes according to the method described by Michelmore et al. (1991). A total of 150 UBC 10-mer primers (University of British Columbia, Canada) were used to carry out RAPD reaction in a total volume of 20 ll containing 10 ng of template DNA, 100 lM of dNTPs, 0.2 lM of primer, 0.8 units of Taq DNA polymerase (Perkin Elmer, Waltham, MA, USA), 1.5 ll of 10· PCR buffer and 1.5 mM of MgCl2. Samples were amplified through 40 cycles of 1 min at 92C, 1 min at 35C, 2 min at 72C, followed by a final extension at 72C for 3 min in a PCR-9700 thermal cycler (Perkin Elmer Cetus). SSRs: A set of seventy-three microsatellite primer pairs (Research Genetics, Huntsville, AZ, USA) were used to study cosegregation of the TGMS trait with SSR markers. Amplification reactions were performed in 25 ll volumes containing 10 mM Tris–HCl at pH 8.3, 1.5 mM MgCl2, 200 lM of dNTPs, 0.5 mM of spermidin, 0.8 U of Taq DNA polymerase enzyme (Perkin Elmer), 0.50 ll each of 5 lM forward and reverse primer and 50 ng of template DNA. Amplifications were carried out (PCR-9700; Perkin Elmer) with the first denaturation at 94C for 5 min followed by 45 cycles at 94C for 1 min, annealing at 50C, 55C or 60C (depending upon the sequence and Tm of the primer) for 2 min, extension 1.5 min at 72C followed by a final extension at 72C for 5 min. The PCR-amplified products
An ideal TGMS line for commercial exploitation should combine low critical temperature for fertility transformation (
