Marker Assisted Backcross Breeding of Bg 358 ( Oryza sativa L.) for the Anaerobic Germination Tolerant QTL AG1

: Sustainability of transplanting systems for rice crop establishment in Sri Lanka is threatened due to climatic changes and heavy rains. Tolerance to anaerobic germination (AG) stress is a key trait that will ensure good crop establishment in direct seeded rice (DSR) farming systems. In this study Marker Assisted backcross breeding was carried out to transfer the quantitative trait loci - QTL AG1 (qAG-9-2), in to the local variety Bg 358 for the development of AG stress tolerant elite lines by AG1 donors IR64-AG1 and Ciherang-Sub1AG1. Marker assisted selection (MAS) was carried out through foreground and recombinant selection. Molecular marker optimization for RM24161, RM8300 and RM553 to the QTL AG1 was carried out using BC1F1 populations. DNA was extracted and PCR was performed using simple sequence repeat (SSR) markers and visualized on 2.5% agarose gel. Based on polymorphism exhibited, RM24161 was chosen as the foreground selection marker, RM553 and RM8300 chosen as proximal and distal markers for recombinant selection. It was suggestive through this study to choose flanking markers RM105 & RM219, closer to the qAG-9-2 locus for recombinant selection as the selected recombinant makers were ineffective and exhibited marker segregation distortion. RM24161 was found to be a tightly linked marker to the AG tolerant QTL AG1 (qAG-9-2) and was successfully used for selection of lines by MAS in generation of anaerobic germination tolerant elite lines.


INTRODUCTION
Abiotic stresses including flooding, drought and salinity limit crop production worldwide. The development of cultivars with enhanced tolerance to abiotic stresses has therefore been advocated as a low-cost means to improve productivity in stressful environments ). In the past, efforts to develop such varieties have typically relied entirely on phenotypic selection in target environments, but with the advances made in marker assisted selection (MAS) development of varieties for abiotic stress conditions have being made easier due to the numerous advantages associated with MAS, including rapid identification of quantitative trait loci (QTL) introgression, selection independent to environmental variation and accuracy. Thus, MAS can be used as a potential method of choice in breeding for abiotic stress tolerance.
Modern rice varieties are sensitive to flooding during germination and early growth, a problem commonly encountered in rain-fed areas, but few landraces including KhaoHlan On, Ma-Zhan (red), Khaiyan and Kalongchi are capable of germination under abiotic stress conditions, enabling research in to anaerobic stress tolerance mechanisms (Angaji et al.,2010). Major QTLs for anaerobic germination (AG) including AG1 and AG2 have being identified, and are being targeted for molecular breeding and cloning (Septiningsih et al., 2009;Angaji et al., 2010). In Sri Lanka 95% of rice is grown under direct seeding (Weerakoon et al., 2011). Sustainability of transplanting systems for rice crop establishment in both irrigated and rain-fed conditions in Sri Lanka is threatened due to increasing rainfall, flooding and labor scarcity for rice cultivation. Thereby adaption to mechanization by DSR farming systems and development of rice varieties tolerant to AG stress is a key trait that will ensure good crop establishment. Thus the availability of anaerobic germination stress tolerant varieties will help increase mechanization in farming systems in Sri Lanka. The AG trait can be combined through gene pyramiding with the SUB1 QTL conferring resistance to submergence, to ensure additive tolerance to flooding during the vegetative stage of the rice crop development.
Among the tolerant landraces and the corresponding QTLs for AG stress tolerance identified at IRRI, the AG1 QTL is located in the chromosome 9 (qAG-9-2) having n logarithm of the odds (LOD) score of 20.3 and explaining 33.5% of the phenotypic variation for the trait of anaerobic germination (Angaji et al., 2010). This QTL was found to be a promising target for marker-assisted selection (MAS) to develop varieties with anaerobic germination tolerance and thus suited for direct seeded rice (DSR). Most of the donors for AG1 QTL are old landraces, with poor phenotypic features such as susceptibility to logging, awns and poor yields thus, marker assisted back-crossing (MABC) can be effectively used to transfer the tolerant QTL to popular varieties, hence introgression of AG1 QTL in to local rice varieties with desirable characters will have a major impact on DSR and improved rice yield.
The objective of the present study is the introgression of the AG1 QTL into local rice variety Bg 358 by crossing with AG1 donor parents-IR64-AG1 and Ciherang-Sub1AG1 and subsequent selection through MABC, with foreground and recombinant selection, in the effective identification of the AG tolerant QTL, in a genetic background of the local, recurrent parent Bg 358, which will result in the generation of elite lines tolerant to AG stress.

METHODOLOGY
Anaerobic germination tolerant donor variety IR64-AG1 and Ciherang-Sub1AG1 were acquired from the International Rice Research Institute (IRRI) and was used for introgression of the anaerobic germination QTL AG1, in to local variety Bg 358. Production of F 1 generations was carried out by crossing the respective parents as shown in Table.1 and Fig.1. Subsequently the BC 1 F 1 backcross populations were produced by hot water treatment method of emasculation (Gracia-Yzaguirre and Carreres, 2008) at the Rice Research and Development Institute Bathalagoda (RRDI) and transplanted under field conditions. b. Donor parent: Ciherang Sub1 AG1 DNA extraction was carried out using leaves of 4weeks old rice plants transplanted in the field using a simple method described by Kottearachchi et al., 2008. Three pairs of simple sequence repeat (SSR) markers mapped to the AG1 QTL (qAG-9-2) region (Angaji et al., 2010) were selected based on the mapped positions of the markers to perform foreground and recombinant selection.
Recurrent parent: Bg 358 a. For foreground selection marker RM24161, a marker tightly linked to (qAG-9-2) and for recombinant selection markers RM8300 and RM553 was used as the proximal and distal markers respectively. The Primer sequences, annealing temperatures and expected product size of each primer pair are given in Table 2. The SSR markers RM24161, RM553 and RM800 were assayed on the parents IR64-AG1, Ciherang-Sub1AG1 and Bg 358 and the markers showing polymorphism among the parental combinations was assayed on the respective BC 1 F 1 progeny lines. PCR was performed in 15μl reaction mixture volumes containing 3μl of 50 ng template DNA, 3μl of 10X PCR buffer, 0.9μl of 25 mM MgCl 2, 0.15μl of 2.5 mM Deoxynucleoside triphosphate mixture, 1μl each of 10µM forward & reverse primers and 0.2μl Taq DNA polymerase (5units/μl). The PCR programme consisted of an initial denaturation at 94°C for 3 minutes; followed by 35 cycles denaturation at 94°C for2 minutes, primer annealing at a primer specific temperature given in Table 2 for 1 minute and an extension at 72°C for 1 minute, and a final extension at 72°C for 5 minutes on a thermal cycler (MyCycler, Biorad Inc., USA). Amplified PCR products and 100bp DNA marker ladder (Promega, USA) were separated using agarose gel electrophoresis on a 2.5% agarose gel separated at a voltage of 60V for 2 hours and stained using 0.5 mg/ml ethidium bromide and visualized with a UV trans-illuminator gel documentation system (Euduro, labnet.Int.Inc, USA). a. b. c.

Molecular Marker Analysis
The AG1 QTL is located in the long arm of the chromosome 9 (qAG-9-2) which has a LOD score of 20.3 explaining 33.5% of the phenotypic variation of the trait as explained by Angaji et al. (2010), therefore, having a major contribution in conferring tolerance to anaerobic germination.

Fig.3. Parental polymorphism of markers a. RM 24161, b. RM 553, c. RM 8300
Thereby, foreground and recombinant selection markers were selected and used effectively to screen for the presence of the QTL AG1 assisting the selection of elite lines with tolerance to anaerobic germination and for detection of additional recombinants for fine mapping of the QTL. The marker RM24161 showed length polymorphism between the respective parents, Bg 358 and IR64-AG1 and was chosen as the foreground selection marker to genotype the population Bg 358 / IR64-AG. For recombinant selection RM553 was selected for genotyping the population Bg 358 / IR64-AG1 and RM8300 was chosen to genotype the population Bg358 / Ciherang-Sub1AG1.

Genotyping of BC 1 F 1 Bg 358/ IR64-AG1 with RM24161 -Foreground selection
Foreground selection was utilized to select for the presence of the target AG1 QTL which enabled easy selection of the AG trait that would otherwise be difficult to select through laborious and time-consuming phenotypic screening procedures. The marker RM24161 was selected based on the location of the marker on the long arm of the chromosome 9, as the closest linked marker to AG1 (qAG-9-2), for foreground selection as carried out by Septiningsih et al., 2009. Subsequent screening with RM24161 resulted in generating good quality genotyping results validating the marker to be used in marker assisted selection of the AG1 QTL in future backcross programs and gene pyramiding. A total of 48 BC 1 F 1 plants were genotyped and results showed segregating banding patterns (Fig.4), 29 heterozygous double bands were observed possessing both the respective alleles from the donor (AG1 allele) and recurrent parent (Bg 358) at the expected band size of 279 bp (www.gramene.org). A total of 16 homozygous single bands (280bp) were observed corresponding to the recurrent parent Bg 358 and 3 homozygous single bands (270bp) were observed corresponding to the donor IR64-AG1. Occurrence of homozygous single bands corresponding to the donor parent in a BC 1 F 1 population is not expected according to the expected Mendelian segregation ratio of 1:1 of the heterozygous DR : Recurrent parent RR respectively; the 3 plants that resulted in donor parent genotype was due to selfing that occurred during back crossing with hot water method of emasculation. The 29 individual plants which showed heterozygous double bands will be selected for generation of BC 2 F 1 progeny aiming at increasing the recurrent parent characters by backcrossing with Bg 358, while possessing the QTL AG1 which confers resistance to anaerobic germination.

Genotyping of BC 1 F 1 Bg 358 / IR64-AG1 with RM553-Recombinant selection
The second level of molecular selection is recombinant selection, which involves selecting backcrossed progeny with the target QTL and recombination events between the target locus and flanking markers. Recombinant selection is done to reduce the size of the donor chromosome segment containing the target locus, thus reducing the background effect, which contributes immensely to minimizing linkage drag (Hospital, 2005). Markers selected for recombinant selection included, RM8300 as a proximal marker and RM553 as a distal marker targeting the QTL region AG1 (qAG-9-2), in order to select recombinants for fine mapping of the region.
A sum of 10 individual samples were selected based on foreground selection with the molecular marker RM24161; as individuals possessing heterozygous bands to be genotyped with RM553 for recombinant selection. Upon carrying out PCR reactions clear banding patterns were not observed and proper amplification was not seen. It was inferred that further optimization in PCR reactions and cycling time had to be carried out to. Due to the ineffectiveness of the proximal and distal markers chosen, it was suggestive through this study to choose flanking markers RM105 and RM219 respectively, which are closer to the AG1 (qAG-9-2) locus ( Fig.1) for recombinant selection for fine mapping in the future, which will be used to effectively to select lines with reduced background effect from the donor parent.

CONCLUSION
The molecular marker RM 24161, a tightly linked marker to the anaerobic germination tolerant QTL AG1 (qAG-9-2) was successfully used as a foreground selection marker. The 29 plants that were selected from the foreground marker will be used for backcrossing resulting in BC 2 F 1 generation, which will be used in the development of elite lines tolerant to anaerobic germination. The proximal and distal markers used for recombinant selection was ineffective, thus it was also suggestive through the study to select flanking markers RM219 and RM105 for recombinant selection.