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1 Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2012, 2 (6): ( ISSN : CODEN (USA) : JMBRB4 Molecular Mapping of the Chromosomal Regions Associated with Zinc Content in Grains of Rice (Oryza sativa L.) using Microsatellite Markers L. Madhuri Lalasa, K. Radhika, C. N. Neeraja, V. Ravindra Babu * and G. Usharani Crop Improvement Section, Directorate of Rice Research, Rajendranagar, Hyderabad ABSTRACT Rice is the most important grain with regard to human nutrition and caloric intake, providing more than one fifth of the calories consumed worldwide by the human species. The present study was conceptualized and executed with the prime objective of mapping the chromosomal regions associated with zinc content involving the F 2 populations derived from a cross between Samba Mahsuri and Ranbir Basmati using microsatellite markers derived from the genomic regions associated with zinc metabolism. Out of the 45 microsatellite markers used for the parental polymorphism studies, 16 markers were polymorphic, 8 markers were monomorphic and 21 were not amplified. Three polymorphic markers associated with cation uptake viz., SC 129, SC 135 and SC 141 were used to assay F 2 individual plants. The linkage distance of these three markers, SC 129, SC 135 and SC 141 with their respective genes OsZIP1, OsZIP8 and OsNRAMP7 on chromosomes 3, 5 and 12 were found to be 47.8 cm, 15.2 cm and 44.6 cm, respectively. The methodology of selective genotyping could successfully identify the chromosomal regions associated with zinc content in grains and the association could be made much effective by analyzing large F 2 population using more functional polymorphic markers. Key words: Biofortification, Mapping, Microsatellite, Rice, Zinc INTRODUCTION Rice (Oryza sativa L.) is a unique crop of great antiquity and akin to progress in human civilization and fulfils the nutritional requirements of more than half of the world s population. Nutrient deficiencies including zinc is a serious public health problem concerning about 124 million children worldwide. The task of improving the nutritional quality of rice through conventional breeding involving phenotype-based selection is very difficult and sometimes impossible because of various reasons. Biofortification of rice for enhanced zinc content involving molecular breeding through the utilization of DNA markers is suggested to be the best strategy. The first and foremost prerequisite to use molecular markers in breeding for enhanced micronutrient content in rice is the identification of molecular markers closely linked to the target traits. Of late, the candidate genes involved in the biosynthetic pathways were considered to be the potential targets for identifying gene specific molecular markers [8]. Quantitative Trait Loci (QTL) associated with cation homeostasis was studied using the model plants like rice and Arabidopsis [10] through database searches to identify the sequences homologous to the already characterized proteins. In the present investigation, microsatellite markers located in the vicinity of the putative candidate genes like zinc transport, yellow stripe like, ZIP, FRO, NRAMP, iron stress and uptake related etc. have been selected. Iron 900

2 Regulated Transporter (IRT) showed capacity for zinc transport and led to ZIP denomination of proteins (Zrt/Irt related Proteins) to map the chromosomal regions associated with zinc content in the grains. Considering these points, an attempt has been made in the present study to identify the specific associated regions of the chromosomes with zinc metabolism showing high and low zinc content in grains of F 2 individual plants derived from the cross between Samba Mahsuri and Ranbir Basmati, using microsatellite markers derived from the genomic regions associated with Zn metabolism. MATERIALS AND METHODS A cross was made between Samba Mahsuri, a popular rice variety with fine grain quality having high consumer acceptance, and Ranbir Basmati, a scented zinc rich rice variety and individual plants with high (24 plants) and low (22 plants) zinc content in grains selected in F 2 generation were utilized for mapping the regions associated with Zn content. The DNA was extracted from freshly germinated young seedlings of parental lines and F 2 population using the method of [11]. The purity and concentration of the isolated genomic DNA samples were estimated by UVabsorption spectrophotometer (Beckman DU 650 model) as per the procedure described by Sambrook and Russell [9]. Agarose gel electrophoresis (0.8%) was carried out for confirming the quality and quantity of the isolated DNA using a known concentration of λdna. The genomic DNA was subjected to PCR amplification as per the procedure described by Chen et al. [1]. DNA samples were amplified in 10 µl reaction volumes containing 1X PCR buffer [10 mm Tris HCl (ph 8.3), 50 mm KCl, 1.5 mm MgCl 2, 0.01% (v/v) gelatin] (Bangalore Genei, India), 0.2 mm of each dntps (Bangalore Genei, India), 10 pmol of each primer and 1 U of Taq polymerase (Bangalore Genei, India). A PCR profile consisting of 5 min of initial denaturation at 94 C, 35 cycles of 1 min of denaturation at 94 C, 1 min of annealing at 55 C, 2 min of extension at 72 C and 7 min of final extension at 72 C was followed in a Thermal cycler (Perkin Elmer-Gene Amp PCR System 9700, USA). Table 1. Details of microsatellite markers used and their chromosomal positions Microsatellite markers Nature of the targeted gene Chromosomal Position SC 100 ZT 1 SC 101 ZT 2 SC 102, SC 103, SC 104 ZT 3 SC 105 ZT 4 SC 106, SC 107, SC 108 ZT 5 SC 109, SC 110, SC 111 ZT 6 SC 112 ZT 7 SC 113, SC 114, SC 115, SC116 ZT 8 SC 117 YSl 1 SC 118, SC 119 YSl 2 SC 120, SC 121, SC 122, SC 123 YSl 4 SC124, SC 125 YSl 5 SC 126 YSl 8 SC127, SC 128 FRO 4 SC 129, SC 133 ZIP 3 SC 130 ZIP 6 SC 131 ZIP 4 SC 132 ZIP 8 SC134, SC 135 ZIP 5 SC136 NRAMP 3 SC137 NRAMP 6 SC 138 NRAMP 2 SC 139 NRAMP 7 SC140 NRAMP 1 SC 141 NRAMP 12 SC143 OsFer1 11 SC144 OsFer2 12 SC145 Os Fe stress 2 SC denotes the microsatellite markers developed at Directorate of Rice Research, Hyderabad ZT: Zinc Transport gene, YSl : Yellow Stripe like gene, ZIP : Zrt/Irt related Protein gene, (Zrt/Irt: Zinc/Iron regulated transporter gene), NRAMP: Natural Resistance-Associated Macrophage Protein gene, OsFer1:Oryza sativa Ferric1 gene, OsFer2:Oryza sativa Ferric2 gene, Os Fe stress: Oryza sativa Ferrous stress gene The amplified products were resolved on 3% agarose gels, stained with ethidium bromide and visualized under UV in a gel documentation system (Alpha Innotech, USA). Microsatellite markers located in the vicinity of the putative 901

3 candidate genes were selected for mapping the chromosomal regions associated with zinc rich regions in rice grains. Parental polymorphism was surveyed between Samba Mahsuri and Ranbir Basmati using 45 microsatellite markers derived from the genomic regions associated with iron and zinc metabolism. The details of the microsatellite markers employed and their chromosomal location is enlisted in Table 1. Iron and zinc content of grain samples were estimated by Atomic Absorption Spectrophotometer as suggested by Lindsay and Norvell [5]. The strategy of selective genotyping was carried out with the F 2 plants showing extreme phenotypes exhibiting high and low zinc content in grains individually with all polymorphic markers as suggested by Nandi et al. [7]. Each gel was scored for maternal, paternal and heterozygous banding pattern and scored accordingly. The maternal band was designated as B, parental band as R and heterozygous as band H. Homozygotes were given a value of 0 or 1 based on their phenotype group. Heterozygotes were given a value of 0.5. By using these values, recombination frequency in relation to total sample was calculated in percentage. Kosambi s mapping function [4] was used to convert recombination frequencies into map distances in centimorgans (cm). The genetic distances were located in BAC (Bacterial Artificial Chromosome) contigs consisting of the candidate genes related to Zn metabolism. RESULTS AND DISCUSSION Parental polymorphism between Samba Mahsuri and Ranbir Basmati was studied using 45 markers, of which 16 markers (35.56 %) showed polymorphism, 8 markers showed monomorphism and 21 were not amplified (Table 2). Three polymorphic markers, which are associated with cation uptake viz., SC 129 marker and SC 135 marker based on ZIP (Zrt/Irt related protein) and SC 141 marker based on NRAMP (Natural Resistance-Associated Macrophage Protein), were used to assay 24 and 22 F 2 individual plants showing high and low zinc content in grains, respectively to identify specific regions of the chromosome associated with zinc metabolism. Table 2. Microsatellite markers exhibiting polymorphism between Samba Mahsuri and Ranbir Basmati Polymorphism Polymorphic Monomorphic Not amplified Microsatellite markers SC 101, SC 103, SC 105, SC 112, SC 113, SC 117, SC 120, SC 129, SC 131, SC 132, SC 135, SC 138, SC 139, SC 141, SC 144 and SC 145 SC 114, SC 116, SC 118, SC 121, SC 126, SC 127, SC 130 and SC133 SC 100, SC 102, SC 104, SC 106, SC 107, SC 108, SC 109, SC 110, SC 111, SC 112, SC 115, SC 119, SC 122, SC 123, SC 124, SC 128, SC 134, SC 136, SC 137, SC 140 and SC 143 The markers used for selective genotyping studies amplified the specific allele of Ranbir Basmati in homozygous condition in more F 2 plants having high zinc content (Table 3). With regard to the specific allele of Samba Mahsuri, amplification was observed in homozygous condition in more F 2 plants with low zinc content (Table 4). This situation was very clearly shown with respect to SC 135 marker. With respect to other two markers SC 129 and SC 141, the amplification of the alleles in heterozygous state was observed in majority of the F 2 plants having both high and low zinc contents in grains (Fig. 1). The marker SC 141 failed to show amplification in 5 F 2 plants having high zinc and 2 F 2 plants with low zinc content in their grains. (Fig. 2). Table 3. Selective genotyping in F 2 lines with high zinc in grain SC 129 SC 135 SC 141 Ranbir Basmati specific allele Samba Mahsuri specific allele 3-2 Heterozygous Not amplified Total Table 4. Selective genotyping in F 2 lines with low zinc in grain SC 129 SC 135 SC 141 Ranbir Basmati specific allele Samba Mahsuri specific allele Heterozygous Not amplified Total No

4 Fig. 1. Segregation pattern of SC 129 in F 2 population with high / low zinc content in their grains Fig. 2. Segregation pattern of SC 141 in F 2 population with high / low zinc content in their grains Selective genotyping in F 2 lines with high zinc in grains had shown that with respect to SC 135 marker, the specific allele of Ranbir Basmati was found in homozygous condition in 21 F 2 plants, but specific allele of Samba Mahsuri was not found in homozygous condition in any plant, suggesting the possible involvement of the gene, OsZIP8 in zinc uptake, as SC 135 marker was found to be tightly linked (15.2 cm) with this gene located on chromosome 5 903

5 (Fig. 3). The strategy of selective genotyping was found to be effective in identifying specific associated regions of the chromosomes as suggested by Nandi et al. [7]. The association could be made more effective with analysis of large number of F 2 plants utilizing more functional molecular markers. This methodology of selective genotyping could successfully identify the chromosomal regions associated with zinc content in grains. The genetic distances of the three markers viz., SC 129, SC 135 and SC 141 from their corresponding genes on the chromosomes 3, 5 and 12, respectively were identified. These positions were then estimated in accordance to genetic markers assigned to a BAC contig related to predicted sequences (Table 5). The SC 129 marker was found to be on chromosome 3 at a distance of 47.8 cm Fig. 3. Segregation pattern of SC 135 in F 2 population with high / low zinc content in their grains Table 5. The location of candidate genes related to zinc metabolism SSR Marker Chromosome No. Related genes Linkage distance (cm) BAC contig From To SC OsZIP ACO SC OsZIP AC SC OsNRAMP AL AL away from the gene, OsZIP1 responsible for zinc accumulation in grain. This genetic map was located in a BAC contig AC092262, in which the predicted sequences were found to have bases between and The studies made earlier had shown that OsZIP1 was found to be on chromosome 3 at a map distance of 83.3 cm [3]. There is a molecular evidence for a function in cation transport for Arabidopsis ZIP1, ZIP2 and ZIP3 as they could restore zinc uptake in the yeast double mutant zrt1 and zrt2. The Arabidopsis ZIP members were investigated by reverse genetic methods, which provided further information about the molecular functions of these genes [6]. The SC 135 marker was linked to the gene OsZIP8 located on chromosome 5, at a genetic distance of 15.2 cm. This genetic map was found to be in a BAC contig AC and the candidate genes responsible for Zn content were found to be bases between and The SC 141 marker was located on chromosome 12 and the linkage distance between this marker and the gene OsNRAMP7 was calculated to be 44.6 cm. The gene OsNRAMP7 had two BAC contigs, as the AL sequence had a gap in the regions corresponding to the 5 end of gene. One is AL772426, where the gene sequences responsible for Zn content were found to be bases between and The other BAC is AL and the predicted sequences were found to have bases between and It was reported earlier that OsNRAMP7 was found on chromosome 12 at a distance of 94.6 cm [3]. The evidence for the involvement of plant NRAMP proteins in cation transport was reported earlier by Curie et al. [2] (Fig. 4) More regions associated with zinc content in grains 904

6 can be identified with screening of more F 2 population involving either more microsatellite markers located in the vicinity of the candidate genes or the functional markers. Thus, the findings from the present study indicated that much more regions associated with zinc content in the grains can be identified with the utilization of more microsatellite markers located in the candidate genes associated with zinc metabolism. The knowledge of QTL analysis and the information of DNA in identified genes on mineral accumulation are helpful in the identification of interesting alleles of relevant genes. Most of the markers studied in the mapping experiment have shown a clear association with the trait despite the less number of F 2 samples analyzed. Thus the mapping results in the present study suggest the strategy of identification of microsatellite markers in the vicinity of candidate genes involved in the cation metabolism and their use in mapping to be very appropriate. Utilization of these novel gene sources is underway in rice breeding programs in the years to come. Fig. 4. Positions of the microsatellite markers in relation to their candidate genes REFERENCES [1] X Chen; S Temnykh; Y Xu; Y Cho; SR Mccouch, Theor. Appl. Genet., 1997, 95, [2] C Curie; JM Alonso; M Jean; JR Ecker; JF Brait, Biochem. J., 2000, 347, [3] G Jeferson; JS Ricardo; AG Fett- Neto; JP Fett, Genet. Mol. Biol., 2003, 26, 4, [4] DD Kosambi, Am. Eugen., 1994, 12, [5] WL Lindsay; WA Norvell, Soil Sci.Soc. Am. J., 1978, 42, [6] P Maser; S Thomine; JI Schroeder; JM Ward; K Hirschi; H Sze; IN Talke; A Amtmann; FJ Maathuis; D Sanders; JF Herper; J Tehieu; M Gribskov; MW Persans; DE Salt; SA Kim; ML Guerinot, Plant Physiol., 2001, 126, [7] S Nandi; PK Subudh; D Senadhira; NL Manigbas; S Sen-Mandi; N Huang, Mol. Gen. Genet., 1997, 255, 1-8. [8] S Pflieger; V Lefebvre; M Causse, Mol. Breed., 2001, 7, [9] J Sambrook; DW Russell, Molecular cloning-a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, [10] D Vreugdenhil; MGM Arts; M Koornneef; H Nelissen; WHO Ernst, Plant Cell Environ. 2004, 27, [11] KL Zheng; B Shen; HR Qian, Rice Genet. Newslet., 1991, 8,