Simarouba Glauca) for IMPROVED PRODUCTION L

J.Res. ANGRAU 38(1&2)1-5, 2010 RAPD AND SCAR MARKER FOR DETERMINATION OF SEX IN SIMAROUBA (Simarouba glauca) FOR IMPROVED PRODUCTION L. PRASANTHI, B. V. BHASKARA REDDY, K. REKHA RANI, P. MAHESWARA REDDY and K.RAJA REDDY Regional Agricultural Research Station, Tirupathi, ANGR Agricultural University, Andhra Pradesh, India. ABSTRACT A total of 250 random oligonucleotide primers were surveyed to identify sex of Simarouba glauca, a tree born oilseed crop. Primer OPA-08 produced a consistent marker, differentiating female from male and andromonoecious sex plants. An identified random amplified polymorphic DNA (RAPD) marker, OPA-08, linked to male sex in Simarouba was cloned and sequenced. Their end sequences were used to design an allele-specific sequence characterized amplicon region (SCAR) primer SCAR (12f/r), which was promising in male and andromonoecious sex. The marker designed was amplified at a specific site of 915 bp only in male and andromonoecious plants. This would help to identify the sex of tree born oilseed crop Simarouba. Simarouba glauca is a tree born oilseed crop. It belongs to the family Simaroubaceae and is a native of El Salvador (Central America). The seeds of Simarouba are economically very important since they contain 55-60% edible oil (Armour, 1959). As demand for edible oil is steadily increasing and production being stagnated it is inevitable to resort to the tree born oilseed crop which can be successfully grown in the available non arable lands. Simarouba, the oil tree is one such ideal evergreen versatile crop which can grow even in waste lands. It is being cultivated in Bihar, Orissa, Gujarat, Rajasthan, Karnataka, Tamilnadu and Andhra Pradesh etc. Simarouba is polygamodioecious with three types of plants (Fig.1, 2, 3) namely pistillate (Female flowers), Staminate (Male flowers) and andromonoecious (male dominated bisexual flowers) (Joshi and Hiremath, 2000). Flowering is annual, beginning in December and continuing up to next February and bears fruits during March –April and fruits can be collected in the month of May. The inflorescence is a compound panicle with main axis showing racemose pattern and ultimate branches having dichasial/ monochasial cymes. The time taken from bud initiation to anthesis is about 15 days. The male inflorescence has a longer peduncle than the female. The length of the panicle is 20-45 cm in pistillate whereas it is 50-60 cm in andromonoecious and staminate inflorescences. The bisexual flowers form 0.5-5% of the total flowers in an andromonoecious panicle. Bisexual flowers are the largest with 12-15mm diameter followed by male flowers with 9-12 mm diameter and the female flowers are the smallest with 6-8 mm diameter. email : [email protected] 1 PRASANTHI et al. The male inflorescence bear maximum of 2500-6250 flowers, the andromonoecious will have 1200-5500 flowers and the female bear 200-650 flowers (Fig.4). In male and bisexual flowers, androecium has 10 stamens in two whorls. In female flowers the androecium is represented by 10 staminodes (sterile stamens) with poorly developed anthers not producing any pollen grains. The pollen is creamy and light, easily carried by wind and insects for pollination. Simarouba usually flowers after 3-4 years after planting .The waiting time from sowing to flowering is long, hence growers need to ensure that a seedling is a female for good harvest.The determination of the sex of Simarouba seedling prior to the flowering stage would avoid the need for removing undesired sex types from the field as only five percent male or andromonoecious plants in a field are sufficient for pollination.By cultivating male and female plants, planting space, fertilizers and water could be optimized. However, they cannot be distinguished at the seedling and vegetative stages of growth. Identification of sex types prior to propagation, especially in polygamodioecious plant species with a long juvenile cycle such as Simarouba, would result in higher fruit production and increased profitability. Molecular markers like ISSR and RAPD markers were developed by Deputy et al., (2002) and Gangopadhyay et al., (2007) to determine sex in papaya in pre flowering stage. Similar studies were initiated to determine sex at seedling stage in Simarouba for improved yield and RAPD method was successfully developed to identify the gene related to sex of the plant. MATERIALS AND METHODS The leaf material of Simarouba glauca was collected from the trees of 5 years old grown at Forest Research Station, Kukkaladoddi, Tirupati Division, Andhra Pradesh. DNA extraction: Tender and fresh leaf samples of 32 male and 27 female known plants were collected and DNA was extracted using CTAB method (Sambrook et al., 1989). 400 mg of leaves were completely homogenized with liquid nitrogen. Extraction buffer(100 mM Tris HCl (pH 8.0), 20 mM EDTA, 1.4 M NaCl, 3% CTAB, 1% PVP, 1% â-mercaptoethanol was added in 2 ml eppendorff tubes filled with leaf powder to the volume 1.5 ml and mixed well. The tubes were incubated at 65°C for 1/2 h with repeated shaking. Equal volume of chloroform : isoamyl alcohol mix(24:1) was added and mixed thoroughly followed by centrifugation at 10000 rpm for 10 minutes at room temperature. Supernatant was transferred to another sterile 2 ml eppendorff tube and chloroform: isoamyl alcohol step was repeated until a clear supernatant was obtained. 5 M NaCl (0.5v/v) was added and mixed gently followed by addition of 0.8 volume of ice cold isopropanol to the aqueous layer and incubated overnight at -20°C. The content was centrifuged at 13000 rpm for 20 minutes at 4ºC and supernatant was discarded. 2 RAPD AND SCAR MARKER FOR DETERMINATION OF SEX IN SIMAROUBA The pellet was washed with 70% ethanol and air dried. Then the pellet was dissolved in 200 µl of TE/distilled water. The DNA was incubated with 10 mg/ml of RNase for 1 h at 37°C. To this equal volume of phenol: chloroform (25:24) was added and centrifuged at 1000 rpm for 10 minutes at room temperature. This step was repeated thrice followed by washing with an equal volume of chloroform: isoamyl alcohol twice. DNA was precipitated with 0.1 volume of 3M sodium acetate and twice the volume of ice cold ethanol at -20°C for one hour. The resultant pellet obtained after centrifugation was washed with 70% ethanol, air dried and dissolved in 50 µL of TE/ distilled water. The quality and quantity of DNA were checked through 1% agarose gel by electrophoresis. The DNA obtained was quantified using Nanodrop spectrophotometer. RAPD analysis: Genomic DNA was used as template for PCR amplification as described by Williams et al., (1990). Amplification was carried out with 25 µL reaction volume containing 1X Assay buffer, 1.5 mM Mg Cl2, 0.2 mM dNTP, 5 pmoles primer (Operon Technologies Inc.), 25-30 ng of genomic DNA and 1 unit of Taq DNA polymerase. Amplification was performed in 0.2 ml thin walled tubes using a thermocycler (Corbett, Australia) programmed for initial denaturation at 94 ºC for 5 minutes, followed by 45 cycles of denaturation at 92 ºC for 1 minute, annealing at 35 ºC for 2 minutes, primer extension at 72 ºC for 2 minutes and a final extension at 72 ºC for 10 minutes .PCR amplified products were subjected to electrophoresis on 1% agarose gel in TBE buffer. The electronic image of ethidium bromide stained gels were captured using Alpha imager system. Cloning and sequencing of RAPD markers RAPD primer OPA-08 (Operon Technologies Inc., Alameda, CF, USA) was used to amplify genomic DNA of male Simarouba plant. Amplified fragments were separated on a 1% (w/v) low melting point agarose gel before being excised and purified by means of the QIAquick Gel Extration Kit (Qiagen, Valencia, CA, USA). The purified DNA fragments were ligated and transformed with the pTZ57R/T Easy Vector System. Cloned RAPD fragments were identified via Colony PCR with RAPD primer of OPA-08 and confirmed by restriction digestion with EcoRI (Fig. 6). SCAR primer design and amplification On the basis of the sequence of cloned RAPD product, oligonucleotide primer pairs of 12 and 14 bases were designed using Oligo Explorer 2.0 software for specific amplification of the loci identified by selected RAPD marker. Each primer contained the original 10 bases of the RAPD primer sequence plus the next 2 and 4 internal bases (Table 1). Care was taken 3 PRASANTHI et al. to avoid possible primer dimer or secondary structure formation. Primers were synthesized by Operon Technologies, Inc. SCAR amplification of female, male and andromonoecious genomic DNA was performed in 25 µL reaction volumes containing the same components used for RAPD analysis. PCR amplification consisted of 35 cycles of 1 minute at 92 °C, 1 minute at 51 °C, and 2 minute at 72 °C followed by a final extension of 10 minute at 72 °C. Agarose electrophoresis was performed as described for RAPD analysis. RESULTS AND DISCUSSION About 250 random primers were screened to select marker showing good amplifications which were further used to identify regions associated with sex linked marker. Specific amplification with 900bp band in males and andromonoecious (male dominated bisexual flowers) was produced by OPA 8 (Fig.5). Reproducibility of the primer was tested by repeating the PCR amplification thrice under similar conditions. The amplified products were separated on 1% agarose gel and were analyzed by comparing them with 1Kb DNA ladder. From the sequence of 915bp, two SCAR primers were designed with 12, and 14 bp (Table-1). SCAR 12f/r with consistent results was utilized in screening of all simarouba lines (Fig 7) and gave specific band at 915bp in males and andromonoecious plants only A major problem associated with RAPD technology is the reproducibility of the profiles and it has been the subject of considerable debate among various investigators (Melotto, et al 1996 Zhag and Stommet, 2001).

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