SPAD chlorophyll meter reading
The third leaf from the apex was selected to record the chlorophyll content. Selected leaf of groundnut was clamped avoiding the mid rib region into the sensor head of SPAD meter. A gentle stroke was given to record the SPAD reading and the average of such four strokes was considered. Since groundnut has tetra foliate leaf, SCMR was recorded in all the four leaflets and the average value was recorded.
Quantification of D13C in leaf samples
About five to six third fully expanded leaves were collected and dried at 80°C for three days. The dried leaves were powdered in a mortar and pestle. The D
13C analysis was done at the National facility for quantification of stable isotopes in the Department of Crop Physiology, UAS, Bangalore. Carbon isotope ratios (
13C/
12C) in comparison with the Pee and Dee Belemnite standard were measured using continuous flow isotopic mass spectrometer (IRMS). The IRMS facility consists of flash elemental analyzer (CE-EA 1112), for sequential combustion of biomass samples and open slit interference (coulo 3). Finely powdered dry leaves samples were accurately weighed in the range of 1.0 to 1.2 mg into silver capsules. The crimped capsules with the sample were placed sequentially in the caraousel of the auto sampler. The samples are dropped at specific interval of time along with a pulse of pure O
2 in to the oxidation reactor.
The combustion (oxidation) reactor contains chromic oxide and silvered cobaltous-cobaltic-oxide heated to 105°C. The biomass is completely oxidized to produce CO
2, N
2O and H
2O. These gases were swept into the reduction furnace using helium carrier gas. The reduction column contains reduced copper in quartz tubes heated to 680°C. In this reaction, the N
2O, is reduced to N
2 and the excess O
2 is absorbed. The resultant gases are then flushed through scrubbers to trap CO
2 and water. The pure CO
2 and N
2 gas after passing through a GC column (5° A molecular sieve) and a thermal conductivity detector (TCD) into the ion source of IRMS. At the source, CO
2 is ionized by electron impacts ionization to produce molecular radicals (CO
+). When accelerated radicals pass through a strong magnetic field it is deflected with the radius of deflection being proportional to the molecular mass of the radicals. These deflecting
12CO
2 and
13CO
2 are collected by the Faraday cups and the signal is amplified and transmitted to the computer and displayed.
DNA isolation
Total genomic DNA was isolated from the groundnut seedlings of 10-15 day old using the cetyl trimethyl ammonium bromide (CTAB) method as described by
(Saghai-Maroof et al., 1984) with some modifications. Freshly harvested groundnut leaves (200 mg) were grounded to fine powder in pre-chilled mortar using liquid nitrogen and transferred into 2 ml centrifuge tubes containing 750 µl extraction buffer (0.1 M Tris Hcl (pH 8.0), 0.02 M EDTA (pH 8.0), 2% CTAB (w/v), 1.4 M NaCl, 0.2% β-mercaptoethanol (v/v) and 2% polyvinyl pyrrolidone (w/v)) and incubated in water bath at 65°C for 30 min, for DNA purification 5 µl of RNase (10 mg/ml) was added and incubated again at 65°C for 10 min. Tubes were cooled down to room temperature and equal volume of chloroform: isoamyl alcohol (24:1) was added, mixed thoroughly and centrifuged at 13,000 g for 10 min at room temperature. Supernatant was carefully transferred to fresh sterile 1.5 ml centrifuge tubes and 0.7 volume of ice-cold isopropanol was added to precipitate DNA and incubated at -20°C for 2 h. Tubes were then centrifuged at 13,000 g for 10 min at 4°C, the pellet was washed twice with 70% ethanol. Tubes were air dried at room temperature and DNA was dissolved in 100 µl 1X TE buffer and stored at 4°C. Quantification of DNA was performed by measuring the absorbance at 260 nm in Nanodrop (Thermoscientific) quality of the DNA was checked on 1% agarose gel.
Screening Parental lines with SSR markers
Parents, NRCG12568 and NRCG12326 were screened for polymorphism by using 350 SSR markers (
Hopkins et al.,1999;
He et al., 2003;
Ferguson et al., 2004;
Moretzsohn et al., 2004 and
Mace et al., 2007) which are polymorphic in other mapping population available at ICRISAT. Out of 350 SSR markers 145 were dye labeled and 205 were unlabelled SSR markers. List of all dye labeled and unlabelled SSR markers are presented in supplementary data Table 1 and Table 2 respectively.
Polymerase chain reaction
Polymerase Chain Reaction (PCR) was performed by using a Touch - Down PCR. DNA amplification was performed in 5 µl reaction mixture using Gene Amp® PCR system 9700. The reaction mixture contained 5 ng/µl template DNA, 4-5 pM / µl SSR primers pair (Forward and Reverse), 25 mM Mgcl
2, 2 mM DNTP’s, 10X PCR buffer and 1U/µl Taq DNA polymerase (Bioline) for unlabeled primers and for labeled primers 5 ng/µl template DNA, 4-5 pM/µl SSR primers pair (Forward-labelled and Reverse), 25 mM Mgcl
2, 2 mM DNTP’s, 10X PCR buffer and 1U/µl Taq DNA polymerase (sib enzyme).The PCR product was separated in 6% poly acrylamide gel at 800 voltages for 2 hours and visualized by silver staining
(Kolodny 1984). Before loading PCR Products in the sequencing gel, amplification was checked on 1.2 per cent agarose gel. For the separation of DNA fragments, non-denaturing polyacrylamide gelelectrophoresis (PAGE) and capillary electrophoresis were used.
Non-Denaturing Polyacrylamide Gel Electrophoresis (PAGE)
After PCR amplification, 2µl of orange dye was added to 5 µl reaction mixture. Then 2 µl of this reaction mixture was loaded on each lane of 96-track of 6% non-denaturing PAGE and as the base pair marker, 100bp DNA ladder was loaded on both the corners of the gel. Recipe for 6% gel consisted of 52.5 ml of distilled water, 7.5 ml of 10X TBE, 15 µl of acrylamide-methylbisacrylamide 29:1 (V/V), 100µl of TEMED and 450 µl of ammonium per sulphate (APS). Electrophoresis was run at 800 volts for 2 hours in 0.5X TBE running buffer, using BIORAD sequencing gel unit.
PCR products were visualized by using silver staining protocol (
Kolodny 1984). Initially, the gel was rinsed with distilled water for 5 min with gentle shaking followed by soaking in 0.1% CTAB for 20 min (1.5 g in 1.5 litre of water) then kept in 0.3% liquid ammonia for 15 min (19.5 ml of 25% liquid ammonia solution in 1.5 litre of water) and later placed in 0.1% silver nitrate solution (1.5 g of silver nitrate + 6 ml of 1M NaOH in 1.5 litre of water and then titrated with ammonia solution till it became colorless) followed by rinsing in water for 1 min. After this gel was kept for developing in solution (22.5 g sodium carbonate and 400 µl of formaldehyde in 1.5 ml of water) till bands became conspicuous. The gel was kept in water for 5 min to stop the staining reaction and fixed in 3% glycerol. After staining the gel, bands were scored to check the polymorphism in two parents NRCG12568 and NRCG12326.
Capillary electrophoresis (ABI 3100 genetic analyzer)
Capillary electrophoresis (ABI3100 genetic analyzer) is used for markers which show 2 to 3 base pair difference which could not be resolved in poly acrylamide gel. Amplified products were separated by using capillary electrophoresis. Total volume of 14.5 µl contains 1.5 µl PCR products of TAMARA, HEX and FAM-labeled products were mixed separately with 7 µl of Hidi, 0.15 µl of Rox-500 size standard and 2.85 µl of double distilled water (adjusted as per dye and number of primers used for multiplexing). Then the samples were kept for denaturation for 5 min at 94°C and chilled on ice for 5 min. Before placing plates containing samples were centrifuged at 900 rpm for 1 minute and size fractioned using capillary electrophoresis on an ABI 3100 DNA Genetic Analyzer (Applied Biosystems). The “G5” dye set, “Genescan POP4” run module and GeneScan™ 500 LIZ® (Applied Biosystems), analysis module were employed and the fragments were separated in 36cm capillary array. After completion of the run, the A and B peak patterns were sized using software GeneMapper v3.5 (Applied Biosystems).
