Hybrid Testing In Pigeonpea Using DNA Fingerprinting By SSR-Markers

DOI: 10.21276/haya.2018.3.7.4 Abstract: Pigeonpea (Cajanus cajan (L.) of Fabaceae family belongs to genus Cajanus usually grown in semi-arid tropics of Asia and Oceania, Africa and America. This crop has been a best source for improving food and soil quality amongst farmers. However, its seed have been always questioned for purity. This problem is managed by using polymorphic SSR markers. In present study, a DNA fingerprints generated by seven SSR markers and hybrid testing is performed on Pigeonpea test samples along with parental lines. The seed samples of pigeonpea were germinated in laboratory and three week old leaves samples were used for DNA isolation by CTAB method. A total of 9 alleles were observed in three test samples using three primers out of seven primers. The screening of the allelic data associated with the three cultivated varieties, revealed markers (CcM0246) displayed unique allelic profiles for one variety. Yet, the genetic fingerprinting data is not well resolved to potentially distinguished two bands of hybrid that are merely of 4-8 bp to confirm hybrid testing of seed. Hybrid Testing of pigeonpea may be confirmed including more SSR primers prepared from genomic DNA of pigeonpea.


INTRODUCTION
Pigeonpea, Cajanus cajan [L] is a drought tolerant crop and one of the most important legumes grown in the Tropic and sub tropic region, popularly known as red gram (arhar dal / tur dal). It is largely used as supplement cereal because it is a rich source of protein for humans.
Pigeonpea is a major food legume in South Asia and East Africa. However, India is the world's largest producer (3.3 mha). Globally the cultivation of pigeonpea is about 4.92 mha which got sixth rank following other legume plants [1]. The protein percentage in seeds is about 20-22% in this crop with enough amount of essential amino acids. Pigeonpea fixes atmospheric nitrogen and improves the quality and structure of soils because of deep root system while the perennial type of pigeonpea provide fuel wood, food and fodder. But the crop suffers from several biotic and abiotic stress, production suffers greatly from mixing of low quality and contaminated seeds. So, there is need to include new genetic resources in pigeonpea breeding with the help of modern tools of Biotechnology to overcome the yield constraints. Marker assisted breeding is one of the solution to achieve higher yield. This plays vital role in assessing seed purity [1,2].
Microsatellites or simple sequence repeats (SSRs) are short tandem repeats of 1-6 neucleotides evenly separated in genome and are present in all eukaryotes. This are standard DNA markers for evolutionary and genetic studies. SSR markers has multi-allelic nature, co-dominant transmission, relative abundance, extensive genome coverage, high reproducibility, simple detection and only a small amount of template DNA require to the extraordinary increase of interest in SSRs in many organisms [1,2]. As compare to other cereals and legumes, the development and use of molecular markers in pigeonpea is limited. Thus, in present study, we used SSR markers on pigeonpea varieties to provide evidence of their purity.

Plant material
The test pigeonpea samples, parental lines and control hybrid were obtained from Seed Testing Laboratory, Nagpur. The cultivar name is ICPH 2740 (Hybrid) along with parental lines (ICPA2047 and ICPR 2740) [A-line (female) and R-line (male)] and three sample set: sample 1: Lot no. AP-DSR-1609-16H, sample 2: Lot no. 01-P39-08-03-07 and sample 3: Lot no. 01-P39-08-03-11 to test hybridity. Individual seeds from each sample, reference hybrid and parental lines were kept for germination in seed germination paper.
Which were used for DNA extraction, PCR experimentation.

SSR-marker Primers
The SSR primers used in the present study were obtained from ICRISAT and details of SSRmarkers such as sequences, melting temperature and expected amplified DNA size in hybrid and parental lines are represented in Table-1.

DNA extraction
The genomic DNA was extracted from three week old leaves of the individual seedling from the control hybrid, parental lines and test samples of pigeonpea to be tested, using CTAB (cetyl trimethylammonium bromide) DNA isolation method as follow:  Fresh 100-500 mg of young leaf samples of pigeonpea were taken.  The leaf samples were grinded in mortal with the pestle in 1 ml CTAB buffer (liquid nitrogen not used).  The solution is then transfered to 2 ml eppendorf tubes and incubated in water bath at 65-70°C for 1 hr.  The solution was allow to attain room temperature, equal volume of chloroform: isoamyl alcohol 24:1 (equal volume to CTAB buffer) was added to it and centrifuged at 12,000 g for 10 minutes.  The supernatant was transfered carefully in fresh 2 ml eppendorf tube & remaining were discarded.  The ice cold isopropyl alcohol (2/3) volume of the supernatant was added in the tube. (Invert slowly thrice to precipitate DNA, small fiber of DNA sitting down was observed) and kept for incubation at 4°C for overnight.  It was then allowed to room temperature and centrifuged at 12,000 g for 10 minutes, supernatant discarded and pellet was collected.  Tubes containing pellet were allowed to air dry for 5-10 minutes and inverted on tissue paper to complete run off any supernatant.  The DNA pellet was washed with 500 μl of 70% ethanol, centrifuged at 12,000 g for 10 minutes.  Later 70% alcohol discarded from tubes and allowed to air dry for 15 min on tissue paper in inverted position. Pellet was dissolved in 100 ul NFW and stored in -20°C for further downstream procedures. The DNA quantity for each sample was assessed on 0.8% agarose gel.

PCR amplification
The amplification of DNA from the samples were carried out by using Polymerase Chain Reaction. The PCR reaction mixture content are as 100ng of DNA dissolved in 2µl was used as template in 20µl of reaction mixture. The dNTP mix 2µl, 10X PCR buffer (with MgCl 2 ) 2µl, primers (F/R) 2µl, Taq DNA polymerase 1µl and total volume is makeup with nuclease free water 11µl. The vial containing all this PCR mix is set in PCR program shown in Table-2.

Gel electrophoresis of PCR products
The amplified PCR products were mixed with DNA loading dye and loaded on 4% agarose gel stained with EtBr (ethidium bromide) and electrophoresis run was set at constant 200 V for 3 hours. After complete run, gel was visualised under UV light in gel documentation unit for visual examination of SSRspecific markers amplified DNA from Pegionpea.

RESULTS
The DNA and SSR-specific markers amplified DNA fragments from Pegionpea test samples are used for spectrophotometer and other techniques for analysis.

DNA quality and quantity
The concentration of DNA was determined by Nano Photometer, in which 1µl of dissolved DNA of test samples was considered. Sample 1: absorbance ratio is 1.671 and concentration is 2.273µg/ml, sample 2: absorbance ratio is 1.543 and concentration is 2.455 µg/ml, sample 3: absorbance ratio is 1.183 and concentration is 2.421µg/ml. The figure 1 shows DNA from Pegionpea samples, hybrid and parental lines.

PCR Amplification of SSR-markers
The DNA of hybrid, parent and test samples were used for SSR-marker specific amplification. In all, seven SSR-primers were used against sample 1, 2, 3, however only three primers were amplified successfully. The amplification of DNA carried out by Polymerase Chain Reaction from test samples uses a programme given in table 2. Briefly 2µl of dissolved amplified DNA was used to check the fingerprinting along with marker ladder. Figure-2 shows SSR profiling for sample 1, 2, 3 each using SSR-primer CcM0246, CcM0516 and CcM0133. -2: SSR profiling in 4% Agarose gel used for four seedling sample 1, 2, 3 each using SSR primer CcM0246, CcM0516 and CcM0133. H stands for hybrid, A and R as parental lines used as reference, L is 50bp ladder.

Allele scoring and analysis
The allelic data was analysed from seven primers out of which 3 SSR-primers shows amplification in sample 1, 2, 3. Yet primer CcM0246 confirms hybrid seed in sample 1 with two separate alleles. The allelic data was analysed after electrophoresis and DNA fingerprinting from sample 1. It was observed that only CcM0246 marker was able to amplify two different loci (see Figure-3). That shows hybrid with two alleles (bands) similar in test sample 1.

DISCUSSION
The Concentration of 1µl DNA of test samples 1, 2, 3 is 2.273µg/ml, 2.455 µg/ml and 2.421µg/ml respectively, which selective states that the adopted protocol is good for DNA isolation from Pegionpea. The PCR products were checked on 1.2% agarose gel but did not showed clear bands instead of that 3% and 4% gel were used for optimisation and good clarity of bands was found on 4% gel.
In total, seven primers were consider for experimentation, nevertheless only three were success in amplification of DNA from Pegionpea samples. Three primer pairs (CcM0246, CcM0516 and CcM0133) which showed polymorphism in three samples. The primer CcM0246 should produced two distinct alleles of different sizes, one at 251bp and the second at 245 bp. Similarly primer CcM0516 should produced two distinct alleles of different sizes, one at 207 bp and the second at 211 bp. Consequently, primer CcM0133 should produced two distinct alleles of different sizes, one at 200 bp and the second at 208bp.
The designed SSR-primers provided by ICRISAT have 4-8bp difference which becomes hurdle for DNA profiling interpretation. Even only one marker is showing hybridity, besides the two bands observed in the hybrid does not seem exactly the same as amplified in respective parental lines. Thus, the SSR-primers that produced more bp differences may solve this drawback. The work of [1] designed SSR-primers and have modified the Tm accordingly on basis of BES sequence obtained from NCBI and have used them successfully in their studies.
The hybrid must produce two alleles of different sizes specific to each SSR-primers (see Figure-2). Among the test samples 1, 2, 3 the PCR amplification of marker CcM0246 produced heterozygous alleles only in samples 1. Thus it is confirmed that marker CcM0246 is highly heterozygous to produced two different alleles and these often differed between individuals. The [4] have designed thirty-five SSR-primers that showed polymorphism among 24 pigeonpea breeding lines. In our work other markers CcM0516 and CcM0133 could not produce two different Loci therefore these markers are not useful for purity and hybrid testing in this germplasm.
The SSR-primer CcM0246 is useful as it has shown similar bands in sample 1 to that of reference hybrid, whereas sample 2 and 3 could not give the confirmation of similarity to that of reference hybrid. Similarly, Primer CcM0516 showed polymorphism but none of the sample gave the confirmation of similarity to the reference hybrid. In contrast, Primer CcM0133 showed similar bands in sample 2 to that of reference hybrid. The sample 1 and 3 cannot give the confirmation of similarity. The band observed in sample 2 have similar intensity, width with hybrid. But, the clear band is not visible which become a cause to labelled it unclear results. The table 3 shows the characteristics of SSR primers. The SSR-markers are extensively used in hybrid testing, genetic mapping and diversity. Recently [5] demonstrated utility of SSR