**2.2 Random amplified polymorphic DNA (RAPD)**

Prior to the availability of complete rice genome sequence, RAPD markers were useful in developing drought tolerant varieties. It is the simplest, cheaper, sensitive and useful technique for the genotype identification, population and pedigree analysis, phylogenetic studies, genetic mapping [26] and analysis of genetic fidelity of commercially micropropagated plants [27]. It is simple since it requires less DNA and simultaneously doesn't require southern blotting and radioactive labeling [28]. Considering the usage of RAPD in wheat, maize, pea-nut, broccoli and cauliflower, RAPD was applied in rice to detect diversity in the low land and upland rice varieties with an aim to identify drought-resistant loci [29] as given below.

Thirteen rice cultivars were grown in a growth chamber at 28°-day temperature, 25°-night temperature and with an irradiance of 800 μmoles m−2 s−1 for 12 h a day. At six leaf stage, leaves were collected and stored in liquid nitrogen and genomic DNA was extracted and purified [22]. Forty-two GC-rich 10 bp random primers were used as RAPD markers. DNA amplification was done in a PCR (Perkin Elmer Cetus) programmed for 45 cycles of1min at 94°C,1 min at 37°C and 2 min at 72°C. The reaction conditions include 25 μl total reaction volume having 10 mM Tris-HC1 (pH 8.3), 50 mM KC1, 2mMMgC12, 50 mM each of dNTP's, 10 ng of a single random primer, 25 ng of genomic DNA and 2 units of Ampli Taq DNA polymerase (Perkin Elmer Cetus) and 50 ml of sterilized mineral oil.

At the end of amplification, 10 μl of each amplification mixture was loaded in either 1.4% agarose (0.5 to 4 kb) or 5% polyacrylamide (<500 bp) gels for electrophoresis in 1 x TBE (89 mM Tris, 89 mM boric acid and 2 mM EDTA). Gels were stained with ethidium bromide and photographed under UV light. Pair-wise comparisons of genotypes, based on the presence (score 1) or absence (score 2) of each marker similarity coefficients were calculated which were used to construct UPGMA tree. One-to-twelve DNA amplicons were observed from each genomic DNA sample. A total of 260 DNA fragments were amplified and 208 (80%) of these showed polymorphisms. Upland cultivars and lowland cultivars were classified into two main clusters-*japonica* and *indica* with seven (Azucena, Rikuto Norin 21, Moroberekan, IAC25, IRAT13, OS4, and 63–83) six (BPI-76 NS, IR20, IR36, CO39, MGL-2 and Salumpikit) cultivars respectively. Later, they screened 2074 rice varieties for drought tolerance where upland varieties were identified with higher score for drought tolerance and were recommended for donors for breeding drought tolerant varieties as well as for developing molecular markers.

However, like RFLP, RAPD is also disadvantageous due to low polymorphism among *japonica* rice, need for hundreds of markers to locate markers in the QTL and absence of RAPD markers for some regions of the chromosomes [30].

## **2.3 AFLP (amplified fragment length polymorphism)**

Restriction enzymes were reported from bacteria. This enzyme identifies foreign DNA in bacterial cells based on the target site and generally, cuts the DNA within

this site if the site is un-methylated. Eventually, the broken DNA cannot show its effect on the host bacterial cell and hence, the natural function of restriction enzymes is to restrict the growth of foreign DNA. Restriction enzymes differ in the length of their target sites (4–8). It is universally accepted that the probability of finding the smaller length of sequence is manifold higher than longer sequences i.e., in other words if the length of the target site is less, then, it can be repeated more frequently in the DNA. In the above example, a 6-base target is rarer to find than the 4-base target site. Adapter /adaptor/linker is a short, chemically synthesized (known sequence), single-stranded or double-stranded oligonucleotide that can be ligated to the ends of other DNA/RNA molecules to convert them to sticky ends of desired sequence.

DNA was isolated from 80 plants of F2 population of the cross 'Labelle' × 'Black Gora' [30] and AFLP was conducted [31]. AFLP involves cutting of genomic DNA with two restriction enzymes that need different lengths of target sites, a 6-base (or "rare") cutter (EcoRI) and a 4-base (or "frequent") cutter (MseI), ligating adapters to the fragment ends, amplifying MseI-EcoRI fragments with primers that match the adapter and contain additional selective nucleotides at the 3′ end and separating the fragments on denaturing polyacrylamide gels. EcoRI and MseI adapters, T4 DNA ligase, DTT and water were added to the restriction reaction-mixture which was incubated for 3 h at 37°C. Then, preamplification of DNA was done with primers which were labeled subsequently with spermidine. The amplicons were separated on 4.5% denaturing (urea) polyacrylamide gels for 90 to 120 min at 120 W. The gels were dried and exposed to X-ray film for 4–7 days.

Bands showing clear polymorphism were scored as present ("1") or absent ("0"). Genetic similarity was computed as the number of common bands divided by the total number of bands of both accessions, and genetic distance was computed as 1 minus this value [32]. These distances were used to construct cluster diagrams by UPGMA method (SAS, PROC CLUSTER) [33]. AFLP is cheaper than RAPD. AFLP is useful to screen small number of samples with large number of markers.

### **3. Second generation markers**

#### **3.1 Inter simple sequence repeats (ISSR)**

ISSR markers are well distributed in the eukaryotic genome [34] more feasible and reproducible than RAPD [35] highly polymorphic, less expensive and independent of sequence information [36]. Polymorphism of 73.02% with RAPD markers and 90.91% with ISSR markers was observed between six rice lines [37]. Seventeen ISSR primers- (8 based on (AG)8, 8 on (GA)8, and 1 on (GATA)4 were used to screen 12 cultivars as presented below [38].

Genomic DNA was isolated from freshly harvested young leaves of each cultivar by Mini prep method. ISSR-PCR was conducted and the amplified products were resolved in 1.8% agarose gel in the presence of ethidium bromide and were documented under ultraviolet light. Polymorphism information content (PIC) was calculated based on the presence (score 1) or absence (score 0) of band. Similarity scores were calculated and un-weighted pair-group method with arithmetic average (UPGMA) dendrogram was generated sub-program of NTSYS-PC software version 2.0at 1000 bootstrap. The three drought tolerant varieties formed one sub-cluster by (GA)8YG primer.
