RESULT AND DISCUSSION RAPD amplification in potato genotypes DNA profiling and polymorphism was studied by RAPD primer in 9 somaclonal variants and 3 popular varieties of potato
RESULT AND DISCUSSION
RAPD amplification in potato genotypes
DNA profiling and polymorphism was studied by RAPD primer in 9 somaclonal variants and 3 popular varieties of potato. Out of 9 RAPD primers, 6 primers showed reproducible and polymorphic amplified bands and these were selected for further amplification (Figure 1). The amplification result of 12 potato genotypes is shown in table 1. The selected six primers were OPA-18, OPB-08, OPC-01, OPD-02, OPF-08 and OPW-01. Each of the primers produced separate RAPD patterns (bands) in 12 potato genotypes. The RAPD amplifications of individual primer were given in figure 2 to 5.These 6 primers generated a total of 54 distinct and differential amplified bands. The average no of DNA bandswas9 per primer and 4.5 bands per potato genotypes. The size of the DNA fragments ranged from 88 to 3265 bp but most of the bands were between 150 to 2000 bp. Out of total 54 bands, 47 bands (87.04%) showed polymorphic amplification and rest of the DNA fragments were monomorphic. The result gave an average of 7.83 polymorphic and 1.16 monomorphic bands per primer.
The primers used in the present study showed different levels of gene frequency and different frequency of polymorphic loci. The gene frequency ranged from 0.083 to 1.00 and the frequency of polymorphic loci ranged from 0 to 0.917. Inter-genotype means of the pair-wise similarity indices (Sij) ranged from 61.59% to 93.55% and the average was 74.31%. The highest similarity indices (93.55%) was present between SVP-55 vs SVP-56 genotype pair. On the other hand, Asterix vs SIP-3 pair showed the lowest inter-variety similarity indices (61.59%). This result is shown in Table 2.
Genetic identity and Nei’s (1972) genetic distance of potato genotypes are given in Table 3. Pair-wise comparisons of Nei’s (1972) genetic distance among 12 potato genotypes ranged from 0.0972 to 0.6217. The highest Nei’s genetic distance (0.6217) was observed in SVP-6 vs SVP-68 genotype pair whereas the lowest genetic distance (0.0972) was estimated in SVP-55 vs SVP-56 genotype pair. Between Cardinal and other somaclonalvariants Nei’s (1972) genetic identity ranged from 0.5741 (at SVP-6) to 0.8148 (at SVP-68), while for Diamant it ranged from 0.5962 (at SVP-6) to 0.7593 (at SVP-68) and in the case of Asterix it ranged from 0.5741 (at SVP-25) to 0.7407 (at SVP-55 and SVP-68). So, SVP-68 is very close to the three varieties among the somaclonal variations.The Nei’s (1972) genetic distance between Cardinal and somaclonal variations ranged from 0.2048 (at SVP-68) to 0.5550 (at SVP-6). Whereas for Diamant, it ranged from 0.2754 (at SVP-68) to 0.5232 (at SVP-6) and in case of Asterix it ranged from 0.3001 (at SVP-55 and SVP-68) to 0.5550 (at SVP-25). So, SVP-6 (to Cardinal and Diamant) and SVP-25 (to Asterix) is distantly linked accessions among the somaclonal variations.
Dendrogram based on Nei’s (1972) genetic distance using Unweighted Pair Group Method of Arithmetic Means (UPGMA) indicated the segregation of 12 genotypes of potato into two main clusters: A and B. The first major cluster (A) had 8 genotypes i.e. Cardinal, SVP-68, SIP-3, Diamant, Asterix, SIP-5, SVP-55 and SVP-56. The second major cluster (B) had 4 genotypes and these were SVP-6, SVP-18, SVP-19 and SVP-25. The cluster A was divided into two sub-cluster: AI and AII. Sub-cluster AI contained 5 genotypes i.e. Cardinal, SVP-68, SIP-3, Diamant and Asterix while Sub-cluster AII contained 3 genotypes i.e. SIP-5, SVP-55 and SVP-56. The above results were shown in Figure 6.
These results suggest that RAPD markers provided substantial information for molecular diversity analysis of somaclonal variants in potato genotypes. The primer sequence determines the number and size of the DNA fragments. Different factors, such as the primer sequence, the type of thermocycler, template quality and quantity and polymerase concentration influenced the reproducibility of the RAPD technique (Afrasiab, H. and Iqbal, J., 2012a). AL-Salihyet al. (2014), Iuliana and Cerasela (2014) screened 6 RAPD primers in case 4 of in vitro propagated potato and 6 RAPD primers in 6 potato genotypes (3 potato cultivars and their 3 somaclonal variants), from which they selected clear and distinct polymorphic bands producing 5 and 3 RAPD primers, respectively. Ahmad et al. (2013) studied 4 RAPD primers in mutant lines of the three potato cultivars, Cardinal, Diamant and Desiree and found bands were present at 50 bp to 1500 bp.Afrasiab and Iqbal (2012b) estimated 123 clear and easily storable bands using 24 RAPD primers in 9 potato genotypes (3 somaclonal variant of cultivar Desiree and 6 gamma mutant lines) ranged from 200 to 3000 bp and average 5.12 bands per primers and 13.67 bands per potato cultivar. They detected 63% polymorphic bands and 37% monomorphic bands, which indicated that their study was almost similar to the present study in respect of bands size ranges but higher in the case of bands per cultivars and percentage of monomorphic bands and lower in the case of the percentage of polymorphic bands.Khatab and El-Banna (2011) used five RAPD primers in 14 somaclonal variants of potato and detected total 61 bands i.e. 12.2 bands per primer and 4.36 bands per cultivar. The size of the bands ranged from 200 to 3050 bp. They revealed 38 (62.29%) polymorphic and 23 (37.71%) monomorphic bands. The result gave an average 7.6 polymorphic and 4.6 monomorphic bands per primer. In a study, Khatab and El-Banna (2011) found the gene frequency ranged from 0.056 to 1.00 and the frequency of polymorphic loci ranged from 0 to 0.944.Kujalet al. (2005) reported pair-wise similarity value of 52% to 86 % (average 65%) among 31 potato genotypes. Iseneggeret al. (2001) studied 64 potato cultivars in Australia and found 67% to 90 % similarity among them. The results of the above two studies were very close to the present study. Again, Gauchanet al. (2012) reported 55.2% to 69% similarity value among 4 Nepali local potato cultivars studied with 10 RAPD primers, which was lower than the present study. This may happen due to cultivar variations. Again, Das et al. (2010) detected a wide range of similarity values (ranged from 29% to 93%) in 30 Indian potato cultivars with 13 RAPD primers.Onamuet al. (2014) estimated a similarity coefficient of 0.55 to 0.89 among 15 potato cultivars (9 cultivars bred in Europe, NorthAmerica and Mexico, and 6 Mexican creole cultivars) which were very close to the present study. Yasmin et al. (2006) reported Nei’s (1972) genetic identity among 6 potato cultivars ranged from 0.6530 to 0.8674 and genetic distance ranged from 0.154 to 0.558. They also reported that Nei’s (1972) genetic identity and genetic distance between Cardinal and Diamant was 0.6530 and 0.558 respectively. Hoqueet al. (2013) studied 12 potatoes with 8 RAPD primers in Bangladesh. They detected Nei’s genetic distance ranged from 0.137 to 0.970. On the other hand, Chakrabartiet al. (2001) detected 0.33 to 0.80 similarity value among 20 potato cultivars with 10 RAPD primers. Fu-cui et al. (2004) reported a high range (0.033 to 0.900) of genetic distance in 67 potato cultivars using 11 RAPD primers in China. Brenna (2004) showed the genetic relationship among 12 potato varieties with a dendrogram. The dendrogram segregated the 12 varieties into 2 main clusters and then different sub-clusters.Hoqueet al. (2013) studied a UPGMA dendrogram based on the Nei’s genetic distances among 12 popular potato varieties in Bangladesh. These varieties were segregated into two main clusters. Then the clusters into sub-clusters. They reported that Cardinal, Diamant and Asterix belonged to the same sub-cluster and their dendrogram was somewhat complex like the present study.
This study revealed a significant amount of relationship and diversity among the studied 12 genotypes. It indicates that the RAPD method can be used as an effective tool for DNA fingerprinting and molecular diversity analysis of various types of potato cultivars and newly created somaclonal variants. However, a larger number of genotypes and a higher number of primers would be necessary to construct an appropriate relationship and diversity but the present type of study is widely accepted in all concerns. Findings of the study can be used as a guideline for future fingerprinting and genetic diversity study of potato varieties. This study suggested some points for future consideration while starting DNA fingerprinting and diversity analysis in potato varieties and its somaclonal variants: To obtain more precious result large number of RAPD primers are needed to be amplified; a large number of varieties and their variant genotypes should be studied; The genotypes and primers can be used in the replication method; Other molecular markers such as SSR, AFLP, SNP etc. should be used for diversity analysis in potato.