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VELICEVICI GIANCARLA, MADOŞĂ EMILIAN, ŞUMĂLAN RADU, CIULCA SORIN,POPESCU SORINA, PETOLESCU CERASELA Table 4. The coefficient values of correlation among RAPD primers under the genetic similarity aspect of the barley cultivars studied Primers P-1 P-16 P-17 P-27 OPA5 OPA11 OPG2 E-6 A-16 P-1 - 0,072 0,178*** 0,334*** 0,246*** 0,205*** 0,170** 0,027 0,182*** P-16 - 0,323*** 0,168** 0,129* 0,317*** 0,101 0,149** 0,090 P-17 - 0,383*** 0,270*** 0,170** 0,355*** 0,392*** 0,192*** P-27 - 0,536*** 0,267*** 0,338*** 0,177*** 0,210*** OPA5 - 0,107* 0,251*** 0,137* 0,269*** OPA11 - 0,152** 0,137* 0,079 OPG2 - 0,387*** 0,183*** E-6 - 0,171** A-16 - r 5% = 0,105; r 1% = 0,138; r 0,1% = 0,176; The six alleles common identified for to all the 19 th genotypes studied (Table 5) could be considered specific markers RAPD for the barley. Table 5. Specific RAPD alleles common for all the barley cultivars studied No. Specific alleles 1 OPG2-400pb 2. P1-400pb 3. P27-600pb 4. P16-530pb 5. P16-650pb 6. P16-850pb Inter simple sequence repeat (ISSR)-PCR is a technique, which involves the use of microsatellite sequences as primers in a polymerase chain reaction to generate multilocus markers. It is a simple and quick method that combines most of the advantages of microsatellites (SSRs) and amplified fragment length polymorphism (AFLP) to the universality of random amplified polymorphic DNA (RAPD). ISSR markers are highly polymorphic and are useful in studies on genetic diversity, phylogeny, gene tagging, genome mapping and evolutionary biology [25] .ISSR markers have been used for studies of hybridization and hybrid speciation [2], population and conservation genetics [7], and systematic investigations in natural populations [20]. Among the tested ISSR primers, only five amplified polymorphic ISSR loci, resulting a rate of polymorphism with values between 57,14% in case of primer 810 and 100% for the first 811, HB12, HB15 (Table.6). The amplified fragments had size among 150 and 600 pb for HB-15 primer, while 810 primer registered the highest amplitude of amplified fragments size (950- 5100 pb). The average number of polymorphic bands per primer was 9.2 with an average rate of polymorphism being 89,13 %. The total of polymorphism generated by a certain primer (PIC) presented values among 0,245 to 811 and 0,391 to 810. The discrimination index (PI) registered values among 1,562 for primer 810 and 2,803 for HB15 primer. Table 6. Polymorphism rate of barley cultivars through ISSR primers No.. Primers Nucleotide No. bands Polymorphis Range of PIC PI sequence Total No of Polymorphic m (%) size bands x ± s x (5’-3’) fragments fragments (pb) 1 HB-12 (CAC)3GC 9 9 100 175-850 0,309+0,044 2,781 2 HB-14 (CTC)3GC 12 10 83.33 200-1400 0,264+0,053 1,806 3 HB-15 (GTG)3GC 9 9 100 150-600 0,311+0,059 2,803 4 810 (GA) 8 T 7 4 57.14 950-5100 0,391+0,111 1,562 5 811 (GA) 8 C 9 9 100 950-2000 0,245+0,053 2,205 7498 Romanian Biotechnological Letters, Vol. 17, No. 4, 2012
The use of RAPD and ISSR markers for genetic diversity among some barley cultivars According the ISSR data, a dendrogram was developed for 19 cultivars and indicates four main clusters; the first cluster divided into two sub clusters, first sub cluster included Romanian cultivars Orizont and Dana presenting a genetic similarity of 75% for ISSR alleles and second sub cluster formed a separate sub cluster with Precoce cultivar, that presenting a difference of 10% face to the cultivars from the first sub cluster. Second sub cluster was further divided in two sub clusters. Among the two sub clusters, first sub cluster formed a separate sub cluster with Romanian cultivars Adi, Regal, Andrei DH 254/10, DH260/18, Madalin, and second sub cluster included foreign cultivars Gerbel, Lyric, Tas, Plaisant, Dina, and Romanian duble haploide lines DH19/1.Cultivar Compact made second cluster manifesting an average genetic similarity of approximate 46% with cultivars Orizont and 55% with Regal and Andrei. Cultivar Secura presented a genetic similarity of 52% with Orizont, making the third cluster. Double haploid lines DH260/12, DH261/22 made a fourth cluster, presenting a genetic difference of approximate 53% face to the genotypes from the first cluster; the highest genetic difference registering face to cultivars Compact (75%), Regal and Secura. ISSR analysis through the 5 primers, attested the existence of a high genetic variability among cultivars involved in this study, which could be efficiently exploited in breeding programs of barley. The results presented in table 7 attested the existence of some significant correlations among ISSR primers, with exception 811 primer. The highest correlation obtained among HB15 and HB14 primers which had capacities very nearby of polymorphism detection to barley genotypes analyzed. Table 7. The coefficient values of correlation among ISSR primers under genetic similarity aspect of barley genotypes studied Primers HB-12 HB-14 HB-15 810 811 HB-12 - 0,338*** 0,331*** 0,333*** 0,192*** HB-14 - 0,401*** 0,219*** 0,383*** HB-15 - 0,258*** 0,063 810 - 0,179*** 811 - r 5% = 0,105; r 1% = 0,138; r 0,1% = 0,176; Orizont Dana Precoce Adi Regal Andrei DH254-10 DH260-18 Madalin Gerbel Lyric Tas Plaisant Dina DH19-1 Compact Secura DH260-12 DH261-22 0.50 0.60 0.70 Similarity coefficient 0.80 0.90 1.00 Fig. 2. UPGMA clustering of barley cultivars using the ISSR primers Romanian Biotechnological Letters, Vol. 17, No. 4, 2012 7499
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