| Abstract |
The study entitled “Identification of molecular markers for resistance<br/>to taro leaf blight in Colocasia esculenta (L.) Schott” was carried out at the<br/>Division of Crop Improvement, ICAR-CTCRI, Sreekariyam, during 2017-18<br/>with the objective to identify molecular markers associated with leaf blight<br/>resistance in taro, sequencing and analysis using BLAST. RAPD, ISSR and<br/>SSR markers were used for the study. A total of 36 taro genotypes were<br/>selected consisting of 18 each susceptible and 18 resistant genotypes. DNA was<br/>isolated by employing the CTAB method of Sharma et al. (2008).<br/>Out of 10 RAPD primers screened, 7 were selected whose annealing<br/>temperature were optimized at 32⁰C and the presence of amplicons were<br/>confirmed in 2% agarose gel. For the selected primers, percent polymorphism<br/>ranged from 50 to 100% where, OPW12 recorded the least polymorphism<br/>(50%) followed by OPW6 (81.8%). The highest was shown by OPW1 and<br/>OPW16. The average percent polymorphism was 86.22%. The PIC values were<br/>highest for OPW8 (0.888) followed by OPW2 (0.886) and OPW1 (0.885) while<br/>least with OPW16 (0.615). Number of alleles per locus varied from 4-8.19 with<br/>the maximum by OPW8 and minimum by OPW5. The He values ranged<br/>between 0.66 (OPW16) to 0.89 (OPW1, OPW2, OPW8) and mostly found to be<br/>>0.8. Dendrogram generated using UPGMA analysis grouped the 36 genotypes<br/>into two major clusters. Cluster I with four susceptible varieties includes Bhu<br/>Sree, Bhu Kripa and Sree Rashmi where, Bhu Sree and Bhu Kripa pooled<br/>together showing 88% similarity. The resistant line, IC310104 and a susceptible<br/>line, L-12 expressed 91% similarity. Similarity index values varied from 0.47 to<br/>0.88 with lowest (0.47) between 557 (S3) and 370 (R13) and between Sree<br/><br/>Pallavi (S9) and 370 (R13) while, the highest similarity index (0.91) was<br/>observed between IC310104 (R11) and L12 (R14).<br/>Fourteen ISSR primers were selected whose annealing temperature was<br/>optimized at 56.3⁰C and amplicons were confirmed in 1.8% agarose gel.<br/>Percent polymorphism of primers varied from 60 to 100% where UBC 827<br/>recorded lowest (60%) while UBC 818 recorded the 80% polymorphism. Rest<br/>of the primers showed 100% polymorphism with an average polymorphism of<br/>95.7%. The PIC values of the primers were highest for UBC 818 (0.862)<br/>followed by UBC 811 (0.861) and UBC 809 (0.857) while UBC 827 (0.709)<br/>recorded lowest PIC content of <0.8. Number of alleles per locus varied from<br/>2.38 - 6.13, where the maximum was shown by UBC 811 and minimum by<br/>UBC 817. He values varied between 0.75 (UBC 827) to 0.87 (UBC 809, UBC<br/>818 and UBC 811). Similarity matrix index values varied from 0.50 to 0.88<br/>with lowest (0.50) shown between Sree Rashmi (S1) and B-2 (SVP) (S18) and<br/>highest (0.88) between Sree Rashmi (S1) and E-10 (R9). A dendrogram was<br/>constructed using UPGMA, which grouped the genotypes into two major<br/>clusters. In the first cluster susceptible variety, Sree Rashmi (S1) pooled<br/>together with resistant, E-10 (R9) and it revealed 88% similarity.<br/>Out of 14 primers tested, the primer (UBC 811) gave an extra band for 7<br/>resistant genotypes (IC012601, IC089624, TCR 429, 679, 370, 84 and 565) out<br/>of the total 18 selected in 1270 bp region and it included the three resistant<br/>lines, 679, 370 and 84 which showed consistency in resistance reaction for the<br/>last four years under artificial screening. For primer UBC836, a resistant<br/>genotype IC089624 expressed a unique band at 1000 bp. The primer (GA)9AC<br/>also produced a unique band for the resistant genotype, IC089624 at 800 bp.<br/>For primer, UBC824, a resistant line, 565 showed a different banding pattern.<br/>From these primers, the one showing unique bands for the maximum genotypes<br/><br/>viz., UBC811 was selected. The band of size 1270 bp was cut and eluted.<br/>However, as the size of the band was very high and concentration was less, it<br/>was reamplified with the same primer. For reamplification, only four resistant<br/>genotypes (IC012601- R2; 370 - R13; 679 - R16 and 84 - R17) were selected<br/>based on the band intensity. This product was then checked in agarose gel,<br/>which gave two bands of which, one was very prominent at approximately 280<br/>bp. This band was isolated and sequenced. Sequence data showed that the<br/>product size ranged from 242 bp, 252 bp, 247 bp and 252 bp, respectively. The<br/>sequences obtained were used for similarity search in BLASTn and 100%<br/>identity and 8% query cover with Arabidopsis lyrata subsp. lyrata disease<br/>resistance protein RML1B (LOC9323997), mRNA was obtained for the DNA<br/>sequence from R13 (370). The following sequence<br/>(TTTGAAGAAGATAGCCT - 17 bp) showed similarity with the above<br/>mRNA.<br/>Nine out of ten SSR primers screened were selected based on their<br/>agarose gel profile whose annealing temperatures were optimized at 56⁰C and<br/>presence of amplicons was confirmed in 2.5% agarose gel. The percent<br/>polymorphism of the nine primers ranged from 33.33% to 100%, Uq 73-164<br/>with the lowest (33.33%) followed by Uq 201-302 (50%). Primers, Ce1 F12<br/>and Uq 97-256 revealed 100% polymorphism with average polymorphism<br/>shown was 71.29%. The PIC values of the primers were highest for Uq 132-147<br/>(0.69) and Uq 201-302 (0.69) followed by Uq 97-256 (0.61) and Uq 73-164<br/>(0.59). Primer, Uq 84-207, recorded the lowest PIC (0.30). Number of alleles<br/>per locus varied from 1.08 - 6.22 with maximum for Uq 97-256 and minimum<br/>with Ce1 B03.<br/><br/>The He values varied between 0.33 (Uq 84-207) to 0.74 (Uq 132-147 and Uq<br/>201-302) whereas similarity coefficient values ranged from 0.49 to 0.89<br/>concentrating between 0.56 to 0.86 with lowest (0.49) between 485<br/>(S11) and 450 (R1) while highest (0.89) between 679 (R16) and TCR 961<br/>(S17).<br/>In present study with three marker systems, the 30 odd primers did not<br/>produce any trait specific band(s) in all the resistant genotypes tested. However,<br/>with ISSR markers, primer UBC 811, expressed unique band in seven resistant<br/>genotypes which was completely absent in the susceptible ones. The specific<br/>band obtained were eluted and sequenced. The sequence showed 100% identity<br/>and 8% query cover with Arabidopsis lyrata subsp. lyrata disease resistance<br/>protein RML1B (LOC9323997), mRNA. This was obtained for the DNA<br/>sequence from R13 (370). The following is the sequence which showed<br/>similarity with the above mRNA - TTTGAAGAAGATAGCCT (17 bp).<br/>Mantel’s test established no correlation between the marker systems employed<br/>since they did not reveal any trait specific marker and only the genetic diversity<br/>was revealed. Therefore, further studies must be performed by employing more<br/>genotypes with increased primers to arrive at a definite consensus. The<br/>sequence obtained can be converted to a SCAR marker and validated with more<br/>resistant genotypes. |
