MSc

The study entitled “Identification of molecular markers for resistance
to taro leaf blight in Colocasia esculenta (L.) Schott” was carried out at the
Division of Crop Improvement, ICAR-CTCRI, Sreekariyam, during 2017-18
with the objective to identify molecular markers associated with leaf blight
resistance in taro, sequencing and analysis using BLAST. RAPD, ISSR and
SSR markers were used for the study. A total of 36 taro genotypes were
selected consisting of 18 each susceptible and 18 resistant genotypes. DNA was
isolated by employing the CTAB method of Sharma et al. (2008).
Out of 10 RAPD primers screened, 7 were selected whose annealing
temperature were optimized at 32⁰C and the presence of amplicons were
confirmed in 2% agarose gel. For the selected primers, percent polymorphism
ranged from 50 to 100% where, OPW12 recorded the least polymorphism
(50%) followed by OPW6 (81.8%). The highest was shown by OPW1 and
OPW16. The average percent polymorphism was 86.22%. The PIC values were
highest for OPW8 (0.888) followed by OPW2 (0.886) and OPW1 (0.885) while
least with OPW16 (0.615). Number of alleles per locus varied from 4-8.19 with
the maximum by OPW8 and minimum by OPW5. The He values ranged
between 0.66 (OPW16) to 0.89 (OPW1, OPW2, OPW8) and mostly found to be
>0.8. Dendrogram generated using UPGMA analysis grouped the 36 genotypes
into two major clusters. Cluster I with four susceptible varieties includes Bhu
Sree, Bhu Kripa and Sree Rashmi where, Bhu Sree and Bhu Kripa pooled
together showing 88% similarity. The resistant line, IC310104 and a susceptible
line, L-12 expressed 91% similarity. Similarity index values varied from 0.47 to
0.88 with lowest (0.47) between 557 (S3) and 370 (R13) and between Sree

Pallavi (S9) and 370 (R13) while, the highest similarity index (0.91) was
observed between IC310104 (R11) and L12 (R14).
Fourteen ISSR primers were selected whose annealing temperature was
optimized at 56.3⁰C and amplicons were confirmed in 1.8% agarose gel.
Percent polymorphism of primers varied from 60 to 100% where UBC 827
recorded lowest (60%) while UBC 818 recorded the 80% polymorphism. Rest
of the primers showed 100% polymorphism with an average polymorphism of
95.7%. The PIC values of the primers were highest for UBC 818 (0.862)
followed by UBC 811 (0.861) and UBC 809 (0.857) while UBC 827 (0.709)
recorded lowest PIC content of <0.8. Number of alleles per locus varied from
2.38 - 6.13, where the maximum was shown by UBC 811 and minimum by
UBC 817. He values varied between 0.75 (UBC 827) to 0.87 (UBC 809, UBC
818 and UBC 811). Similarity matrix index values varied from 0.50 to 0.88
with lowest (0.50) shown between Sree Rashmi (S1) and B-2 (SVP) (S18) and
highest (0.88) between Sree Rashmi (S1) and E-10 (R9). A dendrogram was
constructed using UPGMA, which grouped the genotypes into two major
clusters. In the first cluster susceptible variety, Sree Rashmi (S1) pooled
together with resistant, E-10 (R9) and it revealed 88% similarity.
Out of 14 primers tested, the primer (UBC 811) gave an extra band for 7
resistant genotypes (IC012601, IC089624, TCR 429, 679, 370, 84 and 565) out
of the total 18 selected in 1270 bp region and it included the three resistant
lines, 679, 370 and 84 which showed consistency in resistance reaction for the
last four years under artificial screening. For primer UBC836, a resistant
genotype IC089624 expressed a unique band at 1000 bp. The primer (GA)9AC
also produced a unique band for the resistant genotype, IC089624 at 800 bp.
For primer, UBC824, a resistant line, 565 showed a different banding pattern.
From these primers, the one showing unique bands for the maximum genotypes

viz., UBC811 was selected. The band of size 1270 bp was cut and eluted.
However, as the size of the band was very high and concentration was less, it
was reamplified with the same primer. For reamplification, only four resistant
genotypes (IC012601- R2; 370 - R13; 679 - R16 and 84 - R17) were selected
based on the band intensity. This product was then checked in agarose gel,
which gave two bands of which, one was very prominent at approximately 280
bp. This band was isolated and sequenced. Sequence data showed that the
product size ranged from 242 bp, 252 bp, 247 bp and 252 bp, respectively. The
sequences obtained were used for similarity search in BLASTn and 100%
identity and 8% query cover with Arabidopsis lyrata subsp. lyrata disease
resistance protein RML1B (LOC9323997), mRNA was obtained for the DNA
sequence from R13 (370). The following sequence
(TTTGAAGAAGATAGCCT - 17 bp) showed similarity with the above
mRNA.
Nine out of ten SSR primers screened were selected based on their
agarose gel profile whose annealing temperatures were optimized at 56⁰C and
presence of amplicons was confirmed in 2.5% agarose gel. The percent
polymorphism of the nine primers ranged from 33.33% to 100%, Uq 73-164
with the lowest (33.33%) followed by Uq 201-302 (50%). Primers, Ce1 F12
and Uq 97-256 revealed 100% polymorphism with average polymorphism
shown was 71.29%. The PIC values of the primers were highest for Uq 132-147
(0.69) and Uq 201-302 (0.69) followed by Uq 97-256 (0.61) and Uq 73-164
(0.59). Primer, Uq 84-207, recorded the lowest PIC (0.30). Number of alleles
per locus varied from 1.08 - 6.22 with maximum for Uq 97-256 and minimum
with Ce1 B03.

The He values varied between 0.33 (Uq 84-207) to 0.74 (Uq 132-147 and Uq
201-302) whereas similarity coefficient values ranged from 0.49 to 0.89
concentrating between 0.56 to 0.86 with lowest (0.49) between 485
(S11) and 450 (R1) while highest (0.89) between 679 (R16) and TCR 961
(S17).
In present study with three marker systems, the 30 odd primers did not
produce any trait specific band(s) in all the resistant genotypes tested. However,
with ISSR markers, primer UBC 811, expressed unique band in seven resistant
genotypes which was completely absent in the susceptible ones. The specific
band obtained were eluted and sequenced. The sequence showed 100% identity
and 8% query cover with Arabidopsis lyrata subsp. lyrata disease resistance
protein RML1B (LOC9323997), mRNA. This was obtained for the DNA
sequence from R13 (370). The following is the sequence which showed
similarity with the above mRNA - TTTGAAGAAGATAGCCT (17 bp).
Mantel’s test established no correlation between the marker systems employed
since they did not reveal any trait specific marker and only the genetic diversity
was revealed. Therefore, further studies must be performed by employing more
genotypes with increased primers to arrive at a definite consensus. The
sequence obtained can be converted to a SCAR marker and validated with more
resistant genotypes.