Ss. Lin et al., Heteroduplex mobility and sequence analyses for assessment of variability of Zucchini yellow mosaic virus, PHYTOPATHOL, 90(3), 2000, pp. 228-235
A heteroduplex mobility assay (HMA) was used to analyze the variability amo
ng five isolates of Zucchini yellow mosaic virus (ZYMV; TW-TC1, TW-CY2, TW-
TN3, TW-TNML1, and TW-NT1) collected from cucurbit fields in different area
s of Taiwan. A cDNA fragment of 760 bp covering the variable region of the
N terminal half of the coat protein (CP) gene was amplified by reverse tran
scription-polymerase chain reaction (RT-PCR) and subsequently subjected to
HMA analysis for sequence variation. When TW-NTI combined with any of the o
ther Taiwan isolates, the heteroduplexes obtained migrated much more slowly
than did the heteroduplexes obtained in combinations among the other four
Taiwan isolates, indicating that TW-TC1, TW-CY2, TW-TN3, and TW-TNML1 share
a high degree of sequence homology, while the TW-NT1 isolate is more disti
nct. The complete nucleotide sequences of the CP genes and the 3' noncoding
regions of the five isolates were determined from PT-PCR-derived cDNA clon
es. A phylogenetic tree derived from the actual sequences of the 760-bp fra
gments of the five Taiwan and another six ZYMV isolates from different geog
raphic areas revealed four genotypes. TW-TNML1, TW-TC1, TWC-Y2, and TW-TN3
were in genotype I, while TW-NT1 and U.S, isolates were in genotype II. The
Singapore and Reunion Island isolates were separated into genotypes III an
d IV, respectively. Comparison of the CP genes of the five Taiwan isolates
indicated that they share 92.8 to 98.7% nucleotide identities and 96.4 to 9
9.3% amino acid identities. The amino acid positions 73, 102, 109, and 149
of the CP gene, where lysine, serine, arginine, and aspartic acid reside, r
espectively, were uniquely conserved for genotype I Taiwan isolates. Thus,
results of HMA agreed well with those of phylogenetic analysis based on the
sequence data of the five Taiwan ZYMV isolates. These five ZYMV isolates o
f known sequence can be used as reference strains for HMA to analyze the va
riability of ZYMV in Taiwan.