Download Detection and Sequencing of the Transposable Element ILS

Plant Physiol. (1994) 106: 803-804
Planf Gene Register
Detection and Sequencing of the Transposable Element ILS- 1
in the lllinois Long-Term Selection Maize Strains
Rudaina Alrefai, Beverly Orozco, and Torbert Rocheford*
Department of Agronomy, AW101 Turner Hall, University of Illinois, Urbana, lllinois 61801
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Restriction fragment-length polymorphism analysis performed on 100 FJ families derived from a cross of the IHP x
ILP maize (Zea mays L.) strains detected a QTL linked with
the Sh2 gene (Bhave et al., 1990; Shaw and Hannah, 1992)
with large additive effects on starch concentration (Goldman
et al., 1993). Sh2 encodes the large subunit of ADP-Glc
pyrophosphorylase, which is involved in the rate-limiting
step of starch biosynthesis in plants (Stark et al., 1992). Since
it is known that major mutations at the Sh2 locus of maize
greatly reduce starch levels in the endosperm through its
effect on the starch synthetic enzyme ADP-Glc pyrophosphorylase, we decided to determine if different Sh2 alleles
were present in the ILP and IHP parents of the mapping
population that exhibited a QTL for starch concentration
at Sh2.
During characterization of the IHP and ILP Sh2 genes, an
insertion in intron 13 of the ILP Sh2 allele was detected. To
characterize this insertion, the 3‘ half of intron 13 of the Sh2
gene from ILP was amplified by PCR, cloned, and sequenced
(Table I). Sequence analysis revealed that this insertion is a
new transposable element, which we have designated ILS-1
because it was isolated from the Illinois Long-Term Selection
Experiment maize strains. The ILS-1 element is inserted 4991
bp downstream of the transcription start site of the ILP Sh2
allele. The ZLS-1 element is 1248 bp in length and has 13-bp
nearly perfect (12 of 13 bp) TIRs. This element is rich in A
(30.6%)and T (37.9%)nucleotides and has at least 15 intemal
inverted repeats with a size range of 7 to 15 bp. The ILS-1
element is associated with a duplication of 11 bp of the Sh2
intron 13 sequence at the target site of insertion, and there is
an additional A nucleotide present immediately 5‘ to the first
TIR. Southem analysis suggests that the ILS-1 element is
present in at least five to seven copies in several different
maize inbreds. Search for an ORF detected two putative ORFs
at nucleotide 184 and 572 encoding proteins of M, 15,559
and 32,486, respectively. However, a search of the protein
data base did not detect any significant homology. The small
size of the two ORFs in ILS-1 and the lack of homology of
the putative protein products with other known proteins
suggest that ZLS-1 is a nonautonomous element and that it
probably does not have bona fide ORFs.
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Table 1. Characteristics of ILS- I transposable element
Organ ism:
Zea mays L., ILP selection strain cycle 76.
Location:
lnserted at nucleotide 1286 of intron 13 of the Sh2 gene.
Techniques:
The 3’ half of intron 13 of the Sh2 gene of the ILP maize strain
was amplified by PCR; the amplified fragment of 2.4 kb was
excised from a low-melting-point agarose gel of 1.2%; this
DNA was cloned into a pBluescript T vector. Doublestranded plasmid dideoxynucleotide sequencing of both
strands was performed using synthetic oligonucleotide
primers. Southern blot analysis was performed for several
maize inbreds to estimate the copy number of the element in
other maize inbreds.
Unique Characteristics:
Presence of 13-bp TIRs (5’-CCCCTGCAAATCG-3’ and 5’CCATTAGCAGCCC-3’); 11-bp duplication of intron 13 of
Sh2 gene at the target of insertion (5’-CATCTGTATCT-3’).
DNA sequence homology searches were performed with
the entire 1248-bp ILS-1 sequence and with just the 13-bp
terminal inverted repeats. No significant homology was detected with any transposable elements in any of the searches.
Although the EnhancerlSuppressor elements have 13-bp TIRs
(Schwarz-Sommer et al., 1985), there was no homology
between the ILS-1 TIRs and EnhancerlSuppressor TIRs. This
suggests that ZLS-1 is a member of a new and/or unique
family of transposable elements. This is supported by the
finding that 11 bp of the Sh2 target sequence is duplicated at
the ILS-1 insertion site. The duplication of this number of bp
of target sequence has not been observed previously for any
plant transposable element. Further molecular and genetic
analyses are planned to better characterize this new element.
Received April 8, 1994; accepted May 5, 1994.
Copyright Clearance Center: 0032-0889/94/106/0803/02.
The GenBank accession number for the sequence reported in this
article is U07956.
LITERATURE CITED
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Corresponding author; fax 1-217-333-9817.
Abbreviations: IHP, Illinois high protein; ILP, Illinois low protein;
ORF, open reading frame; QTL, quantitative trait locus; Sh2,
Shmnken-2; TIR, terminal inverted repeats.
Bhave MR, Lawrence S, Barton C, Hannah LC (1990) Identification
and molecular characterization of Shmnken-2 cDNA clones of
maize. Plant Cell2 581-588
Goldman IL, Rocheford TR, Dudley JW (1993) Quantitative trait
803
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804
Alrefai et al.
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Schwarz-Sommer ZS, Gierl A, Klosgen RB, Wienand U, Peterson
PA, Saedler H (1985) Plant transposable elements generate the
DNA sequence diversity needed in evolution. EMBO J 4 591-597
Plant Physiol. Vol. 106, 1994
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type Shrunken-2 allele of Zea mays. Plant Physiol98: 214-1216
Stark DM,Tmmerman KP, Barry, GF, Preiss J, Kishore GM (1992)
Regulation of the amount of starch in plant tissues by ,4DP glucose
pyrophosphorylase. Science 2 5 8 287-292
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