Transcriptome Sequencing of Chinese Spring Group 1 Deletion Lines using Ion Torrent
PGM
Deletion lines of common wheat cultivar Chinese Spring have been intensely used as a powerful
tool for genetic studies of wheat. The deletion lines have terminal deletions of different sizes at
chromosome arms and are available for all seven homoeologous chromosome groups of
hexaploid wheat. Breakage at various points of a particular chromosome arm of same or different
genome makes deletion lines the invaluable study material for geneticists to determine specific
physical location of markers and genes. Detailed information on the available deletion stocks in
Chinese Spring wheat can be accessed at the following link.
http://www.k-state.edu/wgrc/Germplasm/Deletions/group1.html.
The objective of the research is to identify specific location for wheat genes in homoloeogous
group 1 and determine genome specific sequences of those genes.
Flag leaf tissue of 19 group-1 deletion lines were collected at heading stage (Table 1). Total
RNA is extracted from collected leaf tissue. Extracted RNA is taken through several steps
(fragment the whole transcriptome RNA, construct the amplified transcriptome library, prepare
templated IonSphere particles for sequencing) to prepare for sequencing. After sequencing on
Ion Torrent PGM, the sequences are downloaded in FASTQ format. Based on the sequencing
chemistry used, the sequenced fragment lengths may range from 100 to 400 bp. Sequence
fragments will be filtered for ribosomal and chloroplast sequences. Remaining fragments will be
assembled de novo. Assembled sequences will be compared to sequences from its counterparts
from other two genomes to determine the sequences missing from a deletion line. For example,
Deletion lines 1AS-1, 1BS-10, and 1DS-3 have about half of 1AS (FL 0.47), 1BS (0.50), and
1DS (FL 0.48) arms respectively. However, 1BS-10 and 1DS-3 have complete 1AS arm and can
act as reference for EST sequences missing from 1AS-1. Similarly, 1AS-1 and 1DS-3 have
reference sequences for EST sequences missing from 1BS-10, and so forth.
Expected results
Physical location of genes in wheat homoloeogous group 1 will be determined. As sequences of
deletion at same physical position of different genomes (A, B, and D) will be compared,
genome-specific EST sequences will be determined.
Table 1. Chinese Spring Deletion Lines to be sequenced.
Deletion line
1AS-1
1AS-3
1BS-4
1BS-9
1BS-10
1BS-18
1BS-19
1DS-1
1DS-3
1DS-5
1AL-1
1AL-3
1AL-5
1BL-1
1BL-2
1BL-3
1BL-6
1DL-2
1DL-4
x
Deletions present
in line
1AS-1
1AS-3
4AL-2
1BS-4
1BS-9
1BS-10
1BS-18
1BS-19
6DS-4
1DS-1
1DS-3
1DS-5
1AL-1
1AL-3
4BL-12
1AL-5
7AS-5
1BL-1
1BL-2
6AL-8
1BL-3
6DL-11
5AS-10 may be
present?
1BL-6
5AL-?
7DL-? may be
present
1DL-2
1DL-4
FL Valuex
0.47
0.86
0.75
0.52
0.84
0.50
0.50
0.31
0.79
0.59
0.48
0.70
0.17
0.61
0.41
0.46
0.59
0.47
0.69
0.90
0.85
0.74
(1.00)
0.32
Unknown
Unknown
0.41
0.18
FL value: deletion breakpoints expressed as fraction length (FL) value of the arm retained in the
deletion chromosome