RNA Sequencing-Based Bulked Segregant Analysis Facilitates Efficient D-genome Marker Development for a Specific Chromosomal Region of Synthetic Hexaploid Wheat
Abstract
:1. Introduction
2. Results and Discussion
3. Materials and Methods
3.1. Plant Materials
3.2. Library Construction and RNA Sequencing
3.3. Alignment of RNA-seq Reads to de Novo Assembled Transcripts of the Parental Ae. tauschii Accessions
3.4. Identification of D-genome Specific SNPs and Calculation of ΔSNP-Index
3.5. Molecular Marker Development and Genotyping
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
RNA-seq | RNA sequencing |
NGS | next-generation sequencing |
BSA | bulked segregant analysis |
SNP | single nucleotide polymorphism |
Indel | insertion/deletion |
NR | non-redundant |
SP | Segregating Population |
dCAPS | derived cleaved amplified polymorphic sequence |
Ldn | Langdon |
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Samples | Total Read Pairs | Filtered Read Pairs (%) a | Aligned to the Ae. tauschii Transcripts b (%) c | |
---|---|---|---|---|
KU-2075 | KU-2025 | |||
Synthetic hexaploids | ||||
non-carrier-SP1-1st | 4,202,114 | 2,799,202 (66.61%) | 2,020,405 (72.18%) | 1,930,400.5 (68.96%) |
non-carrier-SP1-2nd | 4,059,840 | 2,858,956 (70.42%) | 2,062,291 (72.13%) | 1,971,110.5 (68.95%) |
non-carrier-SP2-1st | 4,492,358 | 2,953,088 (65.74%) | 2,164,656 (73.3%) | 2,083,120 (70.54%) |
non-carrier-SP2-2nd | 4,115,352 | 2,864,271 (69.60%) | 2,098,383 (73.26%) | 2,020,752.5 (70.55%) |
Net2-SP1-1st | 4,710,499 | 3,148,652 (66.84%) | 2,208,392 (70.14%) | 2,110,452.5 (67.03%) |
Net2-SP1-2nd | 4,403,630 | 3,108,568 (70.59%) | 2,178,403 (70.08%) | 2,082,004 (66.98%) |
Net2-SP2-1st | 4,828,182 | 3,249,596 (67.30%) | 2,420,056 (74.47%) | 2,348,814 (72.28%) |
Net2-SP2-2nd | 5,216,082 | 3,709,478 (71.12%) | 2,763,471 (74.5%) | 2,684,019 (72.36%) |
Tetraploid wheat | ||||
cv. Langdon | 6,316,174 | 4,372,660 (69.23%) | 2,974,277 (68.02%) | 2,661,487 (60.87%) |
Transcripts a | KU-2075 | KU-2025 | ||
---|---|---|---|---|
The Number of SNP | Total | Anchored to the Genome b (%) | Total | Anchored to the Genome b (%) |
Synthetic hexaploids | ||||
non-carrier-SP1 | 277,605 | 275,799 (99.35%) | 262,966 | 261,128 (99.30%) |
non-carrier-SP2 | 276,564 | 274,772 (99.35%) | 269,175 | 267,249 (99.28%) |
Net2-SP1 | 318,046 | 315,859 (99.31%) | 296,819 | 294,739 (99.30%) |
Net2-SP2 | 298,496 | 296,419 (99.30%) | 285,798 | 283,684 (99.26%) |
Tetraploid wheat | ||||
cv. Langdon | 429,346 | 421,957 (98.28%) | 350,871 | 345,657 (98.51%) |
Chr. | D-genome-Specific | Homoeologous | Unclassified | Total |
---|---|---|---|---|
1D | 1674 | 29,307 | 11,760 | 42,741 |
2D | 2295 | 33,822 | 13,975 | 50,092 |
3D | 1611 | 31,932 | 15,532 | 49,075 |
4D | 1698 | 28,781 | 11,776 | 42,255 |
5D | 2936 | 34,961 | 14,336 | 52,233 |
6D | 3730 | 24,534 | 10,966 | 39,230 |
7D | 3983 | 28,593 | 11,606 | 44,182 |
Total | 17,927 | 211,930 | 89,951 | 319,808 |
Marker Name | Primer Sequence (5′ to 3′) | Restriction Enzyme |
---|---|---|
bsa1 | TCATGACCTGCTGGTTTGTT | StyI |
GATTCCAATGTTATTTCTGAACCCT | ||
bsa2 | TCACAACATTCGCAGGTCAT | HpaII |
TGGTTCTGTTGATCTCACTGCC | ||
bsa4 | ACAAGTCGGATATCGCCAAA | HinfI |
CAGCTAAAAACTGTTTGCTTGAGA |
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Nishijima, R.; Yoshida, K.; Sakaguchi, K.; Yoshimura, S.-i.; Sato, K.; Takumi, S. RNA Sequencing-Based Bulked Segregant Analysis Facilitates Efficient D-genome Marker Development for a Specific Chromosomal Region of Synthetic Hexaploid Wheat. Int. J. Mol. Sci. 2018, 19, 3749. https://doi.org/10.3390/ijms19123749
Nishijima R, Yoshida K, Sakaguchi K, Yoshimura S-i, Sato K, Takumi S. RNA Sequencing-Based Bulked Segregant Analysis Facilitates Efficient D-genome Marker Development for a Specific Chromosomal Region of Synthetic Hexaploid Wheat. International Journal of Molecular Sciences. 2018; 19(12):3749. https://doi.org/10.3390/ijms19123749
Chicago/Turabian StyleNishijima, Ryo, Kentaro Yoshida, Kohei Sakaguchi, Shin-ichi Yoshimura, Kazuhiro Sato, and Shigeo Takumi. 2018. "RNA Sequencing-Based Bulked Segregant Analysis Facilitates Efficient D-genome Marker Development for a Specific Chromosomal Region of Synthetic Hexaploid Wheat" International Journal of Molecular Sciences 19, no. 12: 3749. https://doi.org/10.3390/ijms19123749