Map** and Candidate Gene Analysis of the Low-Temperature-Sensitive Albino Gene OsLTSA8 in Rice Seedlings
Abstract
:1. Introduction
2. Results
2.1. Genetic Analysis
2.2. Whole-Genome Resequencing Analysis of Four Mixed Pools
2.3. Map** of Genomic Regions Controlling Rice Seedling Albinism
2.3.1. A-14 vs. B-14 Association Analysis
2.3.2. A-22 vs. B-22 Association Analysis
2.4. RNA-Seq Analysis
2.5. OsLTSA8 Candidate Gene Analysis
2.6. RT-qPCR Validation
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. Determination of Chlorophyll Content
4.3. DNA Extraction and Pooling
4.4. DNA Sequencing and Quality Control
- (i)
- Removal of reads containing adapters;
- (ii)
- Filtering out reads with a nucleotide (N) content exceeding 10%;
- (iii)
- Removal of reads with more than 50% of bases with a quality score below 10.
4.5. Alignment to the Reference Genome
4.6. SNP Detection
- (i)
- Based on the subroutine vcfutils.pl (varFilter w 5-W 10) in bcftools, SNPs within 5 bp near InDel (insertion–deletion) were analyzed.
- (ii)
- ClusterSize 2 clusterWindow size 5 indicates that the number of mutations within a 5 bp window should not exceed 2.
- (iii)
- QUAL < 30, a quality value in phred format, indicates the possibility of variant variation at this locus. Filter out those with a quality value below 30.
- (iv)
- QD (quality by depth) < 2.0: The ratio obtained by dividing the quality of variation by the depth of coverage is the sum of the coverage depths of all samples containing mutated bases at this locus. Filter out those with QD below 2.0.
- (v)
- MQ (map** quality) < 40: The root mean square of the comparison quality values for all reads aligned to that site. Filter out MQ below 40.
- (vi)
- FS > 60.0. The p-value conversion obtained through Fisher’s test describes whether there is significant positive or negative chain specificity for reads that only contain mutations and reads that only contain reference sequence bases during sequencing or alignment. That is to say, there will be no chain-specific alignment results, and FS should be close to zero. Filter out those with FS higher than 60.
4.7. High-Quality SNP Filtering
- (i)
- SNPs with multiple genotypes were filtered out, retaining only biallelic genotype sites;
- (ii)
- SNPs with read support less than 4 in the mixed pools were filtered out;
- (iii)
- SNPs where the genotypes were homozygous and consistent among the mixed pools were filtered out;
- (iv)
- SNPs where the genotypes were homozygous and consistent between the two parents were filtered out;
- (v)
- For the recessive mixed pool, SNPs where the genotype did not originate from the recessive parent were filtered out; for the dominant mixed pool, SNPs where the genotype did not originate from the dominant parent were filtered out.
4.8. Euclidean Distance Method
4.9. SNP-Index Method
4.10. RNA Extraction, Library Construction, and RNA Sequencing
4.11. RT-qPCR Analysis
4.12. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Zhang, Z.; Tan, J.; Shi, Z.; ** of the rice gene OsARVL4 regulating leaf morphology and leaf vein development. Plant Growth Regul. 2016, 78, 345–356. [Google Scholar] [CrossRef]
- He, Y.; Shi, Y.; Zhang, X.; Xu, X.; Wang, H.; Li, L.; Zhang, Z.; Shang, H.; Wang, Z.; Wu, J.L. The OsABCI7 transporter interacts with OsHCF222 to stabilize the thylakoid membrane in rice. Plant Physiol. 2020, 184, 283–299. [Google Scholar] [CrossRef]
- Zeng, X.; Tang, R.; Guo, H.; Ke, S.; Teng, B.; Hung, Y.H.; Xu, Z.; ** of a novel barley Stage Green-Revertible Albino Gene (HvSGRA) by Bulked Segregant Analysis based on SSR assay and Specific Length Amplified Fragment Sequencing. BMC Genom. 2015, 16, 838. [Google Scholar] [CrossRef]
- Ye, S.; Yang, J.; Huang, Y.; Liu, J.; Ma, X.; Zhao, L.; Ma, C.; Tu, J.; Shen, J.; Fu, T.; et al. Bulk segregant analysis-sequencing and RNA-Seq analyses reveal candidate genes associated with albino phenotype in Brassica napus. Front. Plant Sci. 2022, 13, 994616. [Google Scholar] [CrossRef]
- Ke, X.; Shen, J.; Niu, Y.; Zhao, H.; Guo, Y.; Sun, P.; Yang, T.; Jiang, Y.; Zhao, B.; Wang, Z.; et al. Cucumber NUCLEAR FACTOR-YC2/-YC9 target translocon component CsTIC21 in chloroplast photomorphogenesis. Plant Physiol. 2023, 192, 2822–2837. [Google Scholar] [CrossRef]
- Pipitone, R.; Eicke, S.; Pfister, B.; Glauser, G.; Falconet, D.; Uwizeye, C.; Pralon, T.; Zeeman, S.C.; Kessler, F.; Demarsy, E. A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis. Elife 2021, 10, e62709. [Google Scholar] [CrossRef]
- Rojas, M.; Yu, Q.; Williams-carrier, R.; Maliga, P.; Barkan, A. Engineered PPR proteins as inducible switches to activate the expression of chloroplast transgenes. Nat. Plants 2019, 5, 211–505. [Google Scholar] [CrossRef]
- Zhou, G.; Qi, J.; Ren, N.; Cheng, J.; Erb, M.; Mao, B.; Lou, Y. Silencing OsHI-LOX makes rice more susceptible to chewing herbivores, but enhances resistance to a phloem feeder. Plant J. 2009, 60, 638–648. [Google Scholar] [CrossRef]
- Ma, F.; Yang, X.; Shi, Z.; Miao, X. Novel crosstalk between ethylene- and jasmonic acid-pathway responses to a piercing–sucking insect in rice. New Phytol. 2020, 225, 474–487. [Google Scholar] [CrossRef]
- Lin, D.; Zhou, W.; Wang, Y.; Sun, J.; Pan, X.; Dong, Y. Rice TSV2 encoding threonyl-tRNA synthetase is needed for early chloroplast development and seedling growth under cold stress. G3 2021, 11, jkab196. [Google Scholar] [CrossRef]
- Sun, J.; Zheng, T.; Yu, J.; Wu, T.; Wang, X.; Chen, G.; Tian, Y.; Zhang, H.; Wang, Y.; Terzaghi, W.; et al. TSV, a putative plastidic oxidoreductase, protects rice chloroplasts from cold stress during development by interacting with plastidic thioredoxin Z. New Phytol. 2017, 215, 240–255. [Google Scholar] [CrossRef]
- Wu, L.; Wu, J.; Liu, Y.; Gong, X.; Xu, J.; Lin, D.; Dong, Y. The rice pentatricopeptide repeat gene TCD10 is needed for chloroplast development under cold stress. Rice 2016, 9, 67. [Google Scholar] [CrossRef]
- Liu, X.; Zhang, X.; Cao, R.; Jiao, G.; Hu, S.; Shao, G.; Sheng, Z.; ** analysis pipeline for pooled RNA-seq. Genome Res. 2013, 23, 687–697. [Google Scholar] [CrossRef]
- Akagi, H.; Abe, A.; Yoshida, K.; Kosugi, S.; Natsume, S.; Mitsuoka, C.; Uemura, A.; Utsushi, H.; Tamiru, M.; Takuno, S.; et al. QTL-seq: Rapid map** of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J. 2013, 74, 174–183. [Google Scholar]
- Yang, X.; **a, X.; Zeng, Y.; Nong, B.; Zhang, Z.; Wu, Y.; Tian, Q.; Zeng, W.; Gao, J.; Zhou, W.; et al. Genome-wide identification of the peptide transporter family in rice and analysis of the PTR expression modulation in two near-isogenic lines with different nitrogen use efficiency. BMC Plant Biol. 2020, 20, 193. [Google Scholar] [CrossRef] [PubMed]
- Yang, X.; Wang, J.; **a, X.; Zhang, Z.; He, J.; Nong, B.; Luo, T.; Feng, R.; Wu, Y.; Pan, Y.; et al. OsTTG1, a WD40 repeat gene, regulates anthocyanin biosynthesis in rice. Plant J. 2021, 107, 198–214. [Google Scholar] [CrossRef]
Gene | Functional Annotation |
---|---|
LOC_Os08g38460 | zinc finger, C3HC4-type domain containing protein, expressed |
LOC_Os08g38710 | uncharacterized glycosyltransferase, putative, expressed |
LOC_Os08g38720 | cytochrome c oxidase assembly protein COX15, putative, expressed |
LOC_Os08g39420 | uncharacterized protein yeiN, putative, expressed |
LOC_Os08g39430 | thylakoid lumenal 19 kDa protein, chloroplast precursor, putative, expressed |
LOC_Os08g39600 | RNA methyltransferase, TrmH family protein, putative, expressed |
LOC_Os08g39694 | cytochrome P450, putative, expressed |
LOC_Os08g39850 | lipoxygenase, chloroplast precursor, putative, expressed |
LOC_Os08g40580 | methyltransferase domain containing protein, expressed |
LOC_Os08g40610 | 30S ribosomal protein S16, putative, expressed |
LOC_Os08g40850 | mitochondrial carrier protein, putative, expressed |
LOC_Os08g40900 | auxin response factor, putative, expressed |
LOC_Os08g40910 | expressed protein |
LOC_Os08g41730 | peptidase, T1 family, putative, expressed |
LOC_Os08g41820 | exo70 exocyst complex subunit domain containing protein, expressed |
LOC_Os08g41890 | microtubule-associated protein, putative, expressed |
LOC_Os08g41990 | aminotransferase, putative, expressed |
LOC_Os08g42020 | zinc ion binding protein, putative, expressed |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Wei, Y.; Li, X.; Li, D.; Su, X.; Huang, Y.; Li, Q.; Liang, M.; Yang, X. Map** and Candidate Gene Analysis of the Low-Temperature-Sensitive Albino Gene OsLTSA8 in Rice Seedlings. Curr. Issues Mol. Biol. 2024, 46, 6508-6521. https://doi.org/10.3390/cimb46070388
Wei Y, Li X, Li D, Su X, Huang Y, Li Q, Liang M, Yang X. Map** and Candidate Gene Analysis of the Low-Temperature-Sensitive Albino Gene OsLTSA8 in Rice Seedlings. Current Issues in Molecular Biology. 2024; 46(7):6508-6521. https://doi.org/10.3390/cimb46070388
Chicago/Turabian StyleWei, Yu, **aoqiong Li, Dongxiu Li, Xuejun Su, Yunchuan Huang, Qiuwen Li, Manling Liang, and **nghai Yang. 2024. "Map** and Candidate Gene Analysis of the Low-Temperature-Sensitive Albino Gene OsLTSA8 in Rice Seedlings" Current Issues in Molecular Biology 46, no. 7: 6508-6521. https://doi.org/10.3390/cimb46070388