identifying-mutations-high-resolution-melting-tilling-population

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

Zhejiang University

The aim of the present work was to investigate the photosynthetic performance and antioxidant enzyme activities in response to γ-irradiation of an aquatic plant Zizania latifolia. The Z. latifolia seedlings at 6-leaf stage were exposed to 25, 50 and 100 Gy of γ rays from a (60)Co source. The growth parameters, chlorophyll contents, photosynthetic gas exchange, chlorophyll fluorescence, malondialdehyde (MDA) content, antioxidant enzyme activities and antioxidant contents were examined at 1-5 weeks post-irradiation (WPI). The results showed that plant height, leaf number and tiller (branch close to ground) number were significantly suppressed by 50 and 100 Gy irradiation at 5, 3-5 and 4-5 WPI, respectively, but they were not significantly different from control by 25 Gy irradiation. Chlorophyll a, chlorophyll b, and total chlorophyll contents were also found to be significantly decreased by irradiation. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr) generally declined in a dose-dependent manner. As for the chlorophyll fluorescence parameters, maximum quantum efficiency of PSII photochemistry (Fv/Fm), actual photochemical efficiency of PSII (Φ(PSII)) and photochemical quenching (qP) were observed to be significantly decreased compared to the control at 3 WPI, while non-photochemical quenching (NPQ) significantly increased by 100 Gy. γ-irradiation induced substantial increase in MDA content, ascorbate peroxidase (APX) activity, reduced ascorbate (AsA) content and reduced glutathione (GSH) content, suggesting a protective mechanism of Z. latifolia plant against oxidative stress when exposed to γ-irradiation.

Regulation of photosynthetic performance and antioxidant capacity by ⁶⁰Co γ-irradiation in Zizania latifolia plants.

A γ-ray-induced xantha trait is epigenetically controlled by the genomes uncoupled 4 gene with enhanced promoter segment methylation and down-regulated expression in rice. For easy testing and to increase varietal purity, a xantha mutation (xnt), which turns plants yellow and makes them visually distinguishable from normal green rice, has been generated and bred into male sterile lines for hybrid rice production. The xnt locus was previously fine mapped to a ~100-kb interval on chromosome 11, but its identity was unknown. In this study, xnt was further narrowed down to a 57-kb fragment carrying eight opening reading frames (ORFs). All eight ORFs had identical genomic sequences and all but ORF2 (g enomes uncoupled 4, OsGUN4) had similar transcript abundance in the xantha mutant Huangyu B (HYB) and its parental variety Longtefu B (LTB). The expression of OsGUN4, however, was significantly reduced in HYB compared with LTB in terms of both transcript abundance (0.2% that of LTB) and expressed protein level (barely detectable in HYB but greater than the heat shock protein reference in LTB). Therefore, OsGUN4 was identified as the candidate gene underlying the xantha trait. The function of OsGUN4 in the xantha phenotype was confirmed by identification and characterization of new allelic OsGUN4 mutations. Comparative bisulfite genomic sequencing of OsGUN4 revealed increased methylation in a promoter region in the mutant, and the correlation between increased methylation and the xantha phenotype was further verified by demethylation treatment. In summary, we have identified an epi-allele of OsGUN4 as the causal gene of the xantha marker trait and revealed that enhanced methylation in its promoter down-regulated its expression in rice.

A down-regulated epi-allele of the genomes uncoupled 4 gene generates a xantha marker trait in rice.

The cytochrome P450 gene CYP81A6 confers tolerance to bentazon and metsulfuron-methyl, two selective herbicides widely used for weed control in rice and wheat fields. Knockout mutants of CYP81A6 are highly susceptible to both herbicides. The present study aimed to characterize the CYP81A6 expression in rice. Quantitative real-time polymerase chain reaction (PCR) analyses demonstrated that foliar treatment of bentazon (500 mg/L) greatly induced expression of CYP81A6 in both wild-type (Jiazhe B) and its knockout mutant (Jiazhe mB): a 10-fold increase at 9 h before returning to basal levels at 24 h in Jiazhe B, while in the mutant the expression level rose to >20-fold at 12 h and maintained at such high level up to 24 h post exposure. In contrast, metsulfuron-methyl (500 mg/L) treatment did not affect the expression of CYP81A6 in Jiazhe B within 80 h; thereafter the expression peaked at 120 h and returned gradually to basal levels by Day 6. We suggest that a metabolite of metsulfuron-methyl, 1H-2,3-benzothiazin-4-(3H)-one-2,2-dioxide, is likely to be responsible for inducing CYP81A6 expression, rather than the metsulfuron-methyl itself. Use of a promoter-GUS reporter construct (CYP81A6Pro::GUS) demonstrated that CYP81A6 was constitutively expressed throughout the plant, with the highest expression in the upper surfaces of leaves. Subcellular localization studies in rice protoplasts showed that CYP81A6 was localized in the endoplasmic reticulum. These observations advance our understanding of CYP81A6 expression in rice, particularly its response to the two herbicides. 

Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application.

The exploitation of male sterility systems has enabled the commercialization of heterosis in rice, with greatly increased yield and total production of this major staple food crop. Hybrid rice, which was adopted in the 1970s, now covers nearly 13.6 million hectares each year in China alone. Various types of cytoplasmic male sterility (CMS) and environment-conditioned genic male sterility (EGMS) systems have been applied in hybrid rice production. In this paper, recent advances in genetics, biochemistry, and molecular biology are reviewed with an emphasis on major male sterility systems in rice: five CMS systems, i.e., BT-, HL-, WA-, LD- and CW- CMS, and two EGMS systems, i.e., photoperiod- and temperature-sensitive genic male sterility (P/TGMS). The interaction of chimeric mitochondrial genes with nuclear genes causes CMS, which may be restored by restorer of fertility (Rf) genes. The PGMS, on the other hand, is conditioned by a non-coding RNA gene. A survey of the various CMS and EGMS lines used in hybrid rice production over the past three decades shows that the two-line system utilizing EGMS lines is playing a steadily larger role and TGMS lines predominate the current two-line system for hybrid rice production. The findings and experience gained during development and application of, and research on male sterility in rice not only advanced our understanding but also shed light on applications to other crops. 

Workable male sterility systems for hybrid rice: Genetics, biochemistry, molecular biology, and utilization.

Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M rice plants were sequenced; a total of 144-188 million high-quality (Q>20) reads were generated for each M plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10-9.8×10 in the six M rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species.

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing.

CRISPR-S: an active interference element for a rapid and inexpensive selection of genome-edited, transgene-free rice plants.

Rice is one of the world's most important foods, but its production suffers from insect pests, causing losses of billions of dollars, and extensive use of environmentally damaging pesticides for their control. However, the molecular mechanisms of insect resistance remain elusive. Although a few resistance genes for planthopper have been cloned, no rice germplasm is resistant to stem borers. Here, we report that biosynthesis of serotonin, a neurotransmitter in mammals, is induced by insect infestation in rice, and its suppression confers resistance to planthoppers and stem borers, the two most destructive pests of rice. Serotonin and salicylic acid derive from chorismate. In rice, the cytochrome P450 gene CYP71A1 encodes tryptamine 5-hydroxylase, which catalyses conversion of tryptamine to serotonin. In susceptible wild-type rice, planthopper feeding induces biosynthesis of serotonin and salicylic acid, whereas in mutants with an inactivated CYP71A1 gene, no serotonin is produced, salicylic acid levels are higher and plants are more insect resistant. The addition of serotonin to the resistant rice mutant and other brown planthopper-resistant genotypes results in a loss of insect resistance. Similarly, serotonin supplementation in artificial diet enhances the performance of both insects. These insights demonstrate that regulation of serotonin biosynthesis plays an important role in defence, and may prove valuable for breeding insect-resistant cultivars of rice and other cereal crops.

Resistance of rice to insect pests mediated by suppression of serotonin biosynthesis.

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.

High-resolution melting-based TILLING of γ ray-induced mutations in rice.

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK) is encoded by six genes in rice (). A previous study had shown that nucleotide substitutions of  could significantly lower the phytic acid content in rice grains. In the present study, the possibility of establishing a genome editing-based method for breeding low-phytic acid cultivars in rice was explored, in conjunction with the functional determination of OsITPK6. Four  mutant lines were generated by targeted mutagenesis of the gene's first exon using the CRISPR/Cas9 method, one () with a 6-bp in-frame deletion, and other three with frameshift mutations (, _, and _). The frameshift mutations severely impaired plant growth and reproduction, while the effect of  was relatively limited. The mutant lines  and _ had significantly lower levels (-10.1% and -32.1%) of phytic acid and higher levels (4.12- and 5.18-fold) of inorganic phosphorus compared with the wild-type (WT) line. The line  also showed less tolerance to osmotic stresses. Our research demonstrates that mutations of , while effectively reducing phytic acid biosynthesis in rice grain, could significantly impair plant growth and reproduction.

Article	Year
Regulation of photosynthetic performance and antioxidant capacity by ⁶⁰Co γ-irradiation in Zizania latifolia plants. Journal of environmental radioactivity\| PubMed ID: 24355402	2014
A down-regulated epi-allele of the genomes uncoupled 4 gene generates a xantha marker trait in rice. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik\| PubMed ID: 25208645	2014
Expression of cytochrome P450 CYP81A6 in rice: tissue specificity, protein subcellular localization, and response to herbicide application. Journal of Zhejiang University. Science. B\| PubMed ID: 25644466	2015
Workable male sterility systems for hybrid rice: Genetics, biochemistry, molecular biology, and utilization. Rice (New York, N.Y.)\| PubMed ID: 26055995	2014
Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing. Journal of Zhejiang University. Science. B\| PubMed ID: 27921396	2016
CRISPR-S: an active interference element for a rapid and inexpensive selection of genome-edited, transgene-free rice plants. Plant biotechnology journal\| PubMed ID: 28688132	2017
Resistance of rice to insect pests mediated by suppression of serotonin biosynthesis. Nature plants\| PubMed ID: 29735983	2018
High-resolution melting-based TILLING of γ ray-induced mutations in rice. Journal of Zhejiang University. Science. B\| PubMed ID: 30070085	2018
Mutation of Impairs Plant Growth and Phytic Acid Synthesis in Rice. Plants (Basel, Switzerland)\| PubMed ID: 31035443	2019

Jian-Zhong Huang

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