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 ...

Jiaxing Academy of Agricultural Sciences

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.

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.

Hao-Wei Fu

Jiaxing Academy of Agricultural Sciences

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