KRAS-Mutant Non–Small Cell Lung Cancer: Recent Progress

The KRAS mutant population has been undruggable for 40 years. G12C inhibitors and immunotherapy are the beginning of success. It is necessary to summarize the successful experience of the existing treatment model and explore the direction of the next treatment. In this review, we discuss the latest developments in targeted therapy and immunotherapy for KRAS-mutation NSCLC, aiming to provide direction or enlightenment for future treatment strategies.

A High-Resolution Method for the Systematic Detection of EMS-Induced Mutations in a Sequenced Population

The precise detection of causal DNA mutations is very crucial for forward genetic studies. Several sources of errors contribute to false-positive detections by current variant-calling algorithms, and these impact associating phenotypes with genotypes. To improve the accuracy of mutation detection we propose and implemented a high-resolution binning method for the accurate detection of likely EMS-induced mutations in a sequenced mutant population. The approach also incorporates a novel clustering algorithm for detecting likely false negatives with high accuracy. Sorghum bicolor is a very valuable crop species with tremendous potential for uncovering novel gene functions associated with highly desirable agronomical traits. We demonstrate the precision of the proposed method in the detection of likely EMS-induced mutations in the publicly available low-cost sequencing of the M3 generation from 600 sorghum BTx623 mutants. The method detected 3,274,606 single nucleotide polymorphisms (SNPs) of which 96% (3,141,908) were G/C to A/T DNA substitutions, as expected by EMS-mutagenesis action. We demonstrated the general applicability of the method, and showed a high concordance, 94% (3,074,759) SNPs overlap between SAMtools-based and GATK-based variant-calling algorithms. We also implemented a novel clustering algorithm which uncovered evidence for an additional 223,048 likely false-negative shared EMS-induced mutations. The final 3,497,654 SNPs represents an 87% increase in SNPs detected in the previous analysis of the sorghum mutant population. Annotation of the final SNPs revealed 10,263 high impact and 136,639 moderate impact SNPs, including 7,217 stop-gained mutations, and an average of 12 stop-gained mutations per mutant. We have implemented a public search database for this new genetic resource of 30,285 distinct sorghum genes containing medium or high impact EMS-induced mutations. Seedstock for a select 486 of the 600 described mutants are publicly available in the Germplasm Resources Information Network (GRIN) database.

Detecting Genetic Mobility Using a Transposon-Based Marker System in Gamma-Ray Irradiated Soybean Mutants

Transposable elements (TEs)—major components of eukaryotic genomes—have the ability to change location within a genome. Because of their mobility, TEs are important for genome diversification and evolution. Here, a simple rapid method, using the consensus terminal inverted repeat sequences of PONG, miniature inverted-repeat transposable element (MITE)-Tourist (M-t) and MITE-Stowaway (M-s) as target region amplification polymorphism (TE-TRAP) markers, was employed to investigate the mobility of TEs in a gamma-irradiated soybean mutant pool. Among the different TE-TRAP primer combinations, the average polymorphism level and polymorphism information content value were 57.98% and 0.14, respectively. Only the PONG sequence separated the mutant population into three major groups. The inter-mutant population variance, determined using the PONG marker (3.151 and 29%) was greater than that of the M-t (2.209 and 20%) and M-s (2.766 and 18%) markers, whereas the reverse was true for the intra-mutant population variations, with M-t and M-s values, being 15.151 (82%) and 8.895 (80%), respectively, compared with the PONG marker (7.646 and 71%). Thus, the MITE markers revealed more dynamic and active mobility levels than the PONG marker in gamma-ray irradiated soybean mutant lines. The TE-TRAP technique associated with sensitive MITEs is useful for investigating genetic diversity and TE mobilization, providing tools for mutant selection in soybean mutation breeding.

Anthracnose resistance induction in chilli by electron beam irradiation.

Chilli seeds were irradiated with 0.3 kGy at 8 MeV from the electron beam source at the Thailand Institute of Nuclear Technology. M1 seeds were planted in Sukhothai Horticultural Research Centre and from these the line CA1131 was selected as suitable for growing in this area. Thirty anthracnose-resistant M2 chilli plants were selected after the appearance of anthracnose disease, caused by Colletotrichum gloeosporioides, in Sukhothai province from an initial M1 mutant population of 123 individuals. However, chilli fruits from 17 plants showed resistance after laboratory inoculation experiments. These chilli plants were crossed with the 'Hoarue Huaisai', which has large fruit. The F2 progenies were selected for anthracnose resistance and large fruits. Two hybrids with anthracnose resistance (derived from the cross CA1131 × 'Hoarue Huaisai') were identified and used for field anthracnose resistance tests in 2015. Resistant plants with large fruits were discovered in the F3 inbred line no. 6-1-4 grown during the dry season, but this line did not show strong disease resistance in the rainy season. A further 63 F5 inbred lines showed anthracnose resistance in the field experiment. Five samples per line of each of the 63 inbred lines were inoculated in the laboratory at Thailand Institute of Nuclear Technology. The fruits of inbred line no. 32-2-8 showed complete anthracnose resistance and seven lines were segregating as resistant. All eight of these lines are being used in the ongoing chilli project aimed at developing chilli varieties with broad resistance to anthracnose caused by three Colletotrichum species that are prevalent in Thailand.

Gamma-ray induced pedigreed mutant population of tossa jute (Corchorus olitorius L.): a key resource for forward and reverse genetics.

Narrow genetic diversity in available germplasm is a serious limiting factor for academic progress and agronomic improvement of crops like Corchorus olitorius, an economically important bast fibre crop. Mutation breeding, with its proven ability to improve qualitative as well as quantitative traits, can be employed to augment germplasm diversity. In the present study, gamma-rays were used to treat the seeds of two promising varieties, JRO 204 and JRO 8432; LD50 doses for gamma-rays were 200 Gy and 300 Gy for JRO 204 and JRO 8432, respectively. Irradiation of two varieties has resulted in the development of a large number of macro-mutants, such as twisted bark, extreme dwarf, non-abscission leaf, soft stem, hard stem and round pod mutants. Morphological and anatomical studies of these mutants gave new light on secondary growth in the species. In addition to the academic utility, these mutants will prove of immense importance to plant breeders aiming to improve fibre quality. Moreover, novel mutants will help to develop new plant architecture suitable for diversified applications of the genus.

Fast and Inexpensive Phenotyping and Genotyping Methods for Evaluation of Barley Mutant Population

To further develop barley breeding and genetics, more information on gene functions based on the analysis of the mutants of each gene is needed. However, the mutant resources are not as well developed as the model plants, such as Arabidopsis and rice. Although genome editing techniques have been able to generate mutants, it is not yet an effective method as it can only be used to transform a limited number of cultivars. Here, we developed a mutant population using ‘Mannenboshi’, which produces good quality grains with high yields but is susceptible to disease, to establish a Targeting Induced Local Lesions IN Genomes (TILLING) system that can isolate mutants in a high-throughput manner. To evaluate the availability of the prepared 8043 M3 lines, we investigated the frequency of mutant occurrence using a rapid, visually detectable waxy phenotype as an indicator. Four mutants were isolated and single nucleotide polymorphisms (SNPs) were identified in the Waxy gene as novel alleles. It was confirmed that the mutations could be easily detected using the mismatch endonuclease CELI, revealing that a sufficient number of mutants could be rapidly isolated from our TILLING population.