Determination of genotoxic alkyl methane sulfonates and alkyl paratoluene sulfonates in lamivudine using hyphenated techniques

Two highly sensitive methods for the determination of genotoxic alkyl methane sulfonates (AMSs) and alkyl paratoluene sulfonates (APTSs) in lamivudine using hyphenated techniques have been presented. AMSs were determined by GC-MS method using GSBPINOWAX (30 m 0.25 mm 0.25 mm) column. Temperature program was set by maintaining at 100 1C initially for 3 min, then rised to 220 1C at the rate of 15 1C/min and maintained at 220 1C for 16 min. N,N-dimethyl formamide was used as diluent. APTSs were determined by LC-MS using Zorbax, Rx C8, 250 mm 4.6 mm, 5 mm column as stationary phase. 0.01 M ammonium acetate is used as buffer. The mixture of buffer and methanol in 75:25 (v/v) ratio was used as mobile phase A and mixture of buffer and methanol in 5:95 (v/v) ratio was used as mobile phase B. The gradient program (T/%B) was set as 0/28, 16/50, 17/100, 23/100, 27/28 and 40/28. Both the methods were validated as per International Conference on Harmonization guidelines. Limit of quantitation was found 1.5 mg/mL for AMSs and was in the range of 1.0-1.5 mg/mL for APTSs.

Creating large EMS populations for functional genomics and breeding in wheat

Wheat germplasm is a fundamental resource for basic research,applied studies,and wheat breeding,which can be enriched normally by several paths,such as collecting natural lines,accumulating breeding lines,and introducing mutagenesis materials.Ethyl methane sulfonate(EMS)is an alkylating agent that can effectively introduce genetic variations in a wide variety of plant species.In this study,we created a million-scale EMS population(MEP)that started with the Chinese wheat cultivars‘Luyan 128’,‘Jimai 38’,‘Jimai 44’,and‘Shannong 30’.In the M1 generation,the MEP had numerous phenotypical variations,such as>3,000 chlorophyll-deficient mutants,2,519 compact spikes,and 1,692 male sterile spikes.There were also rare mutations,including 30 independent tillers each with double heads.Some M1 variations of chlorophyll-deficiency and compact spikes were inheritable,appearing in the M2 or M3 generations.To advance the entire MEP to higher generations,we adopted a single-seed descendent(SSD)approach.All other seed composites of M2 were used to screen other agronomically important traits,such as the tolerance to herbicide quizalofop-P-methyl.The MEP is available for collaborative projects,and provides a valuable toolbox for wheat genetics and breeding for sustainable agriculture.

An ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5 gene mutation confers light green peel in cucumber

The peel color of fruit is an important commercial trait in cucumber, but the underlying molecular basis is largely unknown. A mutant showing light green exocarp was discovered from ethyl methane sulfonate （EMS） mutagenized cucumber line 406 with dark green exocarp. Genetic analysis showed the mutant phenotype is conferred by a single recessive gene, here designated as lgp （light green peel）. By re-sequencing of bulked segregants, we identified the candidate gene Csa7Go51430 encoding ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5 （ARCS） that plays a vital role in chloroplast division in Arabidopsis. A single nucleotide polymorphism （SNP） causing amino acid alteration in the conserved GTPase domain of Csa7Go5143o showed co-segregation with the altered phenotype. Furthermore, the transient RNA interference of this gene resulted in reduced number and enlarged size of chloroplasts, which were also observed in the Igp mutant. This evidence supports that the non-synonymous SNP in Csa7G051430 is the causative mutation for the light green peel. This study provides a new allele for cucumber breeding for light green fruits and additional resource for the study of chloroplast development.

A New Gene Conferring the Glabrous Trait in Cucumber Identified Using MutMap

Tubercles and spines on fruit peel are important commercial traits in cucumber(Cucumis sativus L.). From an ethyl methane sulfonate cucumber mutant library,we discovered a new glabrous mutant that bears no tubercle or spine on fruit peel and fewer and smaller trichomes on the stem and leaves. The new locus is here designated as glabrous2(gl2). Genome sequencing of the mutant and linkage analysis revealed that a non-synonymous mutation in the Csa1G056960 gene rendered the gl2 phenotype. The mutated gene encodes a C-type lectin receptor-like tyrosine protein kinase. This study provides a novel allele for elucidating the genetic basis of wart and trichome development and a new tool for breeding glabrous cucumber varieties.