Assessment of variety distinctness is important for both the registration and the protection of particular variety. However, the current testing system, which assesses a range of morphological characters of each pair of varieties grown side-by-side, is time consuming and is not suitable for the assessment of hundreds of samples. The objective of this study was to develop a procedure for the assessment of wheat variety distinctness using simple sequence repeat (SSR) markers. A comparison between the molecular and morphological profile of 797 varieties was made. On the basis of the comparison, pairs of varieties with a genetic similarity value (GSV) ≤ 90% were deemed to be distinct, accounting for ~85% of varieties assessed in wheat regional trials. For the remaining ~15% of varieties, GSVs between different varieties were > 90%, among which ~35% were not distinct and the other ~65% were distinct. Therefore, if given a GSV > 90%, the pairs of varieties should be morphologically assessed in the field. To avoid any errors in the assessments, we proposed the elimination of contaminant plants from the sample before comparing the varietal genotypes, scoring of the genotype at each locus with a pair of allele numbers when constructing a molecular profile, and faithfully recording two alleles at a non-homozygous locus. To reduce the workload and cost, a three-grade markers comparison among varieties is suggested. In addition, 80 SSR markers and a technical procedure for assessment of wheat variety distinctness have been proposed. Based on the procedure, the distinctness assessment of ~85% of all wheat varieties is completed in our laboratory annually. Consequently, total field assessment has been reduced considerably.

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Background: The two-spotted spider mite (TSSM), Tetranychus urticae (Acari: Tetranychidae), is a cosmopolitan phytophagous pest in agriculture and horticulture. It has developed resistance to many acaricides by target-site mutations. Understanding the status and evolution of resistant mutations in the field is essential for resistance management. Here, we applied a highthroughput Kompetitive allele-specific polymerase chain reaction (KASP) method for detecting six mutations conferring resistance to four acaricides of the TSSM. We genotyped 3274 female adults of TSSM from 43 populations collected across China in 2017, 2020, and 2021.Results: The KASP genotyping of 24 testing individuals showed 99% agreement with Sanger sequencing results. KASP assays showed that most populations had a high frequency of mutations conferring avermectin (G314D and G326E) and pyridaben (H92R) resistance. The frequency of mutation conferring bifenazate (A269V and G126S) and etoxazole (I1017F) resistance was relatively low. Multiple mutations were common in the TSSM, with 70.2% and 24.6% of individuals having 2-6 and 7-10 of 10 possible resistant alleles, respectively. No loci were linked in most populations among the six mutations, indicating the development of multiple resistance is mainly by independent selection. However, G314D and I1017F on the nuclear genome deviated from Hardy-Weinberg equilibrium in most populations, indicating significant selective pressure on TSSM populations by acaricides or fitness cost of the mutations in the absence of acaricide selection.Conclusion: Our study revealed that the high frequency of TSSMs evolved multiple resistant mutations in population and individual levels by independent selection across China, alarming for managing multiple-acaricides resistance.

BACKGROUND Pesticide resistance is a long‐standing and growing problem in the chemical control of invertebrate pests. Molecular diagnostic methods can facilitate pesticide resistance management by accurately and efficiently detecting resistant mutations and their frequency. In this study, the kompetitive allele specific PCR (KASP) approach, a technology for high‐throughput single nucleotide polymorphism (SNP) genotyping, is validated as a useful method for characterizing genotypes at a pesticide‐resistance locus for the first time. We focus on the spinetoram resistance mutation of G275E in the nicotinic acetylcholine receptor alpha 6 (nAChR α6) subunit gene of Thrips palmi. RESULTS Of the 341 individuals of Thrips palmi tested, 98.24% were successfully genotyped, with 100% concordance with Sanger sequencing results. We then quantitatively mixed genomic DNA of known genotypes to establish 21 DNA mixtures with a resistant allele frequency ranging from 0 to 100% at steps of 5%. The linear discriminant analysis (LDA) showed that 75.8% of original grouped cases were correctly classified; six groups had no overlap in membership (resistant allele frequency: 0%, 5%, 10–75%, 80–85%, 90–95%, and 100%). When we chose 11 pooled samples with 10% steps for LDA, 84.4% of original grouped cases were correctly classified; seven groups had no overlap in membership (0%, 10%, 20–30%, 40–70%, 80%, 90%, 100%). The results indicated that KASP applied to pooled samples may provide a semi‐quantitative estimate of resistance. CONCLUSIONS Our study points to the suitability of KASP for high‐throughput genotyping of genotypes affecting pesticide resistance and semi‐quantitative assessments of resistance allele frequencies in populations. © 2023 Society of Chemical Industry.