| Cereal cyst nematode mainly affects wheat,barley,oats,rye and other grass crops,and is widely distributed worldwide causing serious yield losses.In this study,we investigated the occurrence and distribution of Heterodera avenae in 16 provinces and amplified the r DNA-ITS genes of H.avenae populations in different geographical locations and different hosts in China to clarify the occurrence and genetic diversity of H.avenae.We analyzed the mitochondrial COI sequences of 98 H.avenae populations,traced their origin,clarified their genetic structural characteristics and evolutionary relationships in China;In addition,the mitochondrial genome of H.avenae was sequenced to understand its composition and taxonomic status;The whole genome of 79H.avenae populations in China was sequenced to clarify their genetic differentiation and to identify the molecular mechanisms related to their pathogenesis;Heterodera avenae and H.filipjevi were detected by the combination of CRISPR-Cas12a and RPA technologies in affected field samples which served as an important tool for the early diagnosis of wheat cyst nematode in natural infested field.The main findings of this study are as follows:1.In this study,a total of 821 soil and host root samples were collected from 16 provinces in 2019–2022 to investigate the distribution of the CCN.H.avenae was detected in 56.39%of the total samples,primarily in Hubei,Henan,Hebei,Shandong,Shanxi,Gansu,Beijing,Tianjin,Inner Mongolia,Ningxia,Xinjiang,Qinghai,Anhui,Shaanxi and Jiangsu.H.filipjevi was present in 21 samples,with a detection rate of2.60%,and it was found mainly in Henan,Anhui,Jiangsu,Shandong,Shanxi and Qinghai.A phylogenetic analysis of the internal transcribed spacer（ITS）region（18S-ITS1-5.8S-ITS2）of the r RNA gene indicated that significant evolutionary and genetic differences existed between the Chinese populations and the populations from other countries.Our results indicate that ITS1 can be used as a phylogenetic analysis and genetic target for H.avenae populations.The haplotypes of the ITS1 sequences of H.avenae populations from 14 countries were analyzed,and we speculate that H.avenae originated in a Middle East hotspot,then spread westwards to Europe and the United States and eastwards to China and Australia.Genetic differences between Asian and European populations suggested that the Himalayas and Kunlun Mountains formed a barrier that resulted in the formation of a separate evolutionary group in China.The phylogenetic and haplotype analysis results from different hosts showed that there were significant differences among populations isolated from different hosts,and those isolated from weeds were clearly distinct from those from other hosts.This indicates that the rich genetic diversity of H.avenae populations is related to the large numbers of available hosts.Above all,geographic barriers,time of origin and hosts might explain the current known distribution patterns of Chinese H.avenae populations.2.In this study,we determined the complete mitochondrial（mt）DNA genome sequence of H.avenae.The whole mt genome of H.avenae is circular and 20,957 bp in length.It consists of a total of 34 genes including 12 protein coding genes（PCGs）,two ribosomal RNA（r RNA）genes and 20 transfer RNA（t RNAs）genes.A phylogenetic tree reconstructed by using 30 chloroplast genomes reveaed that H.avenae is more closely related to H.glycines.The mt genome data provided in this study can provide new genetic markers for diagnostics,systematics,population genetics and molecular epidemiology of H.avenae in the future.Furthermore,we amplified and analyzed the mt CO1 genes for a total of 98 populations of H.avenae collected from the major wheat-growing regions in China in addition to six forenigner countries.Forty-one mitochondrial COI haplotypes were identified,suggesting a high genetic diversity and endemism level of H.avenae in China.Phylogenetic analysis showed that H.avenae populations in China were divided into four clades.Significant evolutionary and genetic differences were found between Chinese（except Hubei）and foreign populations.Hap1,the most widely distributed haplotype,was considered to be a separate evolutionary origin in China.The gene flow of H.avenae from the northwestern region to the north China region and Huang-Huai-Hai region was significant,also as the direction between north China and Huang-Huai-Hai region.We speculate that water flowing from the Yellow River and the mechanical harvesters promoted gene exchange among these groups.Our obtained results from the distance-based redundancy analysis showed that these genetic distances observed among H.avenae populations were explained foremost not only by geographic distance but also by temperature and precipitation.This study provides theoretical support for the origin and spread of H.avenae populations in China and elsewhere in the world.3.Information of variant loci,including SNP and In Del,was obtained for 79 different geographical samples of H.avenae in China using H.avenae whole genome as a reference.The distribution types of these loci mainly included intergenic regions,intronic regions,3’and 5’end untranslated regions.The GO analysis results showed that the mutated genes were mainly involved in biological processes,cellular components and molecular functions;COG classification statistics showed that the mutated genes were mainly involved in signal transduction mechanisms,general function prediction.The KEGG enrichment analysis showed that the variant genes were classified into 226 metabolic pathways and 10 of them were significantly enriched（P-value<0.01）.Based on the information of SNP loci with high confidence,the study found that the different geographical groups of H.avenae could be divided into four subgroups by evolutionary tree,population structure and principal component analysis,namely,Northwest subgroup,North China subgroup,Middle and Lower Yangtze River subgroup and East China subgroup;In addition,the increase of nucleoglycan polymorphism in Northwest subgroup further accelerated its LD decay fascination.4.In this study,recombinase polymerase amplification（RPA）combined with clustered regularly interspaced short palindromic repeats（CRISPR）/Cas12a（formerly known as cpf1）was developed for the rapid detection of H.avenae and H.filipjevi in the infested field samples.The RPA reaction was performed at a wide range of temperatures from 35 to 42℃ within 15 min.There was no cross-reactivity between H.avenae,H.filipjevi and the common closely related plant-parasitic nematodes,indicating the high specificity of this assay.The detection limit of RPA-Cas12a was as low as 10-4single second-stage juvenile（J2）,10-5 single cyst,and 0.001 ng of genomic DNA,which is 10 times greater than that of RPA-LFD detection.The RPA-Cas12a assay was able to detect 10-1 single J2 of H.avenae and H.filipjevi in 10 g of soil.In addition,the RPA-LFD assay and RPA-Cas12a assays both could quickly detect H.avenae and H.filipjevi in naturally infested soil,and the entire detection process could be completed within 1 h.These results indicated that the RPA-Cas12a assay developed herein is a simple,rapid,specific,sensitive,and visual method that can be easily adapted for the quick detection of H.avenae and H.filipjevi in infested fields. |
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