Study On Resistance Mechanism Of Digitaria Sanguinalis To Haloxyfop-P-methyl In Cotton Field

Cotton is an important economic crop in China.As the dominant weed,Digitaria sanguinalis restricts the economic benefits of cotton fields.In order to clarify the control effect of an ACCase（acetyl-coenzyme A carboxylase）inhibitor herbicide: haloxyfop-P-methyl in cotton field,the resistance level of Digitaria sanguinalis populations in major cotton-producing areas was monitored for 4 years.By combining determination of target enzyme activity,determination of metabolic enzyme activity,detection of mutation sites,restriction-site associated DNA sequence and RNA-sequencing,the metabolic resistance mechanism of Digitaria sanguinalis to haloxyfop-P-methyl was studied.The main findings are as follows:（1）The resistance levels of 65 Digitaria sanguinalis populations were tested.Among them,8 populations: 13HN1,13HN4,13HN5,13HN6,13HN7,13HN8,14JX2 and 14JX7 were sensitive（RI（resistance index）< 2）;40 populations: 13HN2,13HN3,13HN9,13HN10,13HN11,13HN12,13HN13,13JX1,13HEN1,13HEN2,13HB1,13SX1,13HEB2,14HN3,14HN2,14HN5,14HN6,14HN8,14HN9,14HN12,14HEN1,14HEN2,14JX1,14JX3,14JX4,14HEB2,15HN2,15HN3,15HN4,15HN5 15HEN1,15HEB1,15HEB4,15HEB5,15HEB6,15HEB7,16JX1 and 16JX2 showed low-level resistance（2≤RI≤10）;17 populations: 13HB2,13XJ1,13HEB1,13AH1,14HN1,14HN10,14HB1 16HEB1,16HEB2,16HEB3,16HEB4,16HEB5,16HEB6,16HEB7,16HEB8,16HEB9 and 16JX3 showed moderate resistance（10<RI≤20）.No Digitaria sanguinalis population with RI >20 was found in the experiment.The results showed that the resistance of Digitaria sanguinalis to haloxyfop-P-methyl in cotton field in China was increasing year by year.Taking the results of Hunan Province as an example,among the 13 Hunan populations in 2013,6 populations were sensitive and 7 populations showed low-level resistance,of which 13HN9 had the highest GR50（growth reduction 50）of 16.430 g a.i./hm2.The average resistance index was only 3.88.By 2014,the average resistance index of 9 Hunan populations rose to 7.91.And 7 populations showed low-level resistance,2 populations（14HN10,14HN1）were moderately resistant,of which 14HN1 GR50 is the highest,reaching 28.089 g a.i./hm2.Resistance monitoring in Hebei Province showed a significant upward trend of resistance similarly: among the five Hebei populations in 2015,the average resistance index was only 3.55,and all five populations showed low-level resistance,of which 15HEB4 had the highest GR50 of 8.607 g a.i./hm2.In 2016,among the 9 Hebei populations,the average resistance index rose to 11.75,only one population（16HEB9）showed low-level resistance,and 8 other populations showed moderate resistance.The GR50 of 16HEB6 is the highest,reaching 30.024 g a.i./hm2.（2）Part of the CT domain of ACCase from R14LX3,R14LX7 and S14LX6 was sequenced.The results proved that no genetic mutations occurred at the 7 known amino acid sites: 1781,1999,2027,2041,2078,2088 and 2096.Combined with the detection results of metabolic enzyme activities,it was clear that the resistance of Digitaria sanguinalis to haloxyfop-P-methyl was not target-resistance,but metabolic resistance,probably dominated by GSTs.（3）23,789 M reads data were obtained in the restriction-site associated DNA sequence of the 11 Digitaria sanguinalis populations.The sequencing Q30 was 93.63% and the GC content was 51.74%.Through bioinformatics analysis,a total of 359,585 SNPs were obtained.Through the construction of a phylogenetic tree,population structure analysis and principal components analysis,a group of closely related cultivars（R14LX7 and S14LX6）that are resistant and sensitive to haloxyfop-P-methyl were screened out.（4）Through RNA-sequencing of R14LX7 and S14LX6,a total of 298,025 ROI（Read of insert）of Digitaria sanguinalis were obtained,and 133,492 transcripts were obtained after classifying,clustering,correction and de-redundancy.Using the Basic Local Alignment Search Tool to compare the results to the databases of Nr（NCBI non-redundant protein sequences）,Nt（NCBI nucleotide sequences）,Swiss-Prot,KOG（clusters of eu Karyotic Orthologous Groups）,KEGG（Kyoto Encyclopedia of Genes and Genomes）and GO（Gene Ontology）（E-value < 0.00001）,102,317 full-length transcripts were finally annotated.We also obtained 2,294 sequences with significant differences in expression（FDR < 0.001,log2 Ratio ≥ 1）,among which 5 candidate GSTs sequences with significant differences in expression before application were obtained,and the expression of these 5 sequences increased significantly at 12 h after application（MTPacbio₁-4k_c329986/f6p3/980,MTPacbio₁-4k_c269698/ f10p5/717,MTPacbio₁-4k_c4108/f1p0/586,MTPacbio₁-4k_c270311/f15p5/989 and MTPacbio₁-4k_c270025/f1p3/801）.（5）Through sequencing of RACE（rapid amplification of c DNA end）and TA cloning,the full-length sequences of the 5 GSTs genes were obtained.Then,we constructed overexpression vectors carrying the sequence of the GSTs: p ET-32a（+）-GST1,p ET-32a（+）-GST2,p ET-32a（+）-GST3,p ET-32a（+）-GST4 and p ET-32a（+）-GST5,and laid the foundation for subsequent functional verification.