| The bird cherry-oat aphid, Rhopalosiphum padi(L.)(Hemiptera: Aphididae) is a notorious wheats pest which was widely distributed all over the world. This species can cause yield reduction and quality decreased of the wheat by sap sucking and transmission of barely yellow dwarf virus(BYDV) that leads to serious economic damage to the wheat production. In the past few years, with the influence of global climate, farming systems, wheat varieties, anthropogenic effect and other factors, the damage of R. padi to wheats has been increased and the distribution area has been expanded. R. padi is impotant pest in several main wheat production regions of China.In the current study, five polymorphic microsatellite loci were selected and used for the analysis of the genetic diversity and population structure of 22 geographical populations of R. padi in China, and the effect of chlorpyrifos and isoprocarb selection on the genetic diversity and genetic structure of R. padi populations. In addition, the sublethal effects of the two insecticides to R. padi were analyzed using life-table method. Furthermore, the characteristics of microsatellite loci from Rhopalosiphum padi transcriptome were analyzed. The results were as follows: 1. Amplifying stability and genetic diversity of microsatellite loci in Rhopalosiphumpadi samples from ChinaIn order to screen polymorphic microsatellite loci which can be used for the population genetics research of Rhopalosiphum padi populations in China, the amplifying stability and genetic diversity of eight previously reported microsatellite loci were analyzed on 282 individuals sampled in nine regions of eight provinces in China. The PCR fragments of the microsatellite loci were labeled fluorescently and scanned automatically. The results showed that locus R1.35 could not be amplified in any samples of the nine populations, and the locus R5.29 b could only be amplified in a few samples of seven populations. Loci R2.73, R3.171, R5.10, R5.138, R5.50 and R6.3 could be amplified stably in samples of all the populations. The null allele frequency of the six stably amplified loci was between 0.0044 and 0.2663, the mean number of allele(Na) ranged from 2.9 to 9.3, and the observed heterozygosity(Ho) was from 0.047 to 0.912. Locus R6.3 showed lower observed heterozygosity(0.047) and mean number of allele(2.9) than other loci, indicating it could not be used for further population genetics analysis in China. Loci R2.73, R3.171, R5.10, R5.138 and R5.50 showed high observed heterozygosity and mean number of allele, suggesting they are usable for the genetic diversity and genetic structure research of R. padi populations in China. 2. Genetic diversity and genetic structure of Rhopalosiphum padi geographicalpopulations in China.Five microsatellite loci were used for the analyses of genetic diversity and genetic structure in 22 geographical populations of R. padi. A total of 144 alleles were detected and the average null allele frequency was 0.032. The average number of alleles, effective allele, allele richness and Shannon index were 5.39, 3.19, 4.989 and 1.171. The number of MLG and multi-locus genotypic diversity were ranged from 3- 32 and 15%- 100%. The average observed heterozygosity and expected heterozygosity were 0.756 and 0.604. Most population showed heterozygosity excess and only JLB, QHX and GSL population obey Hardy-Weinberg equilibrium. With microsatellite DNA markers, the genetic similarity among populations ranged from 0.3477 to 0.9966, Nei’s genetic distance ranged from 0.0034 to 1.0564 and the pairwise genetic differentiation ranged between-0.021 and 0.334. Meanwhile, a significant isolation-by-distance effect presents among different populations with the Mantel test. No matter STRUCTURE, PCA or NJ analysis with different genetic distance, 22 R. padi geographical populations were divided into three groups, which includes Group I(Population QHX, GSL, GST, JLB, CQB and SAH), Group II(YNK, XZL, GZG), and Group III(HBB, SXT, SXH, SAX, SAY, NMB, SDZ, SDT, SDH, AHC, HNN, HUZ and HUW). The analysis of molecular variance(AMOVA) showed that most of the genetic variation was from individuals, only a small part from populations and groups. Populations genetics of R. padi could be influenced by the geographical isolation, complex climate, or reproductive mode. 3. Inductive effect of chlorpyrifos and isoprocarb on the genetic diversity andpopulation structure of different Rhopalosiphum padi populationsThe effects of chlorpyrifos and isoprocarb on genetic diversity and population structure of R. padi were analyzed over five polymorphic microsatellite loci. The results showed that, the repesctive resistance index of four R. padi populations were 15.12, 5.46, 11.12 and 5.58 folds after slection by chlorpyrifos for 20 generations, while the resistance index of R. padi populations to isoprocarb were 13.28, 10.18, 8.86 and 9.18 folds after being selected for 20 generations. The PIC and allele frequency of resistant populations was changed comparing to the susceptible population. Under the selection pressure of chlorpyrifos and isoprocarb, the genetic diversity indexes and the genetic similarity of resistance populations were reduced and the genetic distances among resistance and susceptible populations were enlarged, respectively. The STRUCTURE analysis result indicated the selection pressure did not influence the population structure. 4. Effects of sublethal concentrations of chlorpyrifos and isoprocarb on experimentalpopulation of Rhopalosiphum padiTo evaluate the effects of sublethal concentrations of chlorpyrifos and isoprocarb on experimental population of R. padi, the immersion test method was used to test the toxicity of chlorpyrifos and isoprocarb to adult R. padi. In a laboratory experiment, the life-table was used to analyze the effects of two sublethal concentrations of chlorpyrifos(C-LC20, C-LC30) and also two sublethal concentrations of isoprocarb(I-LC20 and I-LC30) on the development and reproduction of R padi F0 and F1 generations. The results showed that the average longevity and average fecundity of R. padi F0 generation significantly decreased after treated with the four sublethal concentrations compared with the control. The respective average longevity of R. padi F0 generation decreased by 14.03%, 36.04%, 12.01% and 26.86% while average fecundity of R. padi F0 generation decreased by 20.55%, 42.27%, 17.13% and 26.00% after treated with C-LC20, C-LC30, I-LC20 and I-LC30. Compared to the control, C-LC20, C-LC30, I-LC20 and I-LC30 treatments significantly increased the average development period of the larval stage of F1 generation by 1.51 d, 1.92 d, 0.9 d and 1.19 d respectively. However, the average longevity of R. padi F1 generation under the four treatments decreased by 21.62%, 33.68%, 15.51% and 23.14%, respectively. The average fecundity of F1 generation decreased by 21.81%, 37.4%, 14.51% and 29.29%, respectively. Compared to the control, sublethal concentrations of chlorpyrifos and isoprocarb decreased the survival rate, the intrinsic rate of increase(rm), the net reproductive rate(R0), the finite rate of increase(λ) and the gross reproduction rate(GRR) of R padi F1 generation, but increased the development duration, the mean generation time(T) and the population doubling time(Dt) of F1 generation. The current results suggested that the sublethal concentrations of chlorpyrifos and isoprocarb decreased population development rate and fecundity rate of R. padi. 5. Analysis and selection of microsatellite loci from Rhopalosiphum padi based ontranscriptome sequencingTo develop microsatellite marker avalible for R. padi, we sequenced the transcriptome of the species. In total, 7936 microsatellite loci were identified from 29467 unigenes, of which, 1296 SSR sequences could be used for primer design. The majority repeat types of these microsatellite loci were trinucleotide and mononucleotide motifs(45.75% an 22.86%, respectively). The AAT/ATT and ACG/CGT microsatellite motif were the most abundant trinucleotide motif. The number of repeats of all microsatellite loci were between 4 and 24. Based on the identified microsatellite loci, 60 pairs of primer were designed by the Primer 3 program and 24 pairs of the loci could be stably amplification, of which 14 loci showed relatively high polymorphism and could be used for population genetics of R. padi. These results showed that transcriptome sequencing of Rhopalosiphum padi can be used to develop microsatelliet of the species. |
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