slaf-seq解密灰飞虱长距离迁徙奥秘(英文)

Study on long-distance migration of small brown planthoppers

Laodelphax striatellus in China using next‐generation sequencing

Wenjing Zheng1*, Zhiqiang Li2,Jiaming Zhao1, Yanzhi Zhang3, Changhua Wang3, Xiaochun Lu1*, Fuyu

Sun2*

1 The Crop Molecular Improving Laboratory, Liaoning Innovation Center of the Academy of Agriculture Sciences, Shenyang, People’s Republic of China.

2 Liaoning Plant Protection Institute of the Academy of Agriculture Sciences, Shenyang, People’s Republic of China.

3 Liaoning Rice Research Institute of the Academy of Agriculture Sciences, Shenyang, People’s Republic of China.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/ps.3992

Abstract

BACKGROUND: The small brown planthopper (L. striatellus) is a wide-spread insect pest of rice in East Asia. Previous studies have shown the long-distance migrations undertaken by L. striatellus, but did not provide molecular evidence to support this.

RESULTS: Long-distance immigration occurred in the northeast coastal rice growing region of China. Using the SALF-seq technique, sequence data for 2.7Gb of an abruptly increased population and 13 L. striatellus local populations from a range of regions in China that have serious rice stripe disease were obtained. A total of 2572 SNPs and 37 Indels were detected and the genotypes of many polymorphism sites were heterozygous in every sample, which indicated that there were rich genetic differences among the populations and the migration of insect pests accelerated the gene flow and increased the heterozygosity of L. striatellus populations. The genetic distance and the polymorphism markers among different populations showed that the abruptly increased population in Liaoning Province is close to several populations that from Jiangsu Province and Shandong Province.

CONCLUSION: The vector that caused rice stripe disease in the northeast of China was an immigrant population; however the population may be formed from several groups from different areas, such as Jiangsu and Shandong Provinces.

Key words: The small brown planthopper; Laodelphax striatellus;migration; SALF-seq; SNP

Email：zwj27@https://www.360docs.net/doc/565163787.html,(WZ); luxiaochun2000@https://www.360docs.net/doc/565163787.html, (XL); laassfy@https://www.360docs.net/doc/565163787.html, (FS) Introduction

The small brown planthopper, Laodelphax striatellus Fallén (Homoptera: Delphacide), is a wide-spread insect pest of rice (Oryza sativa) that causes rice stripe disease by transmitting Rice stripe virus (RSV) in East Asia.1 Heavy infestation has occurred in China2, Japan3-6 and South Korea.7-9 Laodelphax striatellus (L. striatellus) and the viral disease that they transmit are difficult to control, especially in the regions where the damage are mainly caused by immigrant populations.8L. striatellus can cross water and international boarders to form new populations. In western Japan, a large trap catches of L. striatellus with high viruliferous rates

have been recorded on a windy day in early June 2008, and subsequently, rice stripe disease spreaded in those regions.5 The migration source has been estimated to be Jiangsu Province of China, via backward trajectory analysis and insecticide susceptibility test.5 Responding to the mass migration identified in Japan, South Korean scientists set up a monitoring network of 13 net traps for L. striatellus along their western coast in May 2009.4 A mass immigration event was detected from May 30 to June 1, 2009.4 In 2011 in Taean, Gunsan and Buan Counties, South Korean, and Taean in 2012 a similar migration event was detected.Based on the backward trajectory analysis, a possible migration source for these case was found to be Jiangsu Province of China. 4 According to surface weather maps for the times when these migration events occurred, there were low-pressure systems over Bohai Sea in 2009 and 2011, and a high pressure system over the southern Yellow Sea in 2012 that may have caused southwesterly winds to carry the insects to South Korea.4 In addition to the overseas migration, L. striatellus can undertake domestic migration. During the wheat harvesting season, migrant populations of L. striatellus can be found in the provinces that neighbor Jiangsu Province.10 In Shandong Province a large population of L. striatellus was observed using a light trap late at night on the 7 June, 2009, and backward trajectories showed that a possible migrant source was northern Jiangsu Province. In addition, forward trajectories were studied on the 15 June 2010 and they showed a destination of Hebei and Liaoning Provinces.10

Genetic polymorphism among different L. striatellus populations has been studied.11-15 There is debate as to whether L. striatellus populations have significant differences and if the population structure is significantly affected by migration. Using an allozyme polymorphism, the geographic differences among 11 L. striatellus populations from Japan and Taiwan were investigated. The results indicated that significant differences could be found among the tested populations; the long-range dispersal of L. striatellus did not have a large effect on its population structure of L. striatellus due to irregular migration.12 Nevertheless mitochondrial DNA sequences and RAPD analysis showed that there was less differentiation among planthoppers populations from different geographical regions.11,13,15 The genome-sequencing of L. striatellus has not been completed yet, and markers are limited for polymorphism identification. Therefore, sequence data to analyze the differences among different L.

striatellus populations is necessary. With the development of high-throughput sequencing technology, more and more genomes have been successfully sequenced by low-cost sequencing technologies such as SLAF-seq (specific-locus amplified fragment sequencing). The SLAF-seq was developed on the basis of high-throughput sequencing technology, which allows researchers to design an experiment via bioinformatics for fragments of a specific length from the constructed SLAF-seq library.16 SLAF-seq technology has many advantages, such as high throughput, low cost, high accuracy and short cycle. It is possible to directly perform polymorphism analysis and molecular marker development from the sequence data provided by SLAF-seq. The use of SLAF-seq has been reported for genetic mapping, linkage mapping, polymorphism analysis, system evolution and germ plasma resource identification.17-20In this paper, the SLAF-seq technology was used for the first time to seek the molecular evidence of the insects’ migration. The use of SLAF-seq to verify the migration of L. striatellus will enable this technique to be used in future studies.

There were severe rice stripe disease infections along the northeast coast of China between 2006 and 2009. To analyze the source of the virus vectors, we used SLAF-seq technique to sequence 14 L. striatellus populations from different regions of China. By comparing the genetic distance and the number of polymorphism markers, we analyzed the geographic homology of L. striatellus from different regions and sought the molecular evidence of L. striatellus migration.

Materials and Methods

L. striatellus populations

A total of 14 samples of L. striatellus were collected from the test plots of agricultural extension stations in 13 cities, where rice stripe disease has been very serious since 2006. All the samples of L. striatellus were adults. Details of the samples and the collections locations are given in Table 1. An extensive field survey combined with trapping by mesh bags was used for sample collection, and obtained an average of 25 samples per locality. To prevent over-representation of siblings from each locality, each insect was collected from a location at least 1 m from the neighboring one. All the samples were preserved and stored at -80 ℃ in the Crop Molecular Improving Laboratory, Liaoning Academy of Agriculture Sciences (Shenyang).

Rice variety

Liaojing 9, a susceptible rice variety to L. striatellus and RSV, was cultivated to investigate population density of L. striatellus and incidence of rice stripe disease at Dagushan town of Donggang city, Liaoning Province, China from 2006 to 2013. The seeds were sown on the 15 April, and the seedlings were transplanted on the 20 May.

Population dynamics of L. striatellus

There were 10 plots in the test field. Every plot consisted of three rows that were three-meters-long, and the plant spacing was 13.3×33.3 cm. No pesticides were used during the entire growth period. In early June from 2006 to 2013, when the abruptly increased population of L. striatellus was found, the population density of L. striatellus was investigated with the field sampling method.21 The middle row in each plot was sampled by trapping with a mesh bag (30×50cm). Hand-beating caused the L. striatellus to fall into the mesh bags and they were counted on the spot. The number of L. striatellus per meter square was considered to be the population density, which was calculated by taking the average value of ten plots. When the population density of the overwintering L. striatellus generation was investigated, we calculated the number of L. striatellus per meter square by collecting insects on rice stubbles on the 1 April to the 7 April.

Detecting of the incidence of rice stripe disease

Field trials were conducted in randomized complete blocks with three replicates. Every plot consisted of four rows that were four-meters-long, and the plant spacing was 16.7×30 cm. No pesticides were used during the entire growth period. The seeds were sown on the 15 April, and the seedlings were transplanted on the 20 May from 2006 to 2013. The incidence of rice stripe disease was evaluated about 30 days after transplanting. The incidence of rice stripe disease (DI) was calculated according to the disease severity as follows.22

DI = Number of hills with rice stripe disease

×100% Number of the total hills

Detecting RSV-carrying rates of L. striatellus

The L. striatellus samples were collected in early June from 2006 to 2013, and the percentage of virus-containing L. striatellus in the fields was estimated by ELISA analysis.23

Genomic DNA Extraction

The CTAB method 24was used to extract genomic DNA from the L. striatellus samples. DNA quality and concentration were measured by 0.8% agarose gel electrophoresis, and adjustments were made for a final DNA concentration of 100 n g.μl -1.

Genomic DNA Digestion

Genomic DNA (500 ng) were incubated at 37℃ with 0.6U SacI (New England Biolabs (NEB), T4 DNA ligase (NEB), ATP (NEB) and SacI adapters. Restriction-ligation reactions were heat-inactivated at 65℃ and then digested in an additional reaction with the restriction enzyme MseI at 37℃.

SLAF library construction and high-throughput sequencing

Initially, a SLAF pre-design experiment was undertaken in which the training data was used to evaluate the enzymes and restriction fragments. To maintain the sequence depth uniformity of different fragments, a tight length range was selected (about 30,50 bp) and a pilot PCR amplification was performed to check the RRL features in this target length range, which would ordinarily include fragments with similar amplification features on the gel. The pre-design step was repeated to generate a new scheme each time the gel showed non-specific amplified bands.We constructed the SLAF library using the pre-designed scheme. Then, pair-end sequencing was performed according to the selected SLAFs using an Illumina high-throughput sequencing platform (Illumina, Inc; San Diego, CA, U.S.). SNP genotyping and evaluation were then performed.

Sequence Analysis

All SLAF pair-end reads with clear index information were clustered based on sequence similarity. To reduce computing requirements, identical reads were merged together, and then sequence similarity was detected using one-to-one alignment by BLAST .25

Results

Dynamic monitoring of L. striatellus and incidences of rice stripe disease from 2006 to 2013 in Donggang city of Liaoning Province

As shown in Figure 1, the over-wintering L. striatellus population on rice stubbles and that moved to rice were small and stable from 2006 to 2013, However, the L. striatellus population increased significantly in early of June each year, especially in 2006 and 2009

when the number of insects per meter square could reach up to 600 and 800, respectively, which means that almost 25 to 35 small brown planthoppers could be found on a rice hill. In the abruptly increased L. striatellus populations between 2006 and 2009, the rate of viruliferous individuals was between 10 and 17 % (Figure 2), and decreased in the years after this. In 2013, the viruliferous rate had fallen to 3 %. The rice stripe disease followed the fluctuation of the L. striatellus population and its viruliferous rate in early June, in that it was very serious in 2006 and 2009. The incidence of the disease reached 25 % in 2006, but decreased to 10 % in 2008, and then peaked in 2009 at 30 %. In the next four years the L. striatellus population fell as did the viruliferous rate and incidence of rice stripe disease. In 2013, the incidence of rice stripe disease had fallen to 4 % in Donggang city in the northeast of China. Taken together, the incidence of rice stripe disease in Liaoning province seemed to change with the size of the L. striatellus population in early June instead of the over-winter population, which suggested that the disease may not be caused by the local L. striatellus population.

SLAF-seq data statistics and genetic diversity analysis on 14 L. striatellus populations

In 2013, we collected the abruptly increased population in Donggang city and 13 over-winter adult populations of L. striatellus from six provinces where rice stripe disease was severe. Fourteen DNA libraries were sequenced using the SLAF-seq technique, and generated 3,377,827 sequence reads, with each read 80 bp. In all the libraries the GC % was around 40 % (Table 2). Based on the GC content, repetitive sequences and gene characters, the L. striatellus DNA sequences were analyzed and 10396 tags with depth larger than eight were screened. For the L. striatellus populations, out of all the tags identified, 2607 were polymorphic SLAF markers. The 10396 SLAF markers could be divided into five types, and the amount and percentage of each type was shown in Table 3. From Table 3 it can be seen that almost a quarter of the SLAF tags were polymorphic among the different populations of L. striatellus. In total 2572 SNP and 35 Indel markers were identified, which is on average three SNPs for every 1000 bases.

The polymorphic nucleotides from each library were assembled separately. Many heterozygous polymorphic nucleotides were found in every tested sample (Figure 3). Three genotypes locus were identified and called B, D, H and V respectively (Figure 3). Two

genotypes locus were identified and called K, M, R, S and Y (Figure 3). The percentages of three types of base mutations (Figure 3B, D, H and V) in the 14 populations were all lower than the types of base mutations made up of K, M, R, S and Y (Table 4), and there were no significant differences between the percentage of transition (Figure 3 S, Y) and transversion (Figure 3 K, M, R). Further analysis on the sequencing data revealed that heterozygous loci account for more than 15 % of all the polymorphism base sequence in all 14 populations of L. striatellus (Table 4), especially for am, the sample from Jining city of Shandong Province, where the number increased dramatically to 43.4 %. The result suggested that all the L. striatellus populations were mixed groups, especially the am population.

Genetic differences and genetic distances among 14 populations of L. striatellus

The genetic distances from pairwise comparison among the populations were calculated using the Kimura 2-parameter method26 and they are shown in Table 5. The mean pairwise sequence difference among the populations was 0.087, and it ranged from 0.031 to 0.135; however, the geographical distances were not correlated with the genetic distances. As shown in Table 5(below the diagonal), the genetic distance between the overwinter population (aj) and the abruptly increased population (ak) in Liaoning was 0.083, which is bigger than the genetic distance between ak and ab (0.060), ak and ad (0.063), ak and am (0.069), ak and ae(0.070), or ak and ah (0.070).

The genetic distances between the samples and ak or aj were clearly different. For example, between aj and aa, the genetic distance was 0.101, while the value between ak and aa was 0.077. In another example, between aj and ah, the genetic distance was 0.118, and the genetics distance between ak and ah was 0.070. It was observed that between ak and aj there were 62 polymorphism markers, however, there were less than 50 between ak and various other samples from Jiangsu and Shandong Provinces (Table 5 above the diagonal). Therefore, the genetic analysis indicated that there was a significant difference between ak and aj, which was consistent with the results of the biological investigation (Figure1). These data suggested that ak and aj are two totally different populations and that the main vector causing rice stripe disease in Liaoning Province is the immigrant insects rather than the local over-wintering ones.

Phylogenetic trees of 14 L. striatellus populations

Neighbor-Joining method27 and Minimum Evolution method28 were used to group the L. striatellus populations and to construct the dendograms (Figure 4 A and B). A similar structure was generated from both dendograms and they suggested that the abruptly increased population of L. striatellus (ak) had the closest relationship with L. striatellus population in Lianyungang city (ad) instead of the aj, and the over-winter population (aj) of Liaoning had the closest relationship with the insects in Weihai city of Shandong Province (af).However, the supporting data for all the branches were less than 50, except for al and am. If we consider the cut off value for the tree to be 50, and then al and am were clustered to a category, but the 12 remaining populations could not be clustered according to the distribution area of different L. striatellus populations (Figure 4 C), which indicated that the common features of the L. striatellus populations may not be significant enough for them to cluster according to the DNA sequence.

Discussion

Sequence polymorphism and population genetic diversity of L. striatellus population in China are rich but the sequence alignment result was not suitable for classification of different geographical populations.

Extensive studies on the genetic differences among L. striatellus populations from different regions have been conducted and there is a debate as to whether the differences can provide a theoretical basis for geographical division.11-15 No conclusive conclusions have been drawn as to whether different L. striatellus populations are significantly different and whether their structure is affected by migration. In the present study, more than 2500 SNPs were detected between 14 populations of L. striatellus, and a quarter of the SLAF markers were polymorphic, which suggests that the observed genetic diversity of L. striatellus in China is rich. However, low genetic diversity has been indicated by a range of studies in the mitochondrial CO and CO genes.12, 13, 15 One reason might be the genome of mitochondria is evolutionarily conserved in L. striatellus; another possibility is that more diversity would be detected with high-throughput sequencing technology. Species with high levels of genetic variability seems healthy because they have the ability to respond to environmental changes. 29-31 In this work, the high rate of heterozygenity in every population suggested that migration of insect pests accelerated the gene flow and genetic evolution,

which may attribute L. striatellus to adapt their environment.This work found a considerable number of differences among the 14 L. striatellus populations, but when plotted on the condensed tree they were shown to be low. If the cut off value for the condensed tree was set to 50, then al and am were clustered in one branch and the 12 remaining populations were not clustered according to the area distribution of different L. striatellus populations. This result is not consistent with an allozyme study.12 Due to the L. striatellus samples in this test being collected from the regions where rice stripe disease was serious and the vector’s migration was active, it was possible that the gene exchange caused the high rate of heterozygenity in the populations, which resulted in sequence alignments that are not suitable for the classification of different geographical populations of L. striatellus.

Migration of L. striatellus did not have strong effect on the over-winter population size, but caused significant changes to genetic structure of L. striatellus populations. Donggang city in Liaoning Province is located at the edge of the Bohai Sea and the Yellow Sea in the northeast of China. The weather is humid and the number of frost-free days can reach 170. Such weather conditions meet the needs of L. striatellus to hibernate. This study found that the over-winter L. striatellus population from 2006 to 2013 remained below 20 per m2, while in early June (migration time) this increased up to 800. These results indicated that the outbreak of the rice stripe disease in Donggang city was not caused by the over-winter L. striatellus population. Based on the dynamic monitoring data, the migration of L. striatellus did not influence the size of over-winter population, which might be associated with the stability of the over-wintering sites used by L. striatellus. In Liaoning Province, the over-winter fields did not have any green plant material, which meant that sites used by L. striatellus to over-winter were very similar and stable between years, and therefore the over-winter populations were stable between years. Despite this, the genetic structure of the over-winter L. striatellus populations changed significantly between years due to the difference between aj and ak, which might be related to the different sources of the immigrant populations from year to year.

Taken together, the migration of L. striatellus did not have a strong effect on the size of the over-winter population, but the immigration of insects caused significant changes in the genetic structure of L. striatellus and the size of the population abruptly increased.

Immigrant L. striatellus populations in northeast of China may mainly come from Jiangsu province and Shandong Provinces.

As shown in the molecular phylogenetic trees of the 14 L. striatellus populations, ak was most closely related to ad, so it can be inferred that the 2013 abrupt population in Donggang city of Liaoning Province mainly came from Lianyungang city of Jiangsu Province. The af population was the one that was most closely related to aj (Figure 4A).As aj was formed from the immigration population of 2012, it can be presumed that the source of the immigrant population in 2012 was Weihai city of Shandong Province (af). Similar results could be observed from the genetic distances analysis. In this study, the genetic distances were calculated by the Kimura 2-parameter method according to the sequence polymorphisms of 14 DNA pools, and the data indicated that the genetic distances of the immigrant population (ak) were closer to Zhenjiang city, Nantong city and Lianyungang city (ab, ac and ad) of Jiangsu Province as well as Qingdao city, Dongying city of Shandong Province (ae and ah). Therefore, the two methods of data analysis indicated that the most likely sources for the abruptly increased L. striatellus population in Liaoning Province were some regions from Jiangsu Province and Shandong Province. It has been reported that the migratory insects are directed by large-scale wind systems during the long-distance travel transport of migrants. During this process, the air current could collect greater numbers of insects as it followed its path32. Considering the complicated heterogeneity of ak and the migration route from south to north, the immigrant population in Liaoning Province was considered to be a mixed group from several regions. Although much have been known about the immigration event of L. striatellus in Liaoning province, further research is necessary to determine the detailed route of the migration.

Application of SLAF-seq technique in migration studies is suitable for future use

To record the features of planthopper migration, many methods have been proposed, such as light-trap network,33 trap catching,34radar observation,35 insecticide resistance identification36 and trace element content.37 With the rapid developments in biotechnology, molecular markers have been used to verify the pest migration and track the migratory paths; however, these molecular markers are limited by the amount of data and available polymorphisms. Sufficient data for comparisons between populations of L. striatellus can be provided by the

high-throughput sequencing SLAF-seq technique used in this work. Its use would enable the design of multiple molecular markers to distinguish different L. striatellus populations by PCR and electrophoresis.

Author contributions statement

Conceived and designed the experiments: Fuyu Sun, Xiaochun Lu and Wenjing Zheng. Performed the experiments: Zhiqiang Li, Jiaming Zhao, Yanzhi Zhang, Changhua Wang and Wenjing Zheng. Analyzed the data: Wenjing Zheng , Zhiqing Li and Jiaming Zhao. Wrote the manuscript: Wenjing Zheng, Fuyu Sun and Xiaochun Lu.

Acknowledgements

We are grateful to many experts for supplying much important information about the small brown planthoppers and some enthusiastic help during our field surveys. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31301636 and 31471770).

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Figure1 The dynamic monitoring of the small brown planthppers in Donggang city of Liaoning Province from 2006 to 2013

Figure 2 The viruliferous rate of L. striatellus and the incidence of rice stripe disease from 2006 to 2013 in Donggang city of Liaoning Province

Figure3. Part of nucleotide polymorphism of 14 L. striatellus populations The letters in the first column (aa to an) showed L. striatellus samples from 14 regions. “.”=Identical ; “-”=Indel; B=T, C or G; D=A, T or G; H=A, T or C;V=A, C or G; W=A or T;S=C or G;K=T or G

Figure4. Phylogenetic trees of L.striatellus based on sequence variation of partial DNA sequences. A: Neighbor Joining (NJ) tree; B: Minimum Evolution (ME) tree; C: NJ tree that was cut off the value of condensed tree less than 50. The optimal tree with the sum of branch length = 0.53 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method. (Units:number of base substitutions per site). 14 nucleotide sequences were analyzed. Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011).