Genetic Diversity Of Grape Phylloxera In China And Screening Of Genes Related To Wing Differentiation Of Grape Phylloxera

Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a gall-forming aphid species native to North America and feeds on various grapevines (Vitis spp. L.). It is a quarantine pest of China, which was first found in 1892 and found again 2005 in China. In order to control grape phylloxera, it is of great importance for us to understand its biology, ecology and genetics. In this paper, we investigated on the biotype, genetic diversity, phylogenetic systematics of grape phylloxera. Subsequently, its genes related to wing differentiation were screened.Grape phylloxera were reared on excised grape roots maintained in petri-dish. Phylloxera on the resistant varieties (SO4, 5BB, 101-14M) had lower survivor rates than on the susceptible varieties, and the development of phylloxera arrested at the very early stage. Life tables were constructed using 2 susceptible varieties, i.e. Cabernet Sauvignon, Davidii Foex, as well as 2 rootstocks: 140Ru and Beta. The survivorship of SH Kyoho clone was better than other clones on Cabernet Sauvign, but fertility and life history parameters were similary. From the performance of 6 clones on Cabernet Sauvign, Davidii, Beta and 140Ru, it was found that net reproductive rates (Ro), innate capacity for increase (rm) and finite rates of increase (λ) of SH Kyoho were the largest, while double time was the shortest, indicating that SH Kyoho clone is an aggressive strain.In order to investigate the levels of genetic variation and differentiation of grape phylloxera in China, the mitochondrial cytochrome oxidaseⅠ(COⅠ) gene of 35 samples from four populations was sequenced. Of the total analyzed sites, 29 (6.13%) were polymorphic, including 17 (3.59%) parsimony informative and 12 (2.54 %) singleton sites. Nucleotide frequencies were A 34.8%, C 15.8%, G 10.2%, and T 39.2%. In total, 13 haplotypes were identified. The most common haplotypes were H3 (HAP-A) shared by 5 samples from SHJ, and H13 (HAP-C) found in 19 samples from SXX, HNH and LNX populations. The SHJ population was unique and shared no haplotypes with other three populations. Nm ranged from 0.02 to 4.03 and genetic distances varied from 0.001 to 0.040 among different populations. Nm (0.02) was small, while the genetic distances (0.039-0.040) were larger between SHJ and other three populations. Phylogenetic analysis and haplotype network showed that all haplotypes from SHJ formed one cluster, while the other haplotypes from SXX, HNH and LNX were grouped into another one, indicating that there were at least two introduction of grape phylloxera into China.The genetic diversity of 4 grape phylloxera populations from Shanghai, Hunan, Shaanxi, and Liaoning was studied based on 7 microsatellite loci. The mean effective number of allele (Ne) was ranged from 1.7143 to 2.0077, while expected heterozygosity (He) from 0.2435 to 0.2580. In addition, the mean Shannon values were all less than 1. All those data showed that genetic variations of 4 grape phylloxera populations were fairly low. The coefficient of gene differentiation demonstrated 83% of the total genetic differentiation within each population. The Nei's genetic distances between 4 populations ranged from 0.0028 to 0.4694. The genetic distances between SHJ and each of other three populations was larger, ranged from 0.4531 to 0.4694, than those between two of three populations (0.0028-0.0114). The four phylloxera populations were clustered into two groups based on the dendrogram on the basis of Nei's genetic distances. SHJ formed one group, while other haplotypes from SXX, HNH and LNX formed another one. The genetic distances was the lowest between HNH and LNX populations.The difference in gene expression between alate and apterous aphids in 2nd stages of grape phylloxera was investigated using suppression subtract hybidization (SSH). 51 ESTs expressed exclusively in alate larval and 44 ESTs in apterous larval were screened by Dot blot. Furthermore, sequence alignment was conducted with BLASTx and BLASTn. The results showed that 14 known genes were implicated into wing differentiation in alate aphids library, and the other 34 ESTs were unknown genes.Thirteen genes were selected to check the relative expression expression in different age of alate aphids with real time RT-PCR. The result showed that the expression levels of Brat, Apyrase, CHD and RPS15a genes were the highest in the 2nd alate larval, while their expression levels were gradually decreased with the age of alate aphid, which indicated that these genes may be important to wing differentiation.