Identifikation differentiell exprimierter Gene der Gerste (<i>Hordeum vulgare</i> L.) nach BYDV-PAV Infektion anhand von cDNA-AFLP

Abstract

Aphid transmitted Barley yellow dwarf, caused by different strains of Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV) is an important virosis of Poaceae worldwide and causes substantial yield losses. For an economical and ecologically sound production of cereals growing of tolerant cultivars is a prerequisite. For efficient breeding of tolerant barley (Hordeum vulgare L.) cultivars molecular markers are useful tools since artifical inoculation using virus bearing aphids can hardy be efficiently implemented in a barley breeding programme. Therefore, this study aimed at the identification of genes involved in BYDV tolerance by cDNA-AFLP followed by mapping of these genes in a phenotypically well characterized DH-line population derived from the cross `Vixen´ (Ryd2) and `Post´ (QTL on 2HL). In order to achieve information on genes differentially expressed after BYDV infection in general and differentiating tolerant and non-tolerant genotypes, the parental cultivars and doubled haploid (DH) lines derived from their cross carrying all possible combinations of positive and negative alleles at respective loci (Ryd2, QTL on 2HL) as well as the non-tolerant cultivar `Nixe´ were raised in growth chambers (18°C, 70% relative humidity, 16 h light period, illumination level of 10<sup>4</sup> lm·sm<sup>-1</sup>). Artificial inoculation took place in the one-leaf stage with aphids (Rhopalosiphum padi) carrying the BYDV-PAV-ASL-Isolate. At 2 dpi the aphids were removed with a systemic insecticide, and 15 dpi the success of artificial infection was checked by analysing the tip of the third leaf by DAS-ELISA. The rest of this leaf was used for total RNA extraction, based on a method using guanidinium thiocyanate. Selective isolation of mRNA was carried out with magnetic beads and the mRNA of infected and healthy control plants was reverse-transcribed to double-stranded cDNA. AFLP reactions were applied and subsequently analysed by 256 EcoRI+2/MseI+2 primer combinations. Differentiating fragments were cut out of electrophoresis gels and re-amplified by using the corresponding +2 primer combinations. Re-amplified fragments were cloned and sequenced. Sequences were compared to EST and protein databases on the worldwide web. A total of 45 differentially regulated genes could be examined and attributed to functions as follows: 13% carbohydrate metabolism, 11% aminoacid biosynthesis, 11% senescence associated, 7% cell wall synthesis, 2% BYDV, 16% mRNA of other classes, 9% without significant similarities and 31% mRNA of unknown function. Out of seven different fragments STS, CAPS and SNP markers were derived and mapped in the existing DH population `Post´ x `Vixen´. Out of the derived markers of the isolated fragments from the cDNA-AFLP, no one mapped in the DH21 population `Post´ x `Vixen´ at known tolerance loci or QTL regions. Some sequences could be assigned to barley chromosomes (1H-7H) with wheat-barley chromosome addition lines, but the exact position on the repective chromosome can not be determined by this approach. The detected gene fragments, especially from the amino acid biosynthesis pathways, possibly play a role in secondary plant reactions after BYDV infection because the high accumulation of carbohydrates in BYDV infected leaves most probabaly resulted from degeneration of the phloem. Accumulation of sucrose in source-organs is the consequence of a distorted phloem loading. On the one hand the plethora of sucrose gets cleaved in fructose and glucose with manifold feedback processeses in the carbohydrate metabolism and, on the other hand, it results in severe chlorosis as a result of the enrichment of soluble carbohydrates in the leaf. The reason for distorted phloem transport could not be clarified but the identification of down-regulated genes from cell wall synthesis after BYDV infection allow to presume that uncured fragments of the cell wall accumulate in the phloem, block the sieve cells, cause chlorosis and finally lead to plant damage and yield reduction.