Analysis Of Genetic Variation Associated With Three Agronomic Traits In ’Su Shuai’ Apple

Apple (Malus×domestica Borkh.) is one of the most popular and the largest temperate fruit in the world. The apple cultivar’Su Shuai’was derived from a cross between’Golden Delicious’used as male parent, and’Indo’used as female parent. The main characteristics of’Su Shuai’are high yields, short internodes, high resistant to diseases such as Alternaria blotch (Alternaria alternata), lighter flavour and a smooth fruit surface with no rust coloration, etc. compared with its parent plants’Golden Delicious’and’Indo’. In this study, internode length, internode number, chlorophyll content, fruit soluble solids and titratable acid content of’Golden Delicious’,’Indo’and’Su Shuai’were determined. The anatomical structure, leaf epidermis structure and of’Golden Delicious’,’Indo’and’Su Shuai’were studied using paraffin section method, scanning electronic microscope.14 pairs of SSR (Simple Sequence Repeats) primers were used to preliminarily analyze genomic variation of’Sushuai’compared with’Golden Delicious’and’Indo’. Meanwhile, the genetically relationship of the material used in this study’Su Shuai’and’Golden Delicious’as well as ’Indo’was further identified, along with the polymorphic amplification loci of known related traits linkage markers between its parents. At present, the genome sequencing of ’Golden Delicious’have been completed. In this study, genomic DNA of’Su Shuai’and its female parent’Indo’were re-sequenced by High throughput sequencing technology. By using bioinformatic approaches to compare the sequencing data with reference genome of cultivar’Golden Delicious’, multitudes of variable polymorphic loci were obtained. After digging mutated genes at mutant site, three agronomic traits related mutations of’Su Shuai’ were preliminarily screened.In addition,8 genes were selected to be expression analyzed. The main results are illustrated as follows:1. The internode length and internode numbers of’Su Shuai’were all significantly less than that those of’Golden Delicious’and’Indo’. For’Su Shuai’, the relative content of chlorophyll in’Su Shuai’was significantly higher than’Golden Delicious’and’Indo’. There were no obvious differences for soluble solids content between’Su Shuai’and ’Golden Delicious’, but titratable acid content was significantly higher than the’Golden Delicious’. The upper epidermal cells of’Su Shuai’were similar to’Indo’. But there were more wrinkles on the surface of the upper epidermal cells of’Su Shuai’and the bulges of epidermal cells were higher compared with its parents. The length of stomata of’Su Shuai’ was significantly less than’Golden Delicious’and greater than’Indo’. There were no obvious difference for the stomatal width between’Su Shuai’and’Golden Delicious’, but significantly wider than the’Indo’. The stomata density of’Su Shuai’was obviously higher than those of’Golden Delicious’and less than those of’Indo’. The size of palisade tissue cells within the same layer of’Su Shuai’was different and its array was irregular. The cells of different layers were intervened each other, and the border of different layers was not clear. The size of palisade tissue cellswithin the same layer of’Golden Delicious’and ’Indo’was equal and their array was quite regular and close together. The border of different layers was obvious to distinguish. The leaf total thickness, palisade tissue thickness and the ratio of palisade tissue thickness and spongy tissue thickness of’Su Shuai’were obviously higher than those of’Golden Delicious’and’Indo’.2. According to the amplified bands of the 14 used SSR primers, there were not much polymorphic bands between’Su Shuai’,’Indo’and’Golden Delicious’, while the major bands were co-dominant, which in accordance with SSR markers, indicating that’Su Shuai’ has a genetic relationship with’Indo’and’Golden Delicious’and is a hybrid progeny of these two species. In the amplified bands of selected dwarfing gene linked CH01g12 marker which has been reported before, polymorphic bands showed in’Indo’and’Golden Delicious’between 100 bp and 140 bp, but did not show in’Su Shuai’. In leaf spot disease resistance gene linked CH02g09 marker amplified bands, no band showed in’Indo’ between 120 bp and 160 bp, while amplified bands existed in’Golden Delicious’and’Su Shuai’. As for acidity gene linked Hi01e10 marker, a different band appeared at 200 bp of ’Su Shuai’and’Golden Delicious’, and at 240 bp of’Su Shuai’and’Indo’. The polymorphic bands of these three traits related linkage markers amplified from’Su Shuai’ revealed that the variation of related traits in’Su Shuai’were associated with genes linked to these markers.3. To obtain a comprehensive overview of the sequence variation in these three agronomic traits, we re-sequenced the genomes of’Su Shuai’and’Indo’using Next-generation sequencing. Of the 25.73 gigabase (Gb) of raw sequence data, around 10 Gb per individual remained after filtering for paired-end libraries per individual. We detected a wide range of genetic variations with 2,454,406 single nucleotide polymorphism sites (SNPs) in the’Indo’genome, and the 1,874,349 SNPs in the’Su Shuai’genome, 59,547 structural variant sites (SVs) in the’Indo’genome and 50,143 SVs in the’Su Shuai’ genome. The distribution of SNPs and SVs in different components of the genome was determined compared with’Golden Delicious’genome. The majority of the variants were found within intergenic and repeat regions. In’Indo’and’Su Shuai’genome, the number of SNPs hits in exons was 258,383 (accounting for 15.2% of the total) and 214,398(16.4%), respectively. And the number of SVs hits in exons was 3,261 and 2,741 respectively. Approximately both of them were accounted for 6.7% of the total.4. In total,24,449 modified genes were identified in’Indo’based on an alignment of the DNA sequence, to the’Golden Delicious’reference genome, and 22,153 in’Su Shuai’. Approximately 94% of the modified genes (22,863 for’Indo’,20,808 for’Su Shuai’) were annotated by databases. Among the SVs,17 genes related to disease resistance,10 genes related to Gibberellin (GA).19 genes involved in fruit flavour were identified in’Su Shuai’, and the expression patterns of eight differential genes were tested using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) at the transcriptome level. Results of the RT-qPCR analysis indicated that the expression of N(MDP0000272916) and MbR7 (MDP0000233155) was highest in’Su Shuai’. The expression of GA3OX1 (MDP0000233590) and RGL2 (MDP0000300311) was highest in’Golden Delicious’. The expression of gene (MDP0000284679), associated with the negative regulation of GA-mediated signalling, however, was highest in’Su Shuai’, followed by’Indo’and lowest in’Golden Delicious’. As for genes associated with carbohydrate metabolism, the expression of SDH (MDP0000693768) was highest in’Su Shuai’, followed by’Indo’and lowest in’Golden Delicious’. NAD-ME2 (MDP0000453139) had a higher level of expression in’Indo’, and lower levels of expression in’Golden Delicious’and’Su Shuai’. The expression of CINV2 (MDP0000745777) was the highest in’Golden Delicious’and very low in’Indo’and’Su Shuai’. Results of the RT-qPCR analysis were in general agreement with the phenotypic differences that were observed among the three cultivars.