Genotyping of Chinese seedling and hybrid apple rootstocks and it’s progenies for the dwarfing trait

Abstract:【Objective】The Chinese apple industry is the largest in the world at present, both for fruit pro- duction and planted area. Most of the orchards are established with trees on seedling rootstocks, which leads to higher costs for the growers because more time and labour are required to grow good quality ap- ples. Apple rootstocks play a vital role in apple cultivation as suitable rootstocks are important to achieve efficient production in high-yielding apple orchards. Growing dwarf trees in high density plant- ings has been the trend in new orchards globally for a considerable time now and the Chinese apple in- dustry is following suit. Hence, the breeding and selection of dwarfing rootstocks with resistance to abi- otic and biotic stresses will form the basis of the future Chinese apple industry. Because of the long cycle of apple rootstock breeding and its high resource requirements, breeding progress has been slow. Nevertheless, over the past decades in China, multiple initiatives in apple rootstock breeding have re- sulted in several cultivars. Compared with phenotypic identification methods, the application of molecu- lar markers in trait characterization is more accurate and efficient, through identifying genomic differ- ences. Molecular marker-assisted screening for the Dw1 and Dw2 dwarfing genes identified in‘M9’rootstock is being used in early pre-selection of hybrid seedlings as a means to predict the dwarfing per- formance of offspring. In future breeding, we can also purposefully design parental combinations with the selection objectives in mind. The objective of this study was the genetic characterization of Chinese seedling and hybrid apple rootstocks and it’s progenies for the dwarfing genes.【Methods】Leaf samples of 27 rootstocks were obtained from different regions around China including‘M9’‘M116’and‘MM106’as reference (semi-) dwarfing genotypes, and from 147 seedlings of a M. sieversii (Li 2-3)בM9’and 345 seedlings of a M. baccata (Wild 1-2)בM9’family. The first-generation hybrid seeds were obtained from the dwarf apple resource garden of the Zhengzhou Fruit Research Institute of the Chinese Academy of Agricultural Sciences in 2016 and 2017. DNA was extracted using DNA extrac- tion kits (Luoyang Aisen Biological Technology Co. Ltd) for the genotyping of the entire M. sieversii(Li 2-3)בM9’and M. baccata (Wild 1-2)בM9’families and the 27 rootstock accessions. Poly- merase chain reaction(PCR)products containing single nucleotide polymorphisms (SNPs) were per- formed on a LightCycler® 480 instrument. SSR markers Hi01c04 and MDP00024703 were amplified for the Dw1 and Dw2 genes, respectively, using PCR conditions as described by Foster et al. and the PCR products were separated by capillary electrophoresis (Tsingke Biological Technology Co. Ltd).【Results】The SSR marker indicated that 8 out of the 24 rootstocks had the allele for the Dw1 dwarfing gene and 16 did not. SSR marker mdp0024703 for the Dw2 gene was amplified in 5 accessions, show- ing the 167 bp band, and not in the remaining 19 Chinese varieties. Of these 24 Chinese apple root- stocks, three showed both the Dw1 and Dw2 markers:‘Liaoning No. 2’,‘GM 256’, and‘77-34’. In the M. sieversii (Li 2-3)בM9’family, 14 progeny containing the markers for both Dw1 and Dw2 were identified, with a further 34 plants only carrying Dw1. In the M. baccata (Wild 1-2)בM9’, 11 progeny contained the marker for Dw1, but no plants carrying both Dw1 and Dw2 were identified. The ratio of progeny carrying Dw1 dwarfing genes was much higher in the M. sieversii (Li 2-3)בM9’family than in the M. baccata (Wild 1-2)בM9’family (38.8% and 3.3%, respectively). The majority of the dwarf- ing and semi-dwarfing rootstock accessions screened carried marker alleles linked to Dw1 and Dw2. This suggests that most apple dwarfing rootstocks have been derived from the main genetic source car- rying both Dw genes for this trait,‘M9’.【Conclusion】Genetic markers linked to Dw1 and Dw2 were screened over 24 Chinese rootstock accessions that confer a range of vigour effects on scion growth, with 3 of them carrying both Dw genes. Phenotypic records in the fields indicated that the combination of Dw1 and Dw2 has the strongest influence on rootstock-induced dwarfing, and that Dw1 had a stron- ger effect than Dw2. Therefore, genetic characterization of rootstocks for dwarfing can assist breeders in rootstock parent selection as well as progeny selection in segregating families derived from dwarf rootstocks. The latter was applied to two‘M9’families, with the M. sieversii (Li 2-3)בM9’cross having more seedlings with one or two dwarfing genes than the M. baccata (Wild 1-2)בM9’cross. The selection of dwarf apple rootstocks will assist in managing tree vigour, which is associated with achieving higher fruit quality. Denser plantings in apple orchards enabled by dwarfing rootstocks will also im- prove yield per unit area, hence improve the production efficiency and economics of the apple industry in China.