Apple Germplasm Research Articles

Genome-wide association study (GWAS) of leaf wax components of apple

The wax layer of apple leaves plays an important role in improving stress resistance, but relatively little is known about the mechanisms of wax synthesis and transport in apple leaves. In this study, 17 wax components, including alcohols, alkanes, fatty acids and terpenes, were analyzed by gas chromatography-tandem mass spectrometry (GC-MS) from the leaves of 123 apple germplasms. Whole-genome sequencing of these apple accessions yielded 5.9 million high-quality single nucleotide polymorphisms (SNPs). We performed a genome-wide association study (GWAS) on 17 wax components and identified several genes related to wax synthesis and transport, including MdSHN1 (SHINE1), MdLTP4 (LIPID TRANSFER PROTEIN4), MdWSD1 (WAX ESTER SYNTHASE/ACYL-COA DIAC-YLGLYCEROL ACYLTRANSFERASE1), MdRDR1 (RNA-DEPENDENT RNA POLYMERASE1), MdACBP6 (ACYL-COA-BINDING PROTEIN6), MdNLE (NOTCHLESS) and MdABCG21 (ATP-BINDING CASSETTE G21). Moreover, we identified some prominent SNPs that may affect gene expression and protein function. These results provide insights into mechanisms of wax synthesis and transport in apple leaves and broaden the genetic resources and basis for facilitating resistance breeding.

Bacterial Endophyte Community Dynamics in Apple (Malus domestica Borkh.) Germplasm and Their Evaluation for Scab Management Strategies.

The large genetic evolution due to the sexual reproduction-mediated gene assortments and propensities has made Venturia inaequalis (causing apple scab) unique with respect to its management strategies. The resistance in apple germplasm against the scab, being controlled for by more than fifteen genes, has limited gene alteration-based investigations. Therefore, a biological approach of bacterial endophyte community dynamics was envisioned across the apple germplasm in context to the fungistatic behavior against V. inaequalis. A total of 155 colonies of bacterial endophytes were isolated from various plant parts of the apple, comprising 19 varieties, and after screening for antifungal behavior followed by morphological, ARDRA, and sequence analysis, a total of 71 isolates were selected for this study. The alpha diversity indices were seen to fluctuate greatly among the isolation samples in context to microflora with antifungal behavior. As all the isolates were screened for the presence of various metabolites and some relevant genes that directly or indirectly influence the fungistatic behavior of the isolated microflora, a huge variation among the isolated microflora was observed. The outstanding isolates showing highest percentage growth inhibition of V. inaequalis were exploited to raise a bio-formulation, which was tested against the scab prevalence in eight apple varieties under controlled growth conditions. The formulation at all the concentrations caused considerable reductions in both the disease severity and disease incidence in all the tested apple varieties. Red Delicious being most important cultivar of the northwestern Himalayas was further investigated for its biochemical behavior in formulation and the investigation revealed different levels of enzyme production, chlorophyll, and sugars against the non-inoculated control.

Cold storage of in vitro apple germplasm in Kazakhstan

ISHS VIII International Scientific and Practical Conference on Biotechnology as an Instrument for Plant Biodiversity Conservation (Physiological, Biochemical, Embryological, Genetic and Legal Aspects) Cold storage of in vitro apple germplasm in Kazakhstan

Morpho-molecular characterization and genetic diversity analysis across wild apple (Malus baccata) accessions using simple sequence repeat markers

Out of five wild Malus species in the world, Malus baccata (L.) Borkh (MB) is reported from the extremely cold areas of north western Himalayas. These are widely used as a rootstock or source of resistance against various biotic and abiotic stresses due to its ecological adaptability and cold hardiness. The study of genetic diversity is an important aspect to be covered for proper utilization of the wild apple germplasm for breeding purpose. The 29 Malus baccata accessions were used to divulge their genetic diversity using morpho-molecular markers. Based on morphological characters, Mahalanobis D2statistics resolved all accessions in four clusters. A set of 85 SSR were used to study molecular diversity among the accessions. The AMOVA stated genetic diversity between populations of genotypes was lower (7%) compared to that observed within and among the individuals of the populations (93%). The effective allele number (2.63) was found to be lower than mean allele number (3.7), with mean heterozygosity as 0.51. The gene diversity varied between 0.10 for Hi02d05 to 0.79 for CH-Vf1.The expected heterozygosity was greater than observed heterozygosity for total analysed loci of SSR. The average PIC value was found as 0.50. The accessions were segregated into two distinct classes using heirarchical clustering method. Further, population structure also differentiated the 29 accessions into two populations, with sub-population I accounts for 18 accessions and sub-population II consisted of only 9 accessions. MB-5 and MB-20 accessions fall in the admixture group. The differentiated populations were validated and reconfirmed by the PCoA analysis. . The morphological features and molecular markers revealed significant level of genetic diversity along with population differentiation among the maintained accessions.

Polyploidy affects the development of Venturia inaequalis in scab-resistant and -susceptible apple cultivars

Apple (Malus × domestica Borkh.) production is significantly affected by apple scab disease caused by the fungus Venturia inaequalis. Currently, management of the disease is largely based on fungicide applications and less on the inherent resistance in apple cultivars carrying resistance (Rvi) genes. Moreover, the durability of both approaches is questionable, since new virulent strains with resistance to fungicides and/or Rvi genes can develop. Hence, a combination of complementary management strategies is needed to tackle apple scab. To broaden the management options, a viable approach might be the breeding for polyploid cultivars and their implementation in commercial orchards. Apple germplasm is substantially characterized by polyploidy, which throughout the evolution of angiosperms often led to improved adaptation to adverse climatic conditions and abiotic stresses. However, the role of polyploidy in conferring resistance to biotic challenges is less clear. Here, we show that autopolyploidy in two apple cultivars, i.e. the susceptible ‘Gala’, and the resistant ‘Makali’ which carries the Rvi6 scab resistance gene, can reduce severity of apple scab symptoms upon inoculation with two different V. inaequalis isolates. Leaves of tetraploid plants show reduced sporulation symptoms in comparison with their diploid counterparts upon infection with both pathogenic strains, attributed to a reduced presence of V. inaequalis. These results suggest that polyploidy enhances resistance of apple cultivars against different V. inaequalis isolates. However, the degree of the enhanced resistance depends on a plant-pathogen-specific interaction. The knowledge provided here offers a relevant framework for improving our understanding of polyploidy-enhanced resistance to biotic stress in plants and may set the stage for implementing polyploidy breeding for further genetic improvement of apple.

Evaluation of local apple germplasm in Iran: study of sensory and physicochemical characteristics with emphasis on acceptability

Evaluation of local apple germplasm in Iran: study of sensory and physicochemical characteristics with emphasis on acceptability

Pyramiding resistance genes and widening the genetic base of the apple (Malus × domestica Borkh.) crop

Apple breeding is active worldwide and yet the apple crop is in a precarious state as it relies on few dominant cultivars and only the Rvi6 (formerly Vf) gene, that confers resistance to scab, has been extensively exploited in the cultivars entered the market in recent years. However, there are some 20 disease resistance genes described in apple and the apple germplasm includes thousands of accessions in the repositories. In this paper, a breeding programme is described, whereby 36 genotypes, including ancient and contemporary apple cultivars, were crossed to produce a new set of selections that combine extensive genetic resources with pyramided resistance genes to several apple diseases, such as scab and powdery mildew. The 110 cross combinations carried out successfully, of the 260 initially planned, produced 7,876 offsprings, reduced to 2,969 after screening with molecular markers associated with five resistance genes. Selections with three or two resistance genes and good agronomic characteristics were kept for further field observations with the aims of creating new cultivars for the market and new parents for future breeding projects

Genetic origin and climate determine fruit quality and antioxidant traits on apple (Malus x domestica Borkh)

Apples are highly appreciated in terms of flavor, nutritional and health-promoting compounds and are associated with a reduced risk to develop age-related and chronic diseases. In this study, 155 accessions of Malus x domestica Borkh from the apple germplasm collection, located at the Experimental Station of Aula Dei in NE Spain, were analyzed during five consecutive years (2014–2018). Basic fruit quality traits including soluble solids content (SSC), titratable acidity (TA) and the ratio SSC/TA (ripening index - RI) were obtained. In addition, biochemical compounds such as total phenolics content (TPC), flavonoids (TFC), vitamin C (Ascorbic acid - AsA) and relative antioxidant capacity (RAC) were determined. Statistical analysis was used to determine differences in trait values among accessions and years. The well adapted local accessions showed, in general, higher average content of antioxidants and RAC compared with the foreign and commercial ones. A multivariate model was fitted with the accessions and climate features of each year as independent variables. A cluster analysis was then performed on the model coefficients space to classify the 155 accessions within five groups. The cluster analysis showed that foreign cultivars (those not originating from Spain) were concentrated in two groups while local accessions could not be segregated and had very different profiles. Furthermore, they increased the concentration of bioactive compounds tended to decrease with higher temperatures, while increased with higher solar radiation. Statistical analyses emphasized differences between groups and highlighted accessions and climate as main factors affecting metabolite profiles and fruit characteristics.

Phenotypic analysis of fruit quality traits and effect of climate in an apple (Malus × domestica Borkh) germplasm bank of Aragón, Spain

Phenotypic analysis of fruit quality traits and effect of climate in an apple (<i>Malus </i>×<i> domestica</i> Borkh) germplasm bank of Aragón, Spain

Pheno-genetic studies of apple varieties in northern Pakistan: A hidden pool of diversity

Apple (Malus × domestica) is a main fruit crop of cold temperate regions and traded globally. Despite being a comparatively well documented plant cultigen, available germplasm in many remote regions is still unexplored and threatened. Increasing market demands for modern cultivars and farmers’ preference for high yields may lead to the gradual loss of indigenous varieties. Gilgit-Baltistan in northern Pakistan – a mountainous and remote apple growing region – was selected to study local cultigen diversity by assessing varietal richness and diversity, arboriculture activities as well as phenotypic and genotypic characters of available germplasm. In total, 106 individual apple trees were sampled, and 35 tree owners from seven villages of the two valleys Ishkoman and Baltistan were interviewed. Dendrometric parameters, fruit traits (qualitative and quantitative), and varietal diversity in each village were measured. Genetic variation was estimated using eleven SSR markers and synonyms and homonyms were identified based on cluster analysis.Sixty-one multi-locus genotypes (MLG; mean genotype diversity: 58 %) and 18 putative clones (comprising 2 to 9 ramets) were identified. Varietal diversity was highest in Baltistan where even a wild apple relative (Malus baccata) was discovered. Mean genetic diversity was moderate (He = 0.677), while negative inbreeding coefficients indicated excess of heterozygotes in three genetic clusters. A moderate correlation (r = 0.627, p = 0.001) between phenotypic and genetic dendrograms suggested genetically linked fruit traits, which merits further exploration.Considering the high genetic and phenotypic variability, Gilgit-Baltistan appears a promising source of apple germplasm for future breeding programs. The promotion of local varieties may allow farmers to diversify their produce and derived sources of income as well as to maintain a historically important bio-cultural heritage of Gilgit-Baltistan. To this end, comprehensive conservation strategies combined with further participatory research will be needed.

Analyses of structural dynamics revealed flexible binding mechanism for the Agrilus mali odorant binding protein 8 towards plant volatiles.

Volatiles from host plants are an important source of insect pest attractants and repellents. Insect odorant binding proteins (OBPs) have been widely characterized, but the molecular binding dynamics and underlying mechanisms are still not well understood. Thus, we characterized binding characteristics of AmalOBP8 from the apple buprestid beetle (Agrilus mali Matsumura), an unprecedented serious threat to rare apple germplasm resources and local ecosystems. Fluorescence studies demonstrated that the quenching mechanism was clearly static. AmalOBP8 was found to bind with both volatiles at single independent sites. Negative thermodynamic parameters suggested that binding interactions between AmalOBP8 and both volatiles could occur spontaneously. Hydrogen bonding was the key force in AmalOBP8's binding to geranyl formate, for which the amino acid residue Trp106 played a critical role in the binding pocket. Multiple Leu residues in AmalOBP8 created a strong hydrophobic environment, and formed the binding pocket for (Z)-3-hexenyl hexanoate. Compared to classic OBPs, in addition to lack of one disulfide bridge, AmalOBP8 had a small α-helix (α7) at the C-terminus, resulting in greater flexibility and adaptability for this protein to bind with different compound molecules. Key residues of AmalOBP8 in binding interactions with plant volatiles were clarified. AmalOPB8 had a large ligand binding spectrum and great flexibility in binding with plant volatiles, providing good molecular targets for screening insect attractants and repellents. Our results can promote understanding of insects' perception of various odorants, and establish a foundation for discovery of new pest control agents. © 2020 Society of Chemical Industry.

Application of genome-wide insertion/deletion markers on genetic structure analysis and identity signature of Malus accessions

BackgroundApple (Malus ssp.), one of the most important temperate fruit crops, has a long cultivation history and is economically important. To identify the genetic relationships among the apple germplasm accessions, whole-genome structural variants identified between M. domestica cultivars ‘Jonathan’ and ‘Golden Delicious’ were used.ResultsA total of 25,924 insertions and deletions (InDels) were obtained, from which 102 InDel markers were developed. Using the InDel markers, we found that 942 (75.3%) of the 1251 Malus accessions from 35 species exhibited a unique identity signature due to their distinct genotype combinations. The 102 InDel markers could distinguish 16.7–71.4% of the 331 bud sports derived from ‘Fuji’, ‘Red Delicious’, ‘Gala’, ‘Golden Delicious’, and other cultivars. Five distinct genetic patterns were found in 1002 diploid accessions based on 78 bi-allele InDel markers. Genetic structure analysis indicated that M. domestica showed higher genetic diversity than the other species. Malus underwent a relatively high level of wild-to-crop or crop-to-wild gene flow. M. sieversii was closely related to both M. domestica and cultivated Chinese cultivars.ConclusionsThe identity signatures of Malus accessions can be used to determine distinctness, uniformity, and stability. The results of this study may also provide better insight into the genetic relationships among Malus species.

A new one-tube reaction kit for the SSR genotyping of apple (Malus × domestica Borkh.)

Though apple genotyping is mainly used for scientific and breeding purposes, it can also be adopted by national authorities to control the authenticity of apple cultivars. To facilitate the introduction of routine apple genotyping into practice, a new apple simple sequence repeat (SSR) genotyping kit was developed (called the Ap17 in. SSR Genotyping Kit). The kit combines 17 SSR markers including those recommended by the Working Group of the European Cooperative Programme for Plant Genetic Resources (ECPGR), covering all apple linkage groups in a one-tube reaction format, using a fragment analysis method to simplify the genotyping procedure. The kit was successfully tested using 880 unique diploid apple germplasm accessions; the kit can also readily discriminate triploid and tetraploid samples. The total probability of identity for the kit and the sample collection used was calculated to be 1.73 × 10-22. Tables for converting results to enable genotype comparisons between currently-used genotyping systems and the Ap17 in. kit are provided. The kit is ideally suited for validation in laboratories genotyping a large number of apple samples, saving time, costs, and labor, while minimizing technical and human errors.

Molecular, Physico-Chemical, and Sensory Characterization of the Traditional Spanish Apple Variety “Pero de Cehegín”

The “Pero de Cehegín” is an ancient local variety of apple grown in Murcia (Spain). In this study, microsatellites markers showed evidence of a unique profile that has never been reported before in other Spanish apple germplasm collections. Five “Pero de Cehegín” clones were evaluated and compared with two commercial apple varieties, “Fuji” and “Golden Delicious”, to assess its marketing potential. For this, the physical (weight, height, and width of the fruit, moisture content, firmness, and color of the fruit, among others), and chemical (total soluble solids, total acidity, and maturity index) properties of the fruits were evaluated. In addition, the content of bioactive compounds such as total polyphenol content, total antioxidant activity using the ABTS+, DPPH•, and FRAP methods, and the sugar profile were analyzed, and their sensory profile was also evaluated. Physico-chemical differences were found within the “Pero de Cehegín” clones and between the commercial varieties. “Pero de Cehegín” had a high firmness, high total soluble solids, very low total acidity, high FRAP antioxidant capacity, and more sucrose content in comparison with “Fuji” and “Golden Delicious”. These distinctive characteristics and the good appearance of the fruit make this variety a marketable product that will increase the offering of traditional, local, but underutilized fruit varieties.

Viral Small RNA-Based Screening of Malus spp. Reveals Citrus Leaf Blotch Virus Infection of Kaido Crab Apple in China

Viral Small RNA-Based Screening of <i>Malus</i> spp. Reveals Citrus Leaf Blotch Virus Infection of Kaido Crab Apple in China

Correction to: Quantitative variation and heritability estimates of fire blight resistance in a pedigree-connected apple germplasm set

The article was published with errors in Table 1 and Online Resource 1 found in the results section and supplementary material.

Mining new scab resistance alleles in apple (Malus × domestica Borkh.) germplasm of Kashmir: Towards breeding scab free apple cultivars

Apple an important fruit crop in Kashmir ranks first in all the fruits grown here. It is commercially the most important horticultural crop grown in temperate parts of the world. To control scab in Apple a lot of fungicides are being used however, an alternative approach to fungicide application is the use of scab resistant cultivars as a genetic stock. Therefore, there is an urgent need to identify more cultivars resistant to scab with maximum genes and can be used for apple breeding. In view of this, one hundred apple cultivars grown in Kashmir Valley were screened for the presence of Vf gene conferring resistance against apple scab. Eight trait linked markers were used so for and among them three markers like AL07, AM19, and CH05e03 proved to be useful to distinguish the resistant cultivars from the other apple germplasm. These three markers have amplified resistant fragments in five cultivars. Thus out of the total of 100 cultivars investigated in the present study, only five cultivars namely Shalimar I, Shalimar II, Dubhour, Gold Spur, and Crab apple were found to harbour resistant alleles which may be resistant to some of the races of V. inaequalis. Among the five cultivars harbouring the scab resistant alleles for some races of V. inaequalis, three cultivars namely, Shalimar I, Shalimar II, and Dubhour, which contain the maximum number of scab resistance alleles can be used as parents in further breeding programms to develop more resistant apple cultivars with high market acceptance.

Quantitative variation and heritability estimates of fire blight resistance in a pedigree-connected apple germplasm set

Breeding resistant apple cultivars is a long-term solution to fire blight, a devastating bacterial disease caused by Erwinia amylovora. However, most resistance sources have been characterized in wild genetic backgrounds with poor fruit quality. Additionally, strong influences of environment and tree vigor on susceptibility, quantitative resistance, and the disease’s erratic nature make phenotyping challenging. This study examined levels of resistance/susceptibility to fire blight among and within 32 full-sib families (n = 314 seedlings) in a pedigree-connected apple germplasm set. In 2016 and 2017, multiple actively-growing shoots per tree (approximately 3 trees per seedling) were challenged with E. amylovora. Responses were quantified as proportions of current season’s shoot lengths that were blighted (SLB). Within a year, seedling responses were estimated using mean adjusted SLB best linear unbiased predictions (adjusted SLB BLUPs). Responses ranged from highly resistant to highly susceptible (0.04–0.97 adjusted SLB BLUPs across years) and were relatively consistent between years (Spearman’s R2 = 0.55). K-means clustering was used to classify seedlings into resistance/susceptibility groups based on incidence, adjusted SLB BLUPs, maximum SLB, and maximum age of wood infected. Most families, including crosses between susceptible parents, demonstrated quantitative variation for resistance/susceptibility. Families derived from ‘Enterprise’ demonstrated low relative susceptibility. Depending on the year and model, average narrow-sense heritability estimates ranged from 0.22–0.49. Phenotypic information (1) increases understanding of variation and inheritance of resistance/susceptibility to fire blight in germplasm relevant to US apple breeding programs and (2) could be used in pedigree-informed QTL analyses to detect loci associated with resistance/susceptibility.

Introgressing blue mold resistance into elite apple germplasm by rapid cycle breeding and foreground and background DNA-informed selection

Apple blue mold causes significant postharvest economic losses worldwide. A blue mold resistance locus, qM-Pe3.1, was previously identified on chromosome 3 of Malus sieversii PI 613981, a wild accession with inferior fruit quality. Introgression of the resistance allele into elite breeding germplasm is difficult and success of introgression and the effect of the PI 613981 genome on fruit quality cannot be phenotypically evaluated until fruiting, which occurs approximately 5 years from seed. In this study, introgression of the qM-Pe3.1 resistance allele was achieved by rapid cycle breeding, utilizing the transgenic line T1190 constitutively expressing the BpMADS4 early-flowering gene. This was supported by DNA-based diagnostic information that enabled marker-assisted selection for blue mold resistance using a locus-specific DNA test developed to detect the qM-Pe3.1 resistance allele in offspring (foreground selection). Of 75 second-generation ([‘Gala’ × PI 613981] × T1190) offspring carrying BpMADS4, 43 also carried the qM-Pe3.1 resistance allele. DNA tests for other trait loci were used to identify other desirable alleles related to fruit quality in progeny and 6874 genome-wide SNP markers (from an apple 20K Illumina® SNP array) were used to identify undesirable genomic segments of PI 613981 (background selection). Three individuals identified with favorable recombination close to qM-Pe3.1 and less than 25% of M. sieversii unimproved genome were selected as best suited for the elimination of unimproved DNA segments in subsequent generations. Our pipeline for introgression of qM-Pe3.1, facilitated by marker-assisted foreground and background selection, successfully advanced this promising germplasm in readiness for the next generation.

Using whole-genome SNP data to reconstruct a large multi-generation pedigree in apple germplasm

BackgroundApple (Malus x domestica Borkh.) is one of the most important fruit tree crops of temperate areas, with great economic and cultural value. Apple cultivars can be maintained for centuries in plant collections through grafting, and some are thought to date as far back as Roman times. Molecular markers provide a means to reconstruct pedigrees and thus shed light on the recent history of migration and trade of biological materials. The objective of the present study was to identify relationships within a set of over 1400 mostly old apple cultivars using whole-genome SNP data (~ 253 K SNPs) in order to reconstruct pedigrees.ResultsUsing simple exclusion tests, based on counting the number of Mendelian errors, more than one thousand parent-offspring relations and 295 complete parent-offspring families were identified. Additionally, a grandparent couple was identified for the missing parental side of 26 parent-offspring pairings. Among the 407 parent-offspring relations without a second identified parent, 327 could be oriented because one of the individuals was an offspring in a complete family or by using historical data on parentage or date of recording. Parents of emblematic cultivars such as ‘Ribston Pippin’, ‘White Transparent’ and ‘Braeburn’ were identified. The overall pedigree combining all the identified relationships encompassed seven generations and revealed a major impact of two Renaissance cultivars of French and English origin, namely ‘Reinette Franche’ and ‘Margil’, and one North-Eastern Europe cultivar from the 1700s, ‘Alexander’. On the contrary, several older cultivars, from the Middle Ages or the Roman times, had no, or only single, identifiable offspring in the set of studied accessions. Frequent crosses between cultivars originating from different European regions were identified, especially from the nineteenth century onwards.ConclusionsThe availability of over 1400 apple genotypes, previously filtered for genetic uniqueness and providing a broad representation of European germplasm, has been instrumental for the success of this large pedigree reconstruction. It enlightens the history of empirical selection and recent breeding of apple cultivars in Europe and provides insights to speed-up future breeding and selection.