Apple Germplasm Research Articles

Self-incompatibility alleles in important genotypes for apple breeding in Brazil

Abstract: The objective of this study was to identify self-incompatibility (S) alleles of advanced breeding selections of apple (Malus × domestica Borkh.). The S-alleles of 42 apple genotypes were analyzed by markers using allele-specific PCR amplification and amplicons digested with restriction endonucleases. Among the screened genotypes were cultivars, advanced selections, and accessions of the Apple Germplasm Bank of Epagri (Cacador, Santa Catarina, Brazil). Two S-alleles were identified in 36 genotypes, and only one S-allele was determined in the other six genotypes. In all, eleven S-alleles were identified among all the genotypes evaluated. The S3 and S5 alleles were most frequent (30.2% and 19.8%, respectively). The identification of S-alleles using molecular markers in important apple tree genotypes is useful for determination of compatible parents for breeding programs.

THE ANTHOCYANIN BIOSYNTHETIC REGULATOR MDMYB1 POSITIVELY REGULATES ASCORBIC ACID BIOSYNTHESIS IN APPLE

<List> <ListItem><ItemContent> • The contents of anthocyanin and AsA in red-flesh apples are higher than that in non-red-flesh apples. </ItemContent></ListItem> <ListItem><ItemContent> • The anthocyanin biosynthetic regulator MdMYB1 directly activates the expression of dehydroascorbate reductase gene MdDHAR, thus promoting the activity of the DHAR enzyme and the accumulation of AsA. </ItemContent></ListItem> <ListItem><ItemContent> • MdMYB1-MdDHAR module may play a key role in AsA-DHA homeostasis. </ItemContent></ListItem></List> Ascorbic acid (AsA, vitamin C) is involved in the regulation of many aspects of plant growth and development. It is an essential micronutrient for humans and can prevent scurvy, maintain the health of gums and blood vessels, reduce the level of plasma cholesterol and enhance the immune systen. Apple cultivars Orin and Guanghui were crossed to obtain a group of hybrid offspring with and without red flesh in the course of assessing apple germplasm resources. Unexpectedly, the red-flesh apples had higher AsA contents than other apples. Further studies showed that the anthocyanin biosynthetic regulator MdMYB1 directly activates the expression of dehydroascorbate reductase gene MdDHAR, thus promoting the activity of the DHAR enzyme and the accumulation of AsA. This finding reveals the mechanism leading to high AsA levels in red-flesh apples and suggests a new idea to cultivate red-flesh apples with high AsA contents and produce AsA efficiently and without pollution.

Serological and Molecular Detection of Latent Viruses in the Apple Germplasm Bank of Santa Catarina

Abstract The Apple Germplasm Bank (AGB) of Santa Catarina Agricultural Research and Rural Extension Company - Epagri, AGB-Epagri, is the largest of the genus Malus in Brazil. Twenty-eight main accessions of this bank were virus screened through DAS-ELISA, RT-PCR and IC-RT-PCR during two consecutive reproductive cycles, and each accession showed latent mixed infection by at least two species, among ASGV, ASPV and ACLSV. The combined use of diagnostic methods helped overcome inconsistencies commonly found in apple virus detection and was shown essential for the AGB-Epagri can be safely used as a source of genetic variability and for the exchange of virus-free propagative material.

A genome-wide association study for the detection of genes related to apple Marssonina Blotch disease resistance in apples

Apple Marssonina Blotch (AMB), commonly referred to as apple blotch disease, is caused by Diplocarpon mali and represents one of the major diseases in Korean apple orchards. AMB can occur on the leaves and fruit, which lowers fruit quality and leads to early defoliation, reducing tree vigor and growth. In this study, a genome-wide association study (GWAS) was conducted to detect genes that may affect AMB disease infection. A multi-locus random SNP effect mixed linear model (mrMLM) method GWAS analysis was conducted with 192 apple germplasms (Malus spp.) in the Apple Research Institute at the National Institute of Horticultural & Herbal Science, Gunwi, Korea. Highly significant five SNP loci were selected from the results of the GWAS analysis and based on the Haploview 4.2 LD analysis data, candidate genes linked five SNP loci and their predicted functions were identified association to AMB resistance by Basic Local Alignment Search Tool of the National Center for Biotechnology Information. One of these candidate genes was identified include leucine rich repeat structure by BLAST. Highly susceptible and resistant cultivars to AMB were separately distinguished in the high resolution melting analysis using primers from two significant loci. Thus, the candidate genes linked to these two SNP loci are potentially strongly associated with AMB resistance.

How to Discover Traditional Varieties and Shape in a National Germplasm Collection: The Case of Finnish Seed Born Apples (Malus × domestica Borkh.)

Cultivated apple (Malus × domestica Borkh.) is a major crop of economic importance, both globally and regionally. It is currently, and was also in the past, the main commercial fruit in the northern European countries. In Finland, apple trees are grown on the frontier of their northern growing limits. Because of these limits, growing an apple tree from a seed was discovered in practice to be the most appropriate method to get trees that bear fruit for people in the north. This created a unique culturally and genetically rich native germplasm to meet the various needs of apple growers and consumers from the late 1800s to the mid-1900s. The preservation, study and use of this genetic heritage falls within the mandate of the Finnish National Genetic Resources Program. The first national apple clonal collection for germplasm preservation was reorganized from the collections of apple breeders. The need to evaluate the accessions, both in this collection and possible missing ones, to meet the program strategy lead us to evaluate the Finnish apple heritage that is still available in situ in gardens. In this article we use multiple-approach methodologies and datasets to gain well-described, proof-rich samples for the trueness-to-type analysis of old heirloom apple varieties. The approach includes a combination of socio-historic, pomological and genotyping methods and datasets that are all valued as equally important. The main finding was that in addition to the pomological, molecular and genetic evaluation of ex situ apple collections, an extensive historical data and socio-economic conditions research are essential to perform good characterization of accessions. After implementing the results in re-creating the Finnish national apple germplasm collection, the number of Finnish local varieties was more than doubled from 38 accessions to 97.

Eastern Anatolian apples with a unique population structure are genetically different from Anatolian apples

The origin of the apple is known to be the Transcaucasian region. Eastern Anatolia, which is located on the migration routes from Asia to Europe, has a rich and an uncharacterized apple germplasm and the characterization of apple genetic sources from this region is important for both evolutionary studies and apple breeding. In this study, 94 M. domestica accessions originated from seven diverse regions within Eastern Anatolia were studied using 16 SSR (simple sequence repeat) loci. SSR markers we used produced high allele numbers in all loci and CH02d11 (PI: 0.059) with 18 alleles was the most informative locus. In addition, 14 identical accession groups were identified. Most likely due to self-incompatibility, relatively high levels of heterozygosity (Ho: 0.696) was found for Eastern Anatolia apples. Structure Harvester analyses of East Anatolian apple accessions showed that although each group seems to be somewhat distinct, some levels of admixture with other populations might also exist. Due to a significant gene flow between all pairs of seven apple populations, a limited (low) differentiation was found between the populations. Comparisons using 16 common SSR loci revealed that Eastern Anatolia accessions were genetically different from Anatolian accessions. In addition, based on FCA, and Nei’s genetic distance analyses, Eastern Anatolian apples were found to be genetically different from the commercial apple cultivars Golden Delicious and Florina. Our results suggesting that Eastern Anatolia apple populations have a unique structure will be useful for future genetic and evolutionary studies on apples.

Evaluation of 110 Apple Cultivars for Resistance to Alternaria Blotch Caused by Alternaria alternata Apple Pathotype

Apple blotch caused by Alternaria alternata apple pathotype is a severe disease of apple (Malus ×domestica Borkh) occurring throughout the world, especially in eastern Asia. Phenotypic and genetic information about resistance/susceptibility of apple germplasm to this disease will be extremely valuable for selecting and developing new disease resistant cultivars. In this study, 110 apple cultivars obtained from the USDA apple germplasm in Geneva, NY, were evaluated for their resistance/susceptibility to apple blotch by field surveys, and inoculation of detached leaves with a suspension of germinated conidia of A. alternata apple pathotype. Disease incidence were different among the cultivars and categorized into resistant (R), moderately resistant (MR), or susceptible (S). Two molecular markers, S428, a random amplified polymorphic DNA (RAPD) marker associated with disease resistance, and a simple sequence repeat (SSR or microsatellite) marker CH05g07, linked to susceptibility were used to correlate the phenotypes expressed in field surveys and laboratory inoculations. The detection using either the S428 marker or the CH05g07 marker in 50 common breeding cultivars was consistent with R or S traits except for ‘Bisbee’ and ‘Priscilla’. These two cultivars were MR to apple blotch through phenotyping. However, SSR markers were detected, but RAPD markers were not and therefore were considered susceptible. Combined with the record of resistance to fire blight from Germplasm Resources Information Network (GRIN), ‘Dayton’, ‘Mildew Immune Seedling’, ‘Puregold’, and ‘Pumpkin Sweet’ were highly resistant to both diseases and considered as the best choices of parents for stacking resistance to multiple diseases in breeding program.

Assessment of Apple (malus × Domestica Bark.) Germplasm of Kashmir Using RAPD Markers

ABSTRACT The availability of diverse germplasm of any crop is an important genetic resource to mine the genes that may assist in attaining food as well as the nutritional quality of the crop. In the present study, a total of 10 RAPD markers were used for the first time in the Kashmir apple germplasm to elucidate diversity among 19 apple cultivars collected from various parts of the Valley. The discriminatory power of each random primer was observed using various parameters like: polymorphic information content, resolving power and marker index. A total of 70 polymorphic bands were observed with a percentage polymorphism of 83.33. Cultivars differentiated through mutations like Oregon Spur, Reeka Red, and Silver Spur were also included in this study and have shown identical banding patterns among each other. The dendrogram generated by using NTSYS grouped cultivars into two major clusters with various degrees of sub-clustering within the cluster. These findings will be helpful for further research programs on apple and have implications in apple breeding as well as in conservation programs.

Sequencing of a Wild Apple (Malus baccata) Genome Unravels the Differences Between Cultivated and Wild Apple Species Regarding Disease Resistance and Cold Tolerance.

Malus baccata is one of four wild apple species that can hybridize with the cultivated apple species (Malus domestica). It is widely used in high-latitude apple-producing areas as a rootstock and breeding resource because of its disease resistance, and cold tolerance. A lack of a reference genome has limited the application of M. baccata for apple breeding. We present a draft reference genome for M. baccata. The assembled sequence consisting of 665 Mb, with a scaffold N50 value of 452 kb, included transposable elements (413 Mb) and 46,114 high-quality protein-coding genes. According to a genetic map derived from 390 sibling lines, 72% of the assembly and 85% of the putative genes were anchored to 17 linkage groups. Many of the M. baccata genes under positive selection pressure were associated with plant–pathogen interaction pathways. We identified 2,345 Transcription factor-encoding genes in 58 families in the M. baccata genome. Genes related to disease defense and cold tolerance were also identified. A total of 462 putative nucleotide-binding site (NBS)-leucine-rich-repeat (LRR) genes, 177 Receptor-like kinase (RLK) and 51 receptor-like proteins (RLP) genes were identified in this genome assembly. The M. baccata genome contained 3978 cold-regulated genes, and 50% of these gene promoter containing DREB motif which can be induced by CBF gene. We herein present the first M. baccata genome assembly, which may be useful for exploring genetic variations in diverse apple germplasm, and for facilitating marker-assisted breeding of new apple cultivars exhibiting resistance to disease and cold stress.

High-quality, genome-wide SNP genotypic data for pedigreed germplasm of the diploid outbreeding species apple, peach, and sweet cherry through a common workflow.

High-quality genotypic data is a requirement for many genetic analyses. For any crop, errors in genotype calls, phasing of markers, linkage maps, pedigree records, and unnoticed variation in ploidy levels can lead to spurious marker-locus-trait associations and incorrect origin assignment of alleles to individuals. High-throughput genotyping requires automated scoring, as manual inspection of thousands of scored loci is too time-consuming. However, automated SNP scoring can result in errors that should be corrected to ensure recorded genotypic data are accurate and thereby ensure confidence in downstream genetic analyses. To enable quick identification of errors in a large genotypic data set, we have developed a comprehensive workflow. This multiple-step workflow is based on inheritance principles and on removal of markers and individuals that do not follow these principles, as demonstrated here for apple, peach, and sweet cherry. Genotypic data was obtained on pedigreed germplasm using 6-9K SNP arrays for each crop and a subset of well-performing SNPs was created using ASSIsT. Use of correct (and corrected) pedigree records readily identified violations of simple inheritance principles in the genotypic data, streamlined with FlexQTL software. Retained SNPs were grouped into haploblocks to increase the information content of single alleles and reduce computational power needed in downstream genetic analyses. Haploblock borders were defined by recombination locations detected in ancestral generations of cultivars and selections. Another round of inheritance-checking was conducted, for haploblock alleles (i.e., haplotypes). High-quality genotypic data sets were created using this workflow for pedigreed collections representing the U.S. breeding germplasm of apple, peach, and sweet cherry evaluated within the RosBREED project. These data sets contain 3855, 4005, and 1617 SNPs spread over 932, 103, and 196 haploblocks in apple, peach, and sweet cherry, respectively. The highly curated phased SNP and haplotype data sets, as well as the raw iScan data, of germplasm in the apple, peach, and sweet cherry Crop Reference Sets is available through the Genome Database for Rosaceae.

Preliminary Pollen Analysis of Some Apple Cultivars in Kashmir: Towards Understanding the Apple Pollen Morphology

Kashmir Valley, a North Western state in India, represents the richest repository of apple germplasm of about 200 cultivars besides some unknown genotypes of seedling origin. Among the rich germplasm resource, sixteen apple cultivars belonging to two commercially important groups with one group having Delicious as a suffix and other group having Ambri as a suffix were examined for the first time based on pollen grain surface morphology using scanning electron microscopy for genotype differentiation. Most of the air-dried apple pollen grains were elliptical and tricolpate with three germinal furrows extending almost the full length of the pollen grain. However, round and tricolpate pollen grains were observed in cultivars Kullu Delicious, Oregon Spur, Reeka Red, and Silver Spur. Striation pattern of the pollen grains varies from cultivar to cultivar. Mostly, the striations extend from one end of colpi to another end, but in case of Cross Delicious, the striations were irregular and form network-like structures. Pores on the exine surface also showed great variations among the cultivars. The cultivars like Oregon Spur, Silver Spur, and Reeka Red are said to be the sports of Delicious, but from the results of scanning electron microscopy, these resemble Kullu Delicious in terms of pollen shape, striation pattern and pores on the exine surface.

SSR analysis based on molecular characterisation of apple germplasm in Lithuania

SSR analysis based on molecular characterisation of apple germplasm in Lithuania

Genotyping apple (Malus x domestica Borkh.) heirloom germplasm collected and maintained by the Regional Administration of Friuli Venezia Giulia (Italy)

This paper reports the genetic diversity of apple germplasm collected in the Friuli Venezia Giulia region (north-eastern Italy). The collection, maintained in three different locations, was represented by local cultivars which probably originated as chance seedlings together with cultivars introduced from neighbouring countries, the name and origin of most of which has been lost over time. A preliminary procedure described in the paper started with 469 molecular profiles analysed using 15 Simple Sequence Repeat (SSR) markers and allowed identification of the ‘true-to-type’ genotypes among those maintained in multiple locations. The set of the remaining 234 accessions was further reduced to 132 unique profiles by removing 102 synonyms, that is accessions with different name and the same molecular profile. Flow cytometry identified as many as 54 triploids (40.9%), whose status was confirmed by field observations on leaf size and the occurrence of triallelic profiles at several SSR markers. The remaining 78 diploid accessions were analysed for their genetic diversity, that is the number of alleles, observed and expected heterozygosity, polymorphism information content (PIC), the frequency of null alleles and the probability of identity for unrelated and full-sib genotypes. The paper provides a critical evaluation of the SSR markers adopted for the study, discusses the genetic diversity observed in the apple germplasm collection examined as well as its high frequency of triploids compared with other apple collections described in the literature.

Allelic Diversity in Apple Germplasm for Fruit Quality and Scab Resistance Using SSR Markers

The present investigation entitled “Allelic diversity in apple germplasm for fruit quality and scab resistance using SSR markers” was conducted in division of plant biotechnology, SKUAST-K, Shalimar during the year 2018. In the present study, four SSR markers namely Md-ACS-1, Md-ACO-1, ALO-7 and AM-19 were used for screening of 40 apple cultivars presently under cultivation in SKUAST-K for quality and scab resistance. The results revealed that out of 40 cultivars evaluated, 3 were found homozygous (ACS-1-2/2), 17 were heterozygous (ACS-1-1/2) and 10 were homozygous (ACS-1-1/1), while as no amplification of ACS-1 was found for 10 cultivars. Similarly, for Md-ACO-1, 3 cultivars were found homozygous, 35 as heterozygous and 2 did not show any amplification. As far as presence of Vf gene conferring resistance against scab disease, ALO-7 amplified at 820bp and 570bp alleles, 570 bp fragment was observed in resistant genotypes and was absent in susceptible genotypes, while the 820 amplicon was observed in both resistant and susceptible genotypes. The markers AM19 led to the amplification of 520 bp fragment in few cultivars.

Genetic variability in a Brazilian apple germplasm collection with low chilling requirements.

The apple (Malus domestica Borkh) originally evolved to require temperatures below 7.2 °C for the induction of budding and flowering. In Brazil, breeders have overcome the climate barrier and developed the cultivars Anabela, Julieta, Carícia, and Eva, with low chilling requirements and good yield characteristics. These cultivars are grown in many warmer climate countries in South America, Africa, and the Middle East. The apple germplasm collection that originated these cultivars has several genotypes with pedigrees for a low chilling requirement. Knowledge of the variability and genetic relationships among these genotypes may be useful in the development of superior new cultivars. In this work, we first selected the best ISSR (inter-simple sequence repeat) primers for genetic studies in apple, and then we used the selected primers to evaluate the genetic variability of the apple germplasm collection at the Instituto Agronômico do Paraná. The evaluation of 42 ISSR primers in 10 apple genotypes allowed us to select the best nine primers based on the polymorphic information content (PIC) and resolving power (RP) indexes. The primer selection step was robust since the dendrogram obtained with the nine selected primers was the same as the one obtained using all 26 polymorphic primers. Primer selection using PIC and RP indexes allowed us to save about 60% of time and costs in the genetic variability study. The nine ISSR primers showed high levels of genetic variability in the 60 apple genotypes evaluated. The relevance of the primer selection step is discussed from the perspective of saving time and money in germplasm characterization. The high genetic variability and the genetic relationships among the genotypes are discussed from the perspective of the development of new apple cultivars, mainly aiming for a low chilling requirement that can better adapt to current climatic conditions or those that may arise with global warming.

A Ma10 gene encoding P-type ATPase is involved in fruit organic acid accumulation in apple.

SummaryAcidity is one of the main determinants of fruit organoleptic quality. Here, comparative transcriptome analysis was conducted between two cultivars that showed a significant difference in fruit acidity, but contained homozygous non‐functional alleles at the major gene Ma1 locus controlling apple fruit acidity. A candidate gene for fruit acidity, designated M10, was identified. The M10 gene encodes a P‐type proton pump, P3A‐ATPase, which facilitates malate uptake into the vacuole. The Ma10 gene is significantly associated with fruit malate content, accounting for ~7.5% of the observed phenotypic variation in apple germplasm. Subcellular localization assay showed that the Ma10 is targeted to the tonoplast. Overexpression of the Ma10 gene can complement the defect in proton transport of the mutant YAK2 yeast strain and enhance the accumulation of malic acid in apple callus. Moreover, its ectopic expression in tomato induces a decrease in fruit pH. These results suggest that the Ma10 gene has the capacity for proton pumping and plays an important role in fruit vacuolar acidification in apple. Our study provides useful knowledge towards comprehensive understanding of the complex mechanism regulating apple fruit acidity.

Genetic Characterization of the Apple Germplasm Collection in Central Italy: The Value of Local Varieties.

In the last 50 years, intensive farming systems have been boosted by modern agricultural techniques and newly bred cultivars. The massive use of few and related cultivars has dramatically reduced the apple genetic diversity of local varieties, confined to marginal areas. In Central Italy a limited spread of intensive fruit orchards has made it possible to preserve much of the local genetic diversity, but at the same time the coexistence of both modern and ancient varieties has generated some confusion. The characterization and clarification of possible synonyms, homonyms, and/or labeling errors in old local genetic resources is an issue in the conservation and management of living collections. 175 accessions provided by 10 apple collections, mainly local varieties, some of unknown origin, and well-known modern and ancient varieties, were studied by using 19 SSRs, analyzed by STRUCTURE, Ward’s clustering and parentage analysis. We were able to identify 25 duplicates, 9 synonyms, and 9 homonyms. As many as 37 unknown accession were assigned to well known local or commercial varieties. Polyploids made up 20%. Some markers were found to be significantly correlated with morphological traits and the loci associated with the fruit over color were related to QTLs for resistance to biotic stresses, aroma compounds, stiffness, and acidity. In conclusion the gene pool of Central Italy seems to be rather consistent and highly differentiated compared with other European studies (FST = 0.147). The importance of safeguarding this diversity and the impact on the management of the germplasm living collection is discussed.

Seed morphometry is suitable for apple-germplasm diversity-analyses

The main objective of this study was to evaluate the trustworthiness of seed image analysis as an approach to discriminate apple germplasm accessions. Digital images of seeds from 42 apple cultivars, acquired by a flatbed scanner, provided a phenotypic dataset with 106 morphometric variables. Stepwise Linear Discriminant Analysis (LDA) was used to examine this dataset, and the results were compared with available genetic data. The first comparison among cultivars provided a 38.8% cross-validation of correct identifications with a discriminant percentage ranging between 11.7 and 70%. In agreement with the genetic diversity analysis, the LDA could discriminate between the apples cultivars, identifying two main groups that could be further divided into additional subgroups. Based on our findings, we propose that seed image analysis is a valuable and affordable tool to investigate phenotypic diversity among a large number of apple cultivars.

Genetic diversity and core collection of Malus × domestica in northwestern Spain, Portugal and the Canary Islands by SSRs

Abstract The domesticated apple (Malus × domestica Borkh.) is one of the most relevant cultivated fruit crops worldwide, in particular in temperate zones, and the fourth most economically important. In order to avoid loss of genetic diversity, germplasm banks have been established to conserve proper genetic diversity, among them we have evaluated five that covers western Iberian Peninsula (the one from Galicia and three from northern to southern Portugal) and Canary Islands.A total of 634 accessions composed of old and local autochthonous apple germplasm, as well as commercial and reference varieties, plus 16 accessions that were collected as wild apples, were studied. Of the total, 484 local accessions were obtained from Spain and 67 from Portugal. The objective of the present study was to analyse the genetic diversity and genetic structure, and define core collection (a small group of cultivars that preserve the allele diversity of the set), by using 13 microsatellite markers (SSRs). Of the total of 634 accessions studied, corresponding to 370 unique genotypes, were grouped into two reconstructed panmictic populations or RPPs; the first RPP included 506 accessions (304 unique genotypes), which belonged to local and autochthonous varieties; and the second RPP clustered the remaining 128 (66 distinct genotypes), which were commercial and reference varieties. In total, 29 new synonyms (74 accessions) were identified between Galicia and the Canary Islands, which indicated the close historical relationship between these two regions located 2000 km apart. A core collection to preserve the whole allelic diversity of the west of the Iberian Peninsula (Galicia and Portugal) and the Canary Islands was defined keeping 10% of the genotypes (63) with respect to total accessions of the complete study, 14% of them (54) from Galicia with wild accessions or 15% (55) from Galicia without wild accessions, 40% (30) from the Canary Islands and 36% (24) from Portugal. The results obtained will allow update of the germplasm banks with correct identification of cultivars, differentiating the autochthonous ones from those derived from commercial cultivars, with the elimination of repetitions (42%), and proposal of the core collection necessary to preserve allele diversity.

Genetic diversity investigation of the apple germplasm available at the Fondazione Edmund Mach

Genetic diversity investigation of the apple germplasm available at the Fondazione Edmund Mach