1. Introduction

The propagation method has profound effects on both plant cultivation and research. Most of the vegetatively propagated crops are perennial, cross-pollinated, and often self-sterile. There is sometimes a progression from seed propagation of landraces to vegetative propagation of more actively selected or intentionally bred cultivars. Seeds derive from crosses between two heterozygous parents and are not genetically identical to the mother plant. Multiplication of selected genotypes with valuable traits is therefore achieved only through vegetative propagation methods. This also affects the research, breeding, and preservation of valuable germplasm, which must be focused on particular genotypes instead of on seedling families as in seed-propagated crops. Genetic research and development of whole-genome sequences are further complicated by the fact that these crops are often polyploid (e.g., banana, mango, ornamental rose, potato and strawberry) or derive from whole-genome duplication, so-called ancient polyploids (apple and pear).

Especially desirable genotypes of, e.g., apple and grapes, have probably remained unaltered for hundreds of years and have been disseminated across large areas, with profound effects on the amount and distribution of genetic variation. Clonal propagation of long-lived crops also involves the emergence of somatic mutants or “sports”, some of which represent an improvement of a particular trait. In some crops like grape, the growing of local varieties in restricted locations like mountain valleys has created locally adapted clones, which have often spread further under different names ^[1]. The true affiliation of these accessions can only be determined through genotyping. Distinct and valuable clones must be preserved to act as a source of propagation material, but the transmission of traits to the offspring is usually identical to that of the original genotype.

1.1. Preservation of Plant Genetic Resources

Fruit and nut trees are traditionally conserved in ex situ field collections as budded or grafted cultivars, while other trees, bushes, and large herbs are grown from the leaf or stem cuttings, or from suckers harvested at the stem basis. Field-planted collections are required also for tuber crops like potatoes, sweet potatoes and cassava, although tubers may be preserved in cold storage for a limited time. The collections are hosted by a wide range of governmental organizations, universities, private companies, botanical gardens, foundations, public parks and individuals. Many publicly-funded collections are focused on the preservation of heritage accessions and include mostly old and/or local cultivars from a defined geographic area. Other collections are designated mainly for genetic studies and plant breeding and may include both old and new cultivars from various areas, advanced breeding lines and wild relatives. Field collections are sometimes backed up by in vitro cultures and cryopreserved tissue (buds and shoot tips), whereas older and/or local cultivars are sometimes preserved in situ (fields and orchards).

The European Cooperative Program for Genetic Resources (ECPGR) has created a network of plant conservationists and scientists, with working groups designated to specific crops, e.g., berries, Malus/Pyrus, potato, Prunus and Vitis. The Global Crop Diversity Trust has employed specialists to develop global ex situ conservation strategies for some key crops. Thus, Bramel and Volk ^[2] outlined a strategy for apple, which could be useful also for other vegetatively propagated crops: (1) coordinate a global genotyping effort to assess and map diversity within and among accessions in the existing global collections, verify accession identity, confirm redundancies, identify mislabeled accessions, identify unique accessions, and determine the existence of key gaps in the global collections and (2) ensure access to an information platform with an international registry and database for ex situ and in situ collections, including tree- and accession-level phenotype, passport, and genotype information.

Global germplasm exchange is facilitated by the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA), which aims to strengthen the conservation, exchange, and sustainable use of plant genetic resources for food and agriculture. Several public databases for accession-level information sharing are available as the European Search Catalogue for Plant Genetic Resources (EURISCO) and the GRIN-Global database for accessions conserved by the USDA-ARS National Plant Germplasm System. Genesys, initiated by Bioversity International, the Global Crop Diversity Trust, and the secretariat of ITPGRFA, was launched as a global platform in 2011, with cooperation by EURISCO, GRIN, and the System-Wide Information Network for Genetic Resources (SINGER) from CGIAR. Not all crops are, however yet covered by these databases and the information is fast becoming obsolete since accessions continuously enter or leave different plant collections.

1.2. Accession-Level Information

Each preserved accession in a plant collection should have passport data (accession name, genus name, country of origin, acquisition date, code of the institute where the accession is maintained, and a unique accession number). The collection becomes infinitely more valuable if the accessions are accompanied by phenotypic data (descriptors and traits of economical relevance) and data from molecular studies on true-to-type status and markers/genes for traits of interest. Many plant collections were established long before DNA fingerprinting became available. Decisions about what accessions to preserve have therefore relied mainly on morphological traits, information on the ecogeographical origin and/or historical importance. The number of redundant (synonymous genotypes) and mislabeled accessions frequently reaches 30% or even higher when these collections are investigated with molecular markers ^[3].

In order to increase the importance and utilization of the preserved accessions, co-ordinated national and regional efforts are needed to compare collections at the phenotypic and genetic levels and to confirm cultivar identity and identify valuable genes. The scientific community has produced considerable amounts of data for accessions in various plant collections. Several rosaceous fruit and berry crops were thus investigated in recent multinational research projects; the RosBREED projects in 2009–2019 involved many research institutes in the USA and some overseas ^[4]. Another major project was the European Union-funded FruitBreedomics project (2011–2015) involving 28 research institutes and private companies working with apple and/or peach ^[5].

2. Genotyping

Of all DNA fingerprint methods emerging in the early 1990s, only simple sequence repeats (SSR, also known as microsatellites) remain as a major option for screening collections of plant genetic resources, although other single-locus methods like SCoT (start codon targeted) markers, and multilocus methods like ISSR (inter-simple-sequence repeats), IRAP (inter-retrotransposon amplified polymorphism), REMAP (retrotransposon-microsatellite-amplified polymorphism), and SRAP (sequence-related amplification polymorphisms) are occasionally applied. Large-scale analyses are sometimes also carried out with the diversity arrays technology (DArT), which is based on the hybridization of fluorescent DNA probes to target DNAs spotted onto a microarray.

However, with the arrival of next-generation sequencing (NGS) technologies, a rapidly increasing number of studies have instead utilized single nucleotide polymorphisms (SNPs) and various other sequencing-based technologies like DArTseq.

2.1. Simple Sequence Repeats (SSR)

SSR have long been the preferred marker for genotyping vegetatively propagated plants, mainly because of their multiallelic and highly polymorphic nature, high discriminatory potential, and good reproducibility ^[3]. Once the primers for suitable SSR loci are developed, laboratory work and data analysis are straight-forward. A definite bonus is that additional samples can be cost-effectively screened and added into an already existing database for renewed analyses of diversity and relatedness. Combining datasets from multiple laboratories can, however, become problematic due to discrepancies in allele size evaluations, but adjustments are facilitated by comparison of shared reference accessions, sometimes complemented with the regenotyping of a subset of accessions ^[6].

SSR-based analyses usually report very high levels of interaccession polymorphism, and duplicate samples are easily detected. By contrast, somatic mutants, so-called sports or clones, are seldom distinguished from their original genotypes although clone-specific mutations are occasionally identified ^[7]. More than two alleles in the same locus/sample usually indicate polyploidy; 10–30% triploids are commonly reported from germplasm screenings of apple and pear ^{[8][9][10][11][12][13]}. SSR analyses can also indicate which one of two diploid parental genotypes has produced an unreduced gamete resulting in a triploid offspring. Supernumerary bands can, however, also derive from duplication events or somatic mutations generating a chimerical state ^[10].

Comparison of results across studies is often hampered by the use of different sets of SSR loci ^[14]. To remedy this situation, ECPGR has published several sets of recommended SSR loci, e.g., one set for pear ^[15], one for cherry ^[16], and one for plum ^[17]. For pear, a different set of loci (the US Pyrus Genetic Resources set, USPGR) was recently proposed, with longer repeat motifs (3–6 bp) resulting in fewer alleles but also fewer artefacts such as stutters, split peaks, and binning errors, which are common when using dinucleotide repeat loci as in the ECPGR set ^[18]. The ECPGR set for apple has been modified over the years as more information about polymorphism and linkage has become available, and now includes 17 SSR loci that span most of the apple genome ^[6].

A standardized and internationally recognized set of SSR loci is used not only for the coordinated characterization of genetic resources, but also as tools for cultivar identification and protection of Plant Breeders' Rights. Such SSR marker-based genotype data are included in the International Variety Catalog (VIVC) for grapes ^[19] and The European Union Common Catalog (EUCC) for potato ^[20].

2.2. Single Nucleotide Polymorphisms (SNPs)

Several approaches are used to generate SNPs in plant material. Genotyping-by-sequencing (GBS) produces SNPs from the whole genome and is especially valuable for plants where reference genome sequences have not yet been published. One way to reduce the complexity in GBS is RAD-Seq (restriction-site associated DNA sequencing). Using this technique, 1.25 million SNPs were discovered in a panel of 84 mango varieties and used for the development of the genomic resource MiSNPDb ^[21]. SNPs are also commonly mined from expressed sequence tag (EST) or transcriptome gene sequences. Insertion/deletion variants occur frequently and are sometimes also used as markers but their reproducibility is generally lower than for SNPs.

The use of a chip or microarray can reduce the time and cost of genotyping large sets of accessions. SNP arrays have been developed for many commercially important crops, such as the 6K cherry ^[22], 8K apple ^[23], 9K peach ^[24], 18K grape ^[25], and 6K avocado arrays ^[26] implemented on the Illumina Infinium® or Illumina II® platforms. Similarly, a 20K array was developed for apple ^[27] but the screening of diverse germplasm indicated very rapid decay of the linkage disequilibrium (LD) for each apple chromosome. A number of markers had to be increased to obviate this problem, and the Axiom® apple 480K SNP array was subsequently developed ^[28]. Several other Axiom® arrays have been developed, including a 20K array for potato ^[29], a 68K array for rose ^[30], a 700K array for walnut ^[31], and both a 70K array and a 200K array for pear ^[32][33]. For the allo-octoploid strawberry, a 90K array has been produced ^[34] and both a 50K “production” array and an 850K “screening” array, with SNPs chosen according to the different purposes ^[35].

The SNPs must be developed from a genetically relevant material to avoid a biased representation and thus failure to reflect true differentiation among investigated samples. SNP discovery panels can differ considerably between projects aiming to produce a genomic map ^[26] and projects aiming to analyze genetic variation and identify marker-trait associations in germplasm collections ^[27]. Ascertainment bias is probably more common in SNP arrays compared to GBS due to the use of biallelic SNPs and lack of sufficient diversity in the discovery panels. Conversely, GBS often involves a higher amount of missing data, and sequence coverage must be very high to achieve reproducibility between different sets of experiments ^[18].

Highly automated scoring of SNPs can easily generate errors that affect the deductions made from obtained results. A common workflow (using software ASSIsT) was therefore designed for removal of faulty SNPs in apple, peach, and sweet cherry, according to inheritance principles and pre-existing pedigree information ^[36]. SNP-based analyses can also help to identify chromosome number levels. Theoretically, triploid individuals should have a 50% higher level of heterozygosity than diploid individuals, assuming Hardy–Weinberg equilibrium conditions. The difference was, however, only 30% in a collection of apples, probably due to the lack of a proper equilibrium across the investigated samples ^[37].

It has been argued that the use of GBS is overall less expensive than SNP arrays since marker discovery and genotyping can be achieved in a single step in highly diverse crops like apple ^[37] and can be applied also in crops for which a reference genome is still lacking. For smaller projects, SSR is still cheaper since a low number of samples can be genotyped whereas at least 96 samples are needed for cost-effective GBS and SNP array genotyping ^[18].