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Contents lists available at ScienceDirect

Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Next generation breeding

Delfina Barabaschi a , Alessandro Tondelli a , Francesca Desiderio a , Andrea Volante b ,
Patrizia Vaccino c , Giampiero Valè b , Luigi Cattivelli a,∗
a
Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, Genomics Research Centre, Via San Protaso 302, 29017 Fiorenzuola d’Arda, Italy
b
Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, Rice Research Unit, SS 11 to Torino Km 2.5, 13100 Vercelli, Italy
c
Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, Research Unit for Cereal Selection in Continental areas, via R. Forlani, e, 26866 S.
Angelo Lodigiano, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The genomic revolution of the past decade has greatly improved our understanding of the genetic
Received 17 April 2015 make-up of living organisms. The sequencing of crop genomes has completely changed our vision and
Received in revised form 10 July 2015 interpretation of genome organization and evolution. Re-sequencing allows the identification of an
Accepted 11 July 2015
unlimited number of markers as well as the analysis of germplasm allelic diversity based on allele min-
Available online xxx
ing approaches. High throughput marker technologies coupled with advanced phenotyping platforms
provide new opportunities for discovering marker-trait associations which can sustain genomic-assisted
Keywords:
breeding. The availability of genome sequencing information is enabling genome editing (site-specific
Next generation sequencing
Molecular markers
mutagenesis), to obtain gene sequences desired by breeders. This review illustrates how next genera-
Allele diversity tion sequencing-derived information can be used to tailor genomic tools for different breeders’ needs to
Genomic selection revolutionize crop improvement.
Genome editing © 2015 Elsevier Ireland Ltd. All rights reserved.
Plant breeding

1. Introduction into genetic variation through partial or complete re-sequencing

of different accessions [2]. Re-sequencing, leading to arrays of
The development of next generation sequencing (NGS) tech- high-density single-nucleotide polymorphisms (SNPs), is allowing
nologies has made DNA sequencing high throughput and very cost whole-genome scans to identify haplotype blocks that are signifi-
effective. Consequently, many opportunities are being opened to cantly correlated with quantitative trait variation. The distribution
explore the relationships between genetic and phenotypic diver- of low cost sequencing technologies offers new opportunities to
sity with a resolution never reached before. Reference genome shape genetic diversity according to the needs of modern agri-
sequences have been published for many crop species [1] and culture and, in turn, has a number of practical consequences
many more genome sequencing projects are in progress (http:// for plant breeding: i) the analysis of genetic diversity can be
www.ncbi.nlm.nih.gov/genomes/leuks.cgi; http://plants.ensembl. based on genome re-sequencing; ii) genome wide association
org/index.html; http://phytozome.jgi.doe.gov/pz/portal.html). The studies (GWAS) become an attractive approach for quantitative
sequences of crop genomes provide a useful starting point to trait loci (QTLs) mapping in plants since broad genetic resources
explore genome organization and evolution and provide insight can be scanned for marker-trait association without any limi-
tation of marker availability; iii) the great number of markers
support genomic selection; and iv) the genome sequences allow
the targeted modification of specific genes through genome editing
Abbreviations: CRISPR/Cas9, Clustered regularly interspaced short palindromic technologies or identification of suitable mutations within muta-
repeats/CRISPR-associated; GBS, genotyping by sequencing; GEBV, genomic esti- genized populations, resulting in the introduction of new allelic
mated breeding values; GS, genomic selection; GWAS, genome-wide association variants in the genome of cultivated varieties. Conversely, these
studies; LD, linkage disequilibrium; MAGIC, multi-parent advanced generation
achievements highlight new bottlenecks for breeding progress,
intercross; MAS, marker-assisted selection; NAM, nested association mapping; NGS,
next generation sequencing; RIL, recombinant inbreed line; QTL, quantitative trait particularly the phenotyping capacity (in terms of both precision
locus; RAD, restriction-site associated DNA; SNP, single-nucleotide polymorphism; and throughput [3]), and recombination frequency [4].
TALEN, transcription activator-like effector nucleases; TILLING, targeting induced Over the last decades, plant breeding has moved from being
local lesions in genomes; ZFN, zinc finger nucleases.
a completely phenotyping-based process to having an increased
∗ Corresponding author.
reliance on some level of genotype-based selection [5]. This trend is
E-mail address: [email protected] (L. Cattivelli).

http://dx.doi.org/10.1016/j.plantsci.2015.07.010
0168-9452/© 2015 Elsevier Ireland Ltd. All rights reserved.

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expected to increase in the coming years as the NGS-based knowl- plant diversity arose. Extensive insights into plant genome com-
edge will be translated into “Next Generation Breeding”. In this position and organization have been gained from the genome
review, we consider current trends and future prospects for the sequencing and new findings on plant origin and evolution
application of genomic instruments in the improvement of plant (genome duplication, ancestral re-arrangements and polyploidiza-
breeding performance. tion events) have been revealed [2]. An in silico paleogenomic study
based on a deep comparison of monocot and eudicot genomes,
allowed the reconstruction of ancestral protochromosome seg-
2. Genome sequencing and sequence-based markers
ments and a description of the evolutionary dynamics leading to the
present-day genomes, their genome organization and regulation
Molecular markers have been available for more than 25
[25].
years, nevertheless the advent of NGS represented a break-
Since a single reference genome is not enough to represent
through in this field. Before NGS, a typical linkage map was
the diversity within a species [26], the re-sequencing of differ-
based on few hundreds markers. In the age of NGS, thousands
ent cultivars, landraces and wild accessions assumes an important
of markers can be easily included in any map, including in
role to reveal domestication events [27], identify gene diversifica-
species with little a priori genome information available. With
tion and variations [28] and explain heterosis mechanisms [29].
NGS technologies the DNA marker identification has shifted from
A most striking example is the “3,000 Rice Genomes Project”, an
fragment-based (RFLPs, AFLPs, microsatellites) to sequence-based
initiative dedicated to the re-sequencing of 3000 rice accessions
polymorphisms (SNPs). Uniplex or multiplex SNP genotyping
selected to represent the genetic and functional rice diversity avail-
platforms that combine a variety of chemistries, detection meth-
able worldwide [30]. With re-sequencing information of many
ods, and reaction formats are available. Uniplex SNP genotyping
accessions, a strategy can be applied to search for allele diversity
platforms are more suitable for applications requiring small to
at candidate gene loci for which a clear association with specific
moderate numbers of SNPs for a large number of samples.
phenotypic traits is known. This allele mining strategy can help
TaqManTM (http://www.appliedbiosystems.com) and competitive
trace the evolution of alleles, identify new useful haplotypes and
allele-specific PCR (KASPTM , http://www.lgcgenomics.com) are
guide the development of allele-specific markers for use in marker-
among the most popular techniques on the market. Multiplexed
assisted selection (MAS). For instance, following an allele mining
SNP analysis can be run on middle throughput platforms with a
approach several resistance gene homologues and functional resis-
capacity of a few hundred SNPs per run (e.g. Illumina BeadXpress,
tance genes have been isolated in potato [31], wheat [32], rice
Fluidigm EP1) or with high-throughput array-based technologies
[33,34], and barley [35].
capable of generating between a few thousand to over one million
When re-sequencing is applied to TILLING populations
SNPs per run (e.g. Illumina BeadArrayTM , Affymetrix GeneChipTM
(TILLING-by-Sequencing), it allows a fast genome-wide identifica-
technology). The advent of high-density SNP arrays coupled with
tion of mutations [36,37]. Enhanced opportunities for functional
powerful computational pipelines has allowed the fast and easy
genomics come from a genome-wide discovery pipeline of induced
scoring of large set of markers across many genotypes. Medium
mutations based on multiplexed (10- to 30- fold) exome capture
or high density arrays are available for many crop species, e.g.
and sequencing. This method called MAPS (mutations and poly-
grapevine [6], maize [7], tomato [8], peach [9], soybean [10], barley
morphisms surveyor), was used to identify about 18,000 mutations
[11], rice [12], wheat [13] and apple [14].
in 72 independent M2 rice lines, of which >2,600 appeared to be
Nevertheless, the production of a high-quality array requires
detrimental to gene function [38].
a substantial investment of resources, and the SNP panel, opti-
Beside the description of allele diversity, genome re-sequencing
mized for the population used to develop it, might be biased toward
coupled with de novo assembly of the sequences not matching
particular panels of germplasm. To circumvent these limitations,
the reference genome offers the possibility of harnessing the gene
NGS technologies offer the possibility of shifting from array-
repertoire from wild relatives of crops leading to the description
based genotyping assays with pre-defined SNP panels to the direct
of their pan-genomes. Pan genome refers to the full complement
sequencing of the populations of interest [15], producing a genome-
of genes in a group of individuals (e.g. species) and consists of a
wide and unbiased set of markers. These techniques employ
core genome containing DNA sequences shared by all the geno-
a reduced genome representation achieved through restriction
types and of a dispensable genome composed of partially shared
enzyme digestion and subsequent adaptor-mediated PCR ampli-
genomic features (i.e. present in only some genotypes) [39]. For
fication, and require no a priori knowledge of the SNPs being
instance, the re-sequencing of seven accessions of Glycine soja led
interrogated, making them useful for genetic analysis in species
to the identification in the dispensable genome of many genes that
where no reference sequence is available. Among them, restriction-
had structural variant involved in the adaptation to the environ-
site associated DNA sequencing (RAD) [16] and genotyping by
ment (R-genes, flowering time-related genes, genes involved in oil
sequencing (GBS) [17] have been adopted in plants [18–22]. Fur-
and fatty acid content), and which were therefore potentially useful
thermore, a strategy based on low coverage genome sequencing of
in crop breeding [40].
all genotypes from a segregating population (POPSEQ) was recently
Since the observed phenotypic diversity is not completely
employed for the development of high density genetic maps.
explained by variation at the genomic level, the establishment
POPSEQ was used to explore the organization of the gene space
of the pan-genome should also be supported by the analysis of
within the large, complex and highly repetitive barley genome [23],
differences in gene expression and regulation. In maize the whole-
and contributed to the assembling of the hexaploid wheat genome
seedling transcriptome variations among 503 diverse inbred lines
[24].
have been described by RNA-sequencing. Besides the pan-genome,
the analysis of the maize pan-transcriptome helps to explain some
3. Mining plant diversity: from genotype to phenotype components of phenotypic variation (e.g. heterosis) in terms of
variation in abundance of transcripts and their alternatively spliced
Many valuable genes and alleles are stored in seed bank col- forms in inbred lines [41]. In addition to gene regulation, epige-
lections, hidden in cultivars, landraces, mutagenized populations netic regulation represents another crucial factor to be considered
and wild species. The identification of these genes requires both for advanced crop breeding, especially for physiological pheno-
genome information and phenotyping capacities. With the advent types, having a fundamental role in regulating gene expression in
of NGS technologies a different dimension to the exploration of response to developmental and environment changes [42].

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Producing high throughput sequence information is only one lighted the most stable and relevant ones, a simplification of great
component of next generation breeding. A major constraint is relevance for breeding.
the ability to associate all these genomic data with systematic To ensure the accessibility and exploitation of all genome and
and robust characterization of phenotypes for a wide range of phenotypic information from a broader number of researchers
traits and conditions. Innovations in this field are expected from and breeders, it is of great value to have website portals to col-
high-throughput phenotyping platforms that employ remote sens- lect all information available for a given crop, as underway in
ing and imaging techniques (based on visible/near-infrared and rice [30] and wheat (http://wheat-urgi.versailles.inra.fr/Projects/
far-infrared radiation, reflected and emitted by the plants, respec- Wheat-Initiative/Wheat-Information-System). These meta-data
tively), and high-performance data recording and computing to collections are expected to reduce the information redundancy
evaluate plant performance in field or controlled environments. and highlight what is still missing, thereby helping to decipher
Automated greenhouse systems (LemnaTec system, http://www. the genetic variation (SNPs, insertion or deletions, structural vari-
lemnatec.com/) coupled with innovative image acquisition tech- ants) between and within different populations, and to contribute
niques (Phenoscope [43], RootReader3D [44], LemnaTec Scanalyzer in the association mapping effort by enabling the extraction of the
3D, http://www.lemnatec.de/scanalyzer gh.htm), advanced image haplotype structure and haplotype maps based on LD.
analysis pipelines (HTPheno [45]), and specific software applica-
tions (RootNav [46], Integrated Analysis Platform [47]) allow the
non-destructive recording of a wide range of phenotypic traits over 5. Marker-trait associations in large germplasm collections
time (e.g. in barley [48] and tomato [49]). Nonetheless, efforts are
still required to implement high-throughput and cost effective phe- The genetic bases of many traits have been conventionally dis-
notyping in field conditions [50] and there is a great interest in sected by linkage analysis in segregating mapping populations
drones or aircrafts as remote phenotyping sensor platforms able (e.g. Double Haploids -DHs, Recombinant Inbreed lines -RILs) or
to monitor the field trials performance throughout the growing using nearly isogenic lines (NILs) developed using several back-
season (e.g. in maize [51]). crosses [58]. Nevertheless, the estimated effects are specific to the
same or genetically related populations and are often not trans-
ferable to other genetic backgrounds, thus limiting their practical
4. Collecting genetic information through meta-analysis application for breeding purposes [58]. In the last decade, the avail-
ability of high resolution and cost effective genotyping platforms
The statistical combination of a huge amount of molecu- have opened the way to GWAS. By exploiting LD between mark-
lar and phenotypic data, obtained from publications and omics ers and traits across all chromosomes, GWAS aims at genetically
databases, provides opportunities to unravel complex traits in scrutinizing complex phenotypes in natural or ad hoc generated
crops through genome-wide meta-analysis, and is becoming a populations, and it has been widely adopted in different plant
promising approach for crop breeding. Meta-analysis, with the sup- species to overcome some of the constraints inherent to bi-parental
port of dedicated statistical procedures, enables the evaluation of linkage mapping, such as the limited genetic diversity explored
key genetic, genomic and environmental variables and their impact [59,60]. Moreover, the long history of recombination events cap-
on crop agronomic performance, by exploiting and then integrating tured in large germplasm collections, when combined with dense
datasets from different studies using various multiple method- marker coverage, permit increased genetic resolution, sometimes
ologies. Nonetheless, caution is needed in the implementation of to a level that allows a causative sequence variant to be identi-
meta-analysis to avoid biases in the interpretation of the results fied [61,62]. Nevertheless, some drawbacks have to be considered:
arising from inappropriate assumptions [52]. i) LD levels, and hence the mapping resolution, can vary not only
Of particular relevance for breeders are the QTL meta-analyses among species (e.g. selfing vs. outcrossing), but also among pop-
of QTL information, which allows QTL locations to be compared for ulations within one species and among different regions within a
a trait between populations and/or to prioritize candidate genes given genome [63–65]; ii) population structure may lead to spu-
[53]. In rice, the genome sequence was used for a meta-analysis of rious associations; and iii) the effect of rare alleles (even if large)
QTLs involved in partial and complete blast resistance. This work might not be detectable by GWAS analysis. The power of detect-
involved an analysis of the co-localization of blast resistance genes ing significant marker-trait associations depends on the quality
and QTLs and indentification of candidate genes on the reference of the phenotypic data, sample size, the genetic architecture and
genome. A few partial-resistance QTLs active against most of the heritability of the trait [61,66].
strains tested, and observable in several independent experiments, Successful examples of GWAS in crop species have been recently
were identified, and represent worthy targets for future breeding reviewed [60,62]. As a whole, these studies provide breeders
programs [54]. In bread wheat, a meta-analysis for crop height vari- with plenty of marker-traits associations that may be directly
ation loci was applied to four doubled populations with parents exploited for crop design since they are applicable to a much wider
representing a wide diversity of European winter wheat. Out of the germplasm base, provided that high LD is maintained between the
16 meta-QTLs identified in the consensus linkage map, those having causal gene and the significant markers in the breeding materi-
additive effects equivalent to height-reducing alleles (Rht-D1 and als. Despite the high number of GWAS done in crop plants, only in
Rht8) could be exploited in wheat breeding to modify crop stature few cases has the effect of an underlying candidate gene been ver-
and hence yield and biomass [55]. In another study, over 3100 ified. In fact, several independent information pieces of evidence
maize individuals from 18 bi-parental populations were genotyped are often necessary to definitively assign SNP association signals to
with the same SNP platform and evaluated in environments with genes and identify the causal mutation. These pieces of evidence
different levels of available water. The data were then summarized can include loss of function mutants [67], over-expression lines,
into 68 meta-QTLs for grain yield and anthesis silking interval. Four expression data, proteome or metabolome data [68,69], sequencing
meta-QTLs for grain yield detected under different environments of candidate genes in diverse germplasm collections, including crop
and in 6 populations were identified as promising for pyramid- wild relatives [11], and linkage mapping and map-based cloning in
ing into breeding lines [56]. In durum wheat, when more than 80 experimental populations [68].
QTLs and 51 resistance genes for powdery mildew from 62 differ- New crossing schemes and types of experimental populations
ent mapping populations were projected onto the same consensus have been suggested to overcome some of the limitations (e.g. pop-
map, they were summarized into 24 meta-QTLs [57]. This high- ulation structure) that are encountered when GWAS is run with

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panels of natural germplasm,breeding lines or varieties, and to the one that predicts GEBV most accurately, as defined by the cor-
increase the statistical power and mapping resolution with respect relation between the GEBV and the true breeding value [79]. A
to bi-parental populations, hence the ability to identify genes number of statistical models have been proposed, mainly based on
underlying phenotypic variation [70]. Multi-parent Advanced Gen- corrected linear regression, best linear unbiased prediction (BLUP)
eration Intercross (MAGIC) populations are created by intercrossing and Bayesian regression methods [81,82]. The prediction accuracy
multiple parental lines and self-crossing the progeny to generate of the different methods is debated; while in some cases all the
RILs. Multiple founders capture more allelic diversity (including models gave similar accuracy in estimation [83], other studies evi-
rare alleles) whereas the multiple cycles of intercrossing give denced how different population features (LD structure, presence
greater opportunities for recombination and hence, greater pre- of epistasis and relationship between the training and validation
cision in QTL location. In rice 4 multi-parent populations named sets) as well as trait characteristics (genetic architecture, heri-
indica-MAGIC (8 indica parents), japonica-MAGIC (8 japonica par- tability) may influence the relative performance of the prediction
ents), MAGIC-plus (extended indica MAGIC with two extra rounds methods [82,84]. Once the model providing the highest accuracy
of intermating) and Global-MAGIC (16 parents, 8 indica and 8 is identified, GS would allow selection of lines without utilization
japonica) have been developed to explore many desirable traits of phenotypic data through the model predicting the individual
(biotic/abiotic stress tolerance, yield, and grain quality) [71]. Sim- GEBVs [79,81,82,85].
ilarly, a wheat MAGIC population was developed using eight Linkage disequilibrium is a parameter of great importance
winter wheat lines selected for yield capacity, quality and dis- in the designing of a GS approach. Significant LD in outcrossed
ease resistance. The genotypic data of the 1091 F7 lines generated species extends for 0.1–1.5 kb in maize [63] and 15–20 kb for
demonstrate that the population is highly recombined making it a sorghum [86], while it extends for a considerably greater dis-
powerful tool for the genetic dissection of the characters in wheat tances in self-crossed species like rice (from 75 kb in the indica
[72]. In Nested Association Mapping (NAM) populations a number background to 500 kb in temperate japonica), or barley where LD
of founder lines are crossed with the same reference line to develop decays across 5–10 cM, representing approximately 20–40 Mb [87].
sets of related (half-sib) mapping progenies. The advantage of this LD can also vary depending on the genomic region and the pop-
population derives from the the ability to incorporate a large num- ulation structure. For species whose LD decays rapidly among
ber of alleles from the gene pool. In maize, a NAM population was unrelated individuals, a lower number of parental lines can be
developed crossing 25 inbred lines to the B73 reference line [73]. screened without lowering the detection power, and a increased
About 5000 RILs were obtained and investigated for QTLs control- number of markers can be employed. The size of the training and
ling the developmental timing of the juvenile to adult transition breeding population is also a critical issue. While larger training
[74] as well as kernel composition [75]. A similar resource is under populations improve the accuracy of GEBV predictions [88], the
development in barley from crosses of the elite barley cultivar Barke training/breeding population size ratio is suggested to be more
with 25 wild barley donors [76]. The genotyping of the 1420 BC1 S3 crucial. In general, a higher training/breeding population ratio is
lines will simultaneously enable study of the diversity of barley required for accurate GEBV prediction in case of greater genetic
wild relatives, identification of genes controlling agronomic traits diversity, smaller-sized breeding populations, lower heritability of
and the transfer of favorable exotic alleles into the elite barley gene traits and larger numbers of existing QTL [78]. Co-dominant mark-
pool. ers (e.g. SNPs, SSRs) provide a more accurate estimation of GEBV
than dominant markers (e.g. DArT markers) due to the higher LD
detection power and the accuracy can be further improved by con-
6. Genome-wide prediction of breeding value and genomic sidering haplotypes [89]. Bi-allelic markers (SNPs, DArTs, GBS and
selection RAD markers) provide individually less information than multi-
allelic ones (SSRs), and therefore require more data to achieve
There are two main strategies to assist breeding with molecular a similar accuracy. Nevertheless, the cost per data-point of the
selection: to use molecular markers that map near or within spe- different marker types makes SNPs the marker of choice for
cific loci with known phenotypic effects (marker-assisted selection, GS.
MAS) or to exploit all available markers as predictors of breeding GS has been applied to several traits in a variety of species
value (genomic selection, GS). MAS is used to drive the selection of a including maize, barley, bread wheat and rice. (Table 1). Over-
relative small set of genes having major phenotypic effects [77,78], all, these studies suggest a wide applicability of GS even for
and much information on these tools is available also through crop- species with large and/or complex genomes such as bread wheat,
dedicated websites (e.g. in wheat http://maswheat.ucdavis.edu/; sugarcane or maize. In many studies, GS has provided reliable
in barley http://www.barleycap.org/). Nevertheless, frequently the information to an extent comparable or even higher than more
success of new crop varieties is based on particular combinations traditional selection approaches; indeed the correlation between
of many small-effect loci (QTLs). In GS, all locus, haplotype and true breeding value and the GEBV has reached levels of 0.85 even
marker effects are estimated across the entire genome to calculate for polygenic low heritability traits [79]. When the response to
the genomic estimated breeding values (GEBVs) in a population genome-wide selection was assessed in comparison with MAS in
of individuals representative of the breeding program in question maize, the response to GS was significantly better, with a value
(often referred as training populations) for which both phenotypic depending on the heritability and number of considered QTLs [90].
and genotypic data are known [79,80]. Accuracy of GEBV prediction Similarly, Rutkosky et al. [91] compared GS vs. selection based on a
is strongly affected by both the marker density and the rates of LD set of QTL associated markers or vs. phenotypic selection for traits
decay across the genome. The evaluation of the inter-marker coef- related to Fusarium head blight resistance in wheat. In all compar-
ficient of determination, r2 , can provide useful information about isons the prediction accuracy was higher for GS.
the marker density required to obtain sufficient GEBV predictions Interestingly, GS provides a higher accuracy in the estimation of
[79]. The training population is used to estimate model parame- GEBV in plants than in animals, although the number of molecular
ters that will be subsequently used to calculate GEBVs of breeding markers used is generally lower. This is probably due to the nar-
materials for which only genotypic data are available and to select rower genetic base (and consequently a lower genetic diversity) of
the individuals for advancement in the breeding cycle. plant genetic materials, which are derived in many cases from a
Different statistical models are initially tested using the geno- small number of parental varieties and a greater bottleneck in the
typic and phenotypic data from the training populations to find breeding materials [80].

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Table 1
–Examples of genomic selection in plant breeding

Species Trait Reference

Maize (Zea mays L.) Grain yield, anthesis date, and anthesis-silking interval [136]
Anthesis date, grain yield, plant height under normal and water stressed conditions [22]
Barley (Hordeum vulgare L.) Fusarium head blight resistance, DON accumulation [137]
Yield, plant height, fusarium headblight resistance, DON accumulation, morphological traits, virus resistance [138,139]
Wheat (Triticum aestivum L.) Yield, thousand-kernel weight, number of kernels per spike, heading date [140]
Resistance to leaf rust, stem rust, stripe rust [141]
Rice (Oryza sativa L.) Days to heading, culm length, panicle number and length, grain length and width, brown rice length and width [83]
Yield, number of tillers per plant, number of grains per panicle, 1000 grain weight [142]
Grain yield, flowering time, plant height [21]
Oat (Avena sativa L.) !-Glucan percentage, yield, heading date, groat percentage, plant height [143]
Soybean (Glycine max L.) Seed weight [144]
Sugarcane (Saccharum officinarum L.) Sugar contents, digestibility and composition of the bagasse, plant morphology and disease resistance [83]
Sugarbeet (Beta vulgaris L.) Sugar yield and content, root yield, potassium and sodium content, "-amino nitrogen content [145]
Apple (Malus x domestica Borkh.) Fruit quality traits [146]

7. Plant improvement through genome editing for many crops will facilitate genome editing approaches for plant
improvement, although few successful examples are yet known.
Genome editing, i.e. the targeted modification of a gene, allows ZFN-mediated mutagenesis was employed to engineer tobacco
generation of new allelic variants in the genome of cultivated plants in the acetolactate synthase (ALS) genes (SuRA and SuRB)
species; it represents an alternative to standard breeding processes inducing herbicide resistance in transformed plants [97]. ZFNs were
based on recombination and, to some extent, to genetic transforma- also used for the stacking of multiple herbicide resistance genes
tion. Genome editing relies on the induction of double strand breaks (pat, aad1) in maize: the accurate targeting of the ZFN activity
in DNA in a targeted part of the genome using an engineered DNA- allowed the insertion of the two genes in close proximity, and the
binding protein. Sequence-specific nucleases, including zinc finger entire array of transgenes segregated as a single locus in subsequent
nucleases (ZFN), and transcription activator like effector nucleases generations [97]. A variety of applications are reported for TALEN
(TALEN) have been initially proposed for targeted genome editing [98]. Shan et al. [99] targeted 4 rice genes related to morphological
in eukaryotic organisms [92]. More recently, another double strand and quality traits, and 8 Brachypodium genes involved in hormone
breaks-based technology for genome editing, the CRISPR/Cas9 sys- balance and gene regulation were targeted, for TALEN-directed
tem, has been developed based on the bacterial and archaeal mutagenesis. Short deletions were most frequently obtained, but
clustered regularly interspaced short palindromic repeats (CRISPR) the use of multiple TALEN constructs targeting different sites of
adaptive immune system. The system exploits the endonuclease the same gene also allowed large deletions to be obtained. TALEN
activity of CRISPR-associated (Cas) proteins, with sequence speci- was also used to knockout the PROCERA gene involved in gibberellin
ficity directed by CRISPR RNAs (crRNAs) [92,93]. Double-strand signaling in tomato. In the progeny plants, individual carrying the
breaks at specific genomic sites can introduce a mutation at the mutations (e. g. with modified leaf shape, growth rate and intern-
DNA break site via the error-prone non-homologous end-joining ode distance) but which were missing the TALEN construct by
pathway. This is the most common system acting in plants and segregation, were selected [100].
frequently induces small insertions/deletions which result in an The CRISPR/Cas9 system was tested for inducing targeted dele-
array of mutations at the targeted gene. Double strand breaks tions in the inositol oxygenase and phytoene desaturase genes in
in multiple sites can also result in homologous recombination tobacco and wheat suspension cell cultures [101]. The construct
between chromosomal DNA and foreign donor DNA through the was targeted to one or more sites of the two genes in different
homologous recombination pathway. In this way more significant combinations and the plants obtained by cell culture regeneration
modifications of the target sequence are possible (gene stacking, showed typically 20–40 bp deletions or insertions. The deletion of
allele substitutions) [94]. To ensure specificity and a low rate of a complete gene was also possible by inducing simultaneous cleav-
off-target cleaving for this system, the most crucial point appears age in two different sites within the gene sequence, and the method
to be the careful selection of the gRNA sequence [95]. Being also allowed mutations to be introduced in multiple genes using the
based on nucleotide-nucleotide interactions, the target sequence same expression cassette. Knockout mutations in the barley MLO
for CRISPR/Cas9 system can be designed in a more predictable way gene is known to confer broad resistance to powdery mildew [102],
compared to TALEN or ZFN. In this system un-specific mutations therefore equivalent mutations accumulated in the three homeol-
can almost completely be avoided in plants. Other possible strate- ogous MLO genes of the wheat genome were expected to result in
gies to increase specificity are based on the use of dimeric, partially a similar phenotype. Indeed Wang et al. [96] used CRISPR-Cas9 for
inactivated CRISPR/Cas9 complexes, which require two precisely knocking-out the three wheat MLO genes and regenerated powdery
disposed recognition sites on the genome. These methods have mildew resistant plants.
been reported to increase specificity from tens to hundreds of times When the TALENs, ZFNs or CRISPR/Cas9 systems are engineered
compared with fully-active cleavage complexes. The regulation of to inactivate one of the two FokI complexes responsible for the dou-
Cas9 and gRNA expression is also a crucial point; indeed, overex- ble strand break, the enzymatic complex can act rather as a nickase
pression of these molecules is often related to an increased rate to producea single strand cut. Besides being a possible way for
of non-target mutations [95]. Transgenic plant lines transiently or improving specificity [95], this strategy promotes repair by homol-
stably carrying the sequence-specific nucleases (able to induce the ogous recombination as opposed to non homologous end joining,
desired mutations) can be generated using biolistics [96], Agrobac- thus allowing much more complex and accurate modifications in
terium or protoplast based methods of transformation [93]. the target sequence (i.e. gene/allele substitutions) [98,103,104].
Genome editing relies on very accurate genome sequence infor- The capacity to induce specific mutations by means of
mation for the precise determination of the target site particularly sequence specific nucleases would allows the direct modifica-
if the target gene is part of a multigene family or if duplications or tion/introduction of relevant agronomic traits into elite lines for
homeologus copies are present. Availability of genome sequence breeding. Nevertheless, a critical question is whether these tar-

Please cite this article in press as: D. Barabaschi, et al., Next generation breeding, Plant Sci. (2015),
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geted DNA modifications might be regarded as genetic modified damage. The treated F1 hybrid is then self-pollinated or back-
organism (GMO) events for regulation. Although this is still being crossed and the resultant F2 progenies are searched for the desired
debated [105–108], one common sense indication, discussed in recombination events [120]. A further complication in manipu-
detail by Araki and Ishii [109], suggests that genome editing lating homoeologous recombination is caused by polyploidization
events not based on the integration of a heterologous DNA (i.e. [121]. Indeed in polyploid species there are factors that ensure that
small insertions/deletions induced by non homologous end join- chromosome pairing occurs only between homologous chromo-
ing mechanisms), should not be regulated as GMOs. Instead, events somes and not homoeologous chromosomes. This diploidization
generated through homologous recombination with a foreign tem- mechanism also affects the ability to exploit interesting character-
plate sequence, especially in the case of a whole gene, are expected istics from wild species in breeding programs [122]. A well known
to result in an organism containing recombinant DNA, and there- gene affecting homoeologous recombination in wheat is Pairing
fore they should be considered as GMOs. homoeologous 1 (Ph1), which inhibits pairing between homoeol-
When deep and accurate knowledge is available concerning ogous chromosomes [123–125]. In the absence of Ph1, pairing
metabolic pathways of primary importance in crops (including and recombination between homoeologous chromosomes is fre-
information about the molecular mechanisms responsible for and quent, facilitating introgressive hybridization. If the constitutive
regulating these processes), all the technologies grouped under the deletion of Ph1 can over time lead to rearranged chromosomes in
definition of “genome editing” can give a significant contribution the genome, the knock-down of these genes, for example using RNA
in the design and development of “ad hoc” metabolically optimized interference (RNAi) would bring great benefits for plant breeding
crop lines, thanks to synthetic biology approaches [110]. [126].
Although breeding is based on meiotic recombination, in spe-
cific cases there is an interest in blocking recombination to fix
8. The control of genetic recombination aheterozygous state. Many crops are cultivated as heterozygous F1
hybrids, that carry a unique combination of alleles and outperform
Even with all the new available technologies, plant breeding their parents due to hybrid vigour. The allele combinations that
still depends on recombination. New genes/alleles are required to make F1 unique are broken by recombination when the F1 is selfed.
be recombined into advanced lines and despite the great number The application of a new strategy, known as reverse breeding,
of markers available, recombination is still required to give new allows genetic preservation of any selected fertile plant through
allele combinations for tightly linked loci. It is therefore essential seeds even if its genetic composition is unknown and if vegeta-
to develop tools capable of increasing crossover incidence to break tive propagation is not applicable [127]. The method is based on
negative allele associations. reducing homologous recombination in the selected heterozygote
To ensure proper segregation at metaphase I, each pair of chro- by eliminating meiotic crossing-over. This is done using RNAi con-
mosomes have at least one crossover known as an obligatory structs targeting genes for proteins involved in the formation of
crossover. Nevertheless, the presence of a crossover inhibits the crossovers, such as DISRUPTED MEIOTIC CDNA1 (DMC1) [128]. The
formation of a new crossover nearby on the chromosome and elite heterozygote is transformed using the RNAi construct and the
this inhibition decreases with the distance (crossover interference) resulting plant is expected to produce low numbers of viable bal-
[111]. Furthermore, the crossovers are not uniformly distributed anced haploid spores. These are regenerated into doubled haploid,
along chromosome: some regions, termed as hot spots, show much perfectly homozygous, plants. The resulting doubled haploids dif-
higher frequencies of crossover than cold spot areas. In species fer genetically solely as a consequence of the independent parental
such as barley, wheat or maize, the crossovers occur much more in chromosome assortment which occurred during meiosis. There-
the distal part of chromosome than in the pericentromeric regions fore, it is sufficient to make use of one co-dominant, polymorphic
[112–114]. Analysis of the crossover distribution along chromo- marker per chromosome to determine which of the lines should
some 3B of bread wheat showed a meiotic recombination gradient be combined through crossing to reconstruct the genetic composi-
from the centromere to the telomeres on both arms. The two distal tion of the original starting plant. The technique however is limited
regions were characterized by an elevated meiotic recombination to crops in which spores can be regenerated into double haploids.
rate (of 0.60 and 0.96 cM/Mb) which was clearly distinct from the In polyploids or species with high chromosome numbers, another
low meiotic recombination rate (of 0.05 cM/Mb) observed in the hybrid reconstruction method based on plants regenerated from
large proximal region spanning the centromeric–pericentromeric unreduced spores, named Near Reverse breeding, has been pro-
area [115]. Different studies are underway to understand the mech- posed [129].
anisms (and the genes) regulating the formation of crossovers to A reproductive mechanism that can fix favorable allelic com-
allow the control of their frequency and position [116]. For exam- binations obtained through meiotic recombination is represented
ple, it was shown that a mutation of the Arabidopsis FANCM gene by the apomixes, a process producing genetically identical off-
results in a substantial increase of meiotic crossover formation, spring via seed without the intervention of a second parent. This
without negative impacts on chromosome stability [117]. process offers some advantages for fixing desirable complex geno-
Crossover localization is partially determined by the presence types, such as high yielding F1 hybrids [130]. In crops apomixis
of particular chromosome structures, such as tandem repeats, is rare and cross pollination is not suitable for transferring the
short terminal deletions and translocations. For example, when a apomixis trait [131]. Nevertheless the engineering of apomictic
chromosome carries a distal translocation, this causes a strong dis- crops, through targeted manipulations of reproduction, although
similarity at the chromosome end resulting in a shift of crossovers challenging, could have a great value as a breeding technology
to interstitial chromosome segments [118]. Other internal and [132]. Even though the genes controlling apomixis have not yet
external factors can impact crossover incidence. For instance, been identified and many studies are focusing on a possible epige-
barley cultivars with different genetic background (internal fac- netic regulation, some genes identified from natural apomicts may
tor) showed up to 30% difference in crossover frequencies, while switch the sexual pathway of crops to apomixis [130].
chemical/physical treatments (external factor) with actinomycin
D, diepoxybutane and/or radiations gave a large increase (from 9. Conclusions
two- to seven fold) in recombination frequencies in Arabidop-
sis [119,120]. The authors proposed to apply a chemical/physical Current breeding programs rely on integrating phenotypic
treatment to a F1 hybrid that results in DNA modifications or selection in standard breeding schemes (e.g. pedigree, backcross,

Please cite this article in press as: D. Barabaschi, et al., Next generation breeding, Plant Sci. (2015),
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Fig. 1. Schematic representation of the key elements of a “next generation breeding”. The combination of genetic resources, NGS technologies, bioinformatics capacities and
automatic phenotyping facilities will revolutionize the traditional breeding strategies moving to a more efficient genetic improvement.

progeny test for combinatory efficiency) with molecular inputs (e.g. only require some phenotyping to maintain and increase the accu-
MAS and genetic transformation for GM plants). The availability racy of the prediction models.
of NGS, bio-informatics resources and phenotyping platforms is Once genes and alleles responsible for traits are identified,
moving plant breeding a step forward and a next generation breed- molecular and computational tools can be applied to potentially
ing strategies resulting from combining of genetic resources with gain an understanding of the evolutionary processes that have
advanced technologies can be foreseen for the near future (Fig. 1). shaped their current diversity in the genepool. The exploitable
The first effect of the NGS revolution is to drop the marker value of these genes/allele from unadapted germplasm for pre-
cost per data point. SSRs or CAPS markers run on agarose gels breeding purposes could also be determined. For example,
or capillary sequencer are much more expensive than SNPs run knowledge of haplotype of favourable alleles present in elite cul-
on high-throughput platforms. As a result, while in the past only tivars will help to identify other (including superior) alleles from
markers in critical genomic regions were employed to follow really diverse landraces and wild relatives. With the help of agronomists
important traits, nowadays markers are used to assess the inheri- and crop (eco)-physiologists, the optimal combinations of traits, or
tance of as many loci as possible across the entire genome and with so-called ideotype, might be now defined by gene (network) mod-
nucleotide-level precision [5]. elling. The approach of molecular crop design has the opportunity
NGS technologies are giving a relevant contribution for the to improve the speed and efficiency of breeding programs for sev-
characterization of plant genetic resources. A worldwide effort is eral species of agricultural interest. In rice, for example, a number of
currently focused on understanding the genetic bases of agronomic genes have been identified and characterized in detail, which influ-
traits, analyzing allelic variants at the corresponding loci and pro- ence flowering time, reviewed by Matsubara et al., 2014 [133]. It
viding catalogs of allele series for the most important loci, thus has been suggested and observed that different allelic combinations
enabling the breeders to select the most appropriate allele combi- of this kind of genes may influence the geographical distribu-
nations. The massive accumulation of QTL information is paving the tion of this crop [133,134]. Therefore this information might be
way for more accurate and powerful meta-analyses, allowing con- exploited to breed new early flowering rice varieties, in order
sistent genetic determinants of quantitative traits to be identified. to further facilitate the expansion northward of the cultivation
Meanwhile, the availability of low cost markers is facilitating the region.
introgression into elite cultivars of specific loci/QTLs from landraces In disease resistance breeding, mechanisms of resistance-
or wild accessions while limiting the negative effects of linkage drag suppression were frequently observed in several crop species when
[5]. different resistance alleles were stacked in pyramided lines, result-
The exploitation of a genome-wide approach such as GS is ing in a significant limitation of the gene-pyramiding approach
becoming feasible and would help to design the new plant not only [135].
for a few selected traits but for virtually all loci in the genome, Although genetic resources, from elite cultivars to landraces and
in a cost effective and relatively fast way, promising to give a wild relatives, will remain the foundation of any breeding pro-
remarkable contribution to the concept of next generation breed- gram, it is expected that in the near future the application of NGS
ing. Application of the GS procedure would eliminate the need for technologies, bioinformatics and automatic phenotyping tools for
extensive multi-location field trials at each generation, and would the characterization and subsequent exploitation of genetic diver-

Please cite this article in press as: D. Barabaschi, et al., Next generation breeding, Plant Sci. (2015),
http://dx.doi.org/10.1016/j.plantsci.2015.07.010
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sity will revolutionize breeding strategies to achieve more efficient clusters of yield-related loci and orthology with the tomato genome, PLoS
genetic improvement. One 9 (2014).
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This work was supported by FP7 project WHEALBI (Wheat and Jannink, S.R. McCouch, Genomic selection and association mapping in rice
(Oryza sativa): effect of trait genetic architecture, training population
barley Legacy for Breeding Improvement). composition, marker number and statistical model on accuracy of rice
genomic selection in elite, tropical rice breeding lines, PLoS Genet. 11 (2015)
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Glossary genome sequencing is performed on the whole population, and a medium-

density framework genetic map is calculated. SNPs and associated sequencing
CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats/Cas9(CRISPR- contigs are then integrated into the framework map through nearest-neighbor
associated) is a tool based on the reprogramming of CRISPR/Cas9 endonuclease search.
activity normally acting in prokaryote cells to target a specific sequencing, RAD: Restriction-site Associated DNA sequences short genomic DNA regions sur-
using short non-coding RNAs designed on the basis of the targeted sequence. rounding most restriction sites of a given restriction endonuclease. To achieve
Cas9 nuclease is used for genome engineering applications, and a single guide this, the restriction fragments are randomly sheared and fragments with a length
RNA (sgRNA) homologous to a target sequence is used to drive the CRISPR/Cas suitable for the NGS platform of choice are selected after size fractionation and
complex and to induce desired mutations (insertions/deletions). The target subjected to a PCR reaction designed to amplify for sequencing only those frag-
specificity is given by the sgRNA which forms the editing complex with the ments containing the selected restriction site. SNPs are called by comparing DNA
Cas enzyme, and whose sequence is engineered ad hoc. of different genotypes using dedicated bioinformatic pipeline.
GBS: Genotyping By Sequencing is genotyping method involving the digestion of TALEN: Transcription Activator-Like Effector Nucleases are a class of DNA-binding
genomic DNA with a frequent cutter restriction endonuclease and the sequenc- proteins produced by plant pathogens of the genus Xanthomonas, which during
ing of the ends of the resulting restriction fragments with a NGS platform. infection deliver the proteins to plant cells aimed at increasing the susceptibil-
Adaptors containing barcodes and common adaptors (without barcodes) are ity to the pathogen infection. A central portion of TALE proteins that contains
mixed and used in the ligation reaction. Due to the specificity the NGS system as many as 30 tandem repeats of a 33-35-amino-acid-sequence motif responsi-
only small fragments (between 100 and 250 bases) featuring a barcode-common ble for DNA binding, while the nuclease activity is executed by a FokI nuclease
adapter combination are yielded allowing simultaneous marker discovery and domain.
genotyping. SNPs are called by comparing DNA of different genotypes using TILLING: Targeting Induced Local Lesions IN Genomes is a high-throughput reverse
dedicated bioinformatic pipeline. genetic technique for the discovery of mutations in a specific gene. Mutations are
GWAS: Genome-wide association studies aims at the detection and fine mapping induced through chemical mutagenesis (usually by ethyl methane sulfonate -
of quantitative trait loci (QTLs) underlying complex agronomic traits thought EMS) and detected through enzymatic cleavage of heteroduplex DNA molecules
linkage disequilibrium (LD) analysis. GWAS exploits ancestral recombination or through whole genome/exome re-sequencing (TILLING by sequencing).
events that occurred in existing natural populations and takes into account all ZFN: Zinc Finger Nucleases are engineered enzymatic complexes developed by fus-
major alleles present in the population to identify significant marker-phenotype ing the non-specific cleavage domain from the FokI restriction endonuclease
associations. In comparison to LD studies commonly performed on bi-parental (responsible for the induction of DNA double strand cuts) with custom-designed
populations, due to many more historical recombination events occurred in the Cys2-His2 zinc-finger proteins, able to guide the whole complex onto the desired
population, a much higher mapping resolution is achieved. position on the genome.
POPSEQ: Population Sequencing is based on genome sequencing of a segregating
population that allows de novo production of a genetically anchored linear
assembly of the gene space. A high coverage whole-genome shotgun is gen-
erated for one parent, and used to construct a gene space assembly on which
gene models are defined using RNA-seq. In parallel, a low coverage whole

Please cite this article in press as: D. Barabaschi, et al., Next generation breeding, Plant Sci. (2015),
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