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Genome-wide association studies GWAS and linkage mapping are two methods used to establish genotype-phenotype relationships. GWAS relies on differences within a population of diverse, unrelated individuals in order to discover correlations between markers and traits. In comparison, linkage mapping exploits bi-parental crosses to map traits in the resulting progeny. One of the main advantages of GWAS over traditional linkage mapping is its superior mapping resolution. GWAS markers correlated with a phenotype are likely to be very close to the causal locus. In some cases, the likely causal genetic variant itself can be identified through GWAS Migicovsky et al.
In linkage mapping, large genomic intervals, often spanning millions of nucleotides, are identified while the causal genetic variant is unlikely to be pinpointed. GWAS is particularly promising in perennials because of the time and cost required to generate bi-parental crosses. An additional benefit is that GWAS can be applied to germplasm collections that are already in the ground and waiting to be exploited Chitwood et al. The discrepancy in mapping resolution between the two methods is a function of the number of recombination events captured by each method. In GWAS, a large number of unrelated individuals means that a large number of recombination events have occurred in the history of the genetic material being assessed.
In linkage mapping, only the recombination events captured through the generation of the bi-parental cross can be exploited, resulting in relatively large chunks of DNA that share co-ancestry among individuals. The high mapping resolution offered by GWAS is amplified in many perennials because of the relatively rapid linkage disequilibrium LD decay in high-diversity perennial crops.
For example, LD decays within bp in grape Lijavetzky et al. The correlation between a marker and a causal variant is related to the level of LD between the two: While rapid LD decay results in high mapping resolution, it also means that a very high density of markers is required for effective GWAS because the correlation among markers surrounding the causal variant decays so quickly. In some cases, generating sufficient coverage for GWAS by saturating the genome with markers may be prohibitively expensive due to rapid LD decay.
Genome Mapping and Molecular Breeding in Plants
However, the cost of marker discovery and genotyping is likely to continue to decrease, and it will therefore surely be feasible in the future for researchers to acquire the genotype data required for effective GWAS. While GWAS in perennials is an attractive option, it is not always viable. Traits targeted by breeders are often present only within a wild relative species, and are completely absent within cultivated germplasm. Attempts to map such a trait in a population composed of the wild relative and the cultivated germplasm using GWAS would be futile because the trait co-segregates perfectly with ancestry.
The marker you aim to uncover will be present in the wild relative but absent in the cultivated germplasm, but that is also the case for millions of other markers across the genome Figure 3. When the phenotypes are perfectly segregated, GWAS is of no help and a bi-parental cross between the wild and cultivated populations must be made to genetically map the trait. Linkage mapping in the resulting bi-parental population allows for such co-segregating traits to be genetically mapped, because the confounding effects of population structure are broken through crossing.
Thus, when mapping traits of interest found only in wild relatives, linkage mapping studies may be necessary due to co-segregation. However, it is sometimes the case that wild and domesticated germplasm share segregating polymorphism and are not significantly genetically differentiated, as is the case with apple and grape Figure 1. In such instances, the confounding effects of co-ancestry may not be too severe and GWAS may be the genetic mapping option of choice. Additionally, when a phenotype is not perfectly co-segregated with ancestry, but rather differentially expressed in the two populations, it may be possible to perform GWAS using wild and domesticated plants.
In this scenario, including both population structure and the SNP-by-population interaction in the GWAS model would help avoid false positives and ensure that SNPs are consistently associated with the trait across wild and domesticated populations Biscarini et al. For each crop and phenotype of interest, the optimal genetic mapping approach, and the desired genetic composition of the population, will vary.
Comparison of the effectiveness of genome-wide association studies GWAS and linkage mapping for mapping alleles of interest in wild relatives. When an allele of interest is found only in wild germplasm it co-segregates with population structure and cannot be mapped using GWAS. Linkage mapping provides a viable alternative for mapping traits in wild relatives.
However, in the F1 generation, alleles homozygous for alternative states in the wild and cultivated parent will not segregate. Thus, a backcross, or pseudo-backcross, is required to map most alleles of interest. For co-segregating traits, linkage mapping provides a viable alternative to GWAS. In annual crops, it is typically performed through a cross of highly homozygous parents, often as a result of selfing.
In perennials, the severe inbreeding depression and high level of heterozygosity require a mapping design in which parents are not selfed. As an alternative, the two-way pseudo-testcross design, in which two highly heterozygous parents are crossed, has been successfully applied in many perennials, beginning in with an interspecific Eucalyptus cross Grattapaglia and Sederoff, However, the progeny resulting from a two-way pseudo-testcross will not segregate for markers homozygous for alternative alleles in the parental plants Figure 3.
Given that many wild traits of interest will likely fall into this category, mapping will require at least one generation of backcrossing before linkage mapping can be applied. However, many perennials also have high levels of inbreeding depression, so close relatives cannot be used when performing backcrosses. Instead, a cultivar that is not one of the parents from the initial cross should be used to perform pseudo-backcrossing.
The combination of a two-way pseudo-testcross design and pseudo-backcrossing can enable the detection of markers for valuable traits in wild perennial relatives. When introgressing regions of the genome associated with a phenotype, or quantitative trait locus QTL , from wild germplasm, linkage drag may lead to undesirable phenotypes in the resulting progeny.
Linkage drag is the result of unfavorable genes linked to a desirable QTL also being incorporated into the domesticated germplasm Varshney et al. Additional generations of pseudo-backcrossing can reduce the effects of linkage drag. If undesirable loci are tightly linked to the locus of interest, it may be difficult to eliminate the impact of linkage drag through conventional breeding. Fine-mapping of a QTL can allow for the selection of individuals with specific recombination events that minimize linkage drag.
Unfortunately, fine mapping requires generating a large number of crosses for sufficient recombination Khan and Korban, Reduced recombination frequencies have also been reported surrounding loci introgressed for resistance from a related species, such as a fold reduction in poplar Populus spp. As a result, the fine-mapping process is both expensive and time-consuming Khan and Korban, Once a recombinant individual is identified, they can be used as a donor in breeding and backcrossing can continue for several generations using MAS.
In addition to eliminating linkage drag, fine-mapping may lead to the identification of causal alleles which can be subsequently incorporated into the genomes of domesticated crops through genetic modification GM or genome editing techniques. These techniques can be applied directly to the cultivar of interest, immediately incorporating the trait, and do not require multiple generations of backcrossing to eliminate linkage drag.
This is especially valuable in perennial crops with a lengthy juvenile phase or infertile hybrid progeny. Previous work successfully generated transgenic bananas with resistance to Fusarium wilt, the major pathogen threatening banana production Paul et al. Similarly, transgenic plantains Musa spp. The first successfully backcrossed PRSV-resistant papaya was only reported in , after 50 years of attempts Siar et al. Instead, for almost two decades, papaya with transgenic resistance to PRSV have been cultivated in Hawaii Gonsalves et al.
Thus, GM is a valuable tool that can expedite the breeding of disease-resistant cultivars. Clearly, incorporation of desirable traits from wild relatives into perennial crops is not limited to MAS, but can also be achieved through GM. However, the social and regulatory acceptance of GM crops, including papaya outside of Hawaii, is often limited Davidson, Acceptance of GM perennials is especially difficult since many are fruit crops that are consumed fresh.
Additionally, unlike genome editing, MAS remains useful when precise detection of causal loci is not possible and only markers highly correlated with the trait of interest are available. The simple distinction between GWAS and linkage mapping is useful, but experimental designs that blur this distinction, and exploit the benefits of both methods, are uncovering numerous genotype-phenotype associations. The increased level of recombination in the progeny allows for improved precision of mapping using inbred offspring Cavanagh et al.
In perennials, where the creation of inbred lines is often not possible, other designs have been implemented. For example, work in apple made use of a factorial mating design consisting of four female parents and two pollen parents Kumar et al. This family-based design allowed for the discovery of markers for traits such as fruit firmness, internal browning, and titratable acidity, which could be implemented in MAS Kumar et al. Therefore, alternative mating designs are a promising tool for increased mapping resolution when performing linkage mapping between wild and domesticated crops.
The limited diversity—often a single bi-parental cross—exploited in traditional linkage mapping results in a mapping population where many QTL will not segregate and therefore not be detected. Further, due to a potentially small population size, small-effect QTL may not exceed the significance threshold. Significant markers identified are often only relevant to populations that share significant co-ancestry with the parents of the bi-parental mapping population.
Thus, in comparison to GWAS, markers discovered using linkage mapping may not be predictive in diverse collections of germplasm Owens, However, when identifying a marker for a trait from a wild relative, it is only necessary that the marker functions within that population, as a single source can be used as a donor for MAS. For example, while several sources of PD resistance have been used in grape breeding, the most important donor has been from a single V. Given that a single wild individual possessing a desirable trait is often sufficient for introgression into elite cultivars through MAS, transferability is of limited concern when exploiting alleles derived from a single wild relative.
A form of genomics-assisted breeding that is increasingly being used for complex traits is genomic selection GS. GS is particularly useful when the breeder aims to predict a complex trait controlled by numerous QTL. In these cases, a small number of markers will not be sufficient for phenotype prediction. Many economically important traits, such as fruit quality, are polygenic and therefore controlled by a large number of loci.
In comparison, GS uses all marker data as well as phenotype data from a population to predict a genomic estimated breeding value GEBV for an individual. Once a model has been validated, GEBVs can be calculated using only genotype information. However, while particular markers for MAS can be used to track a trait of interest across multiple generations, as breeding populations evolve, GS requires additional rounds of phenotyping in order to maintain an accurate prediction model Varshney et al.
Additionally, in contrast to MAS, GS requires genotyping a large number of markers, which may still be cost-prohibitive in many breeding programs. Such a strategy may benefit many perennial crops when introgressing multiple traits from wild relatives, especially to allow for durable disease resistance Kumar et al.
There are many tools and designs for genetic mapping and implementation of genomics-assisted breeding. The decision of which strategy to employ will vary depending on the genetic architecture of the trait as well as the genetic structure of the mapping and breeding populations. Similarly, the specific tool for introgression of markers is a complex decision that will require weighing factors such as the urgency of developing a new cultivar, the extent of linkage drag, and the acceptance of GM technology.
While the optimal combination of genomic tools will differ by crop, the adoption of genomics-assisted breeding will ultimately enable breeders to efficiently and cost-effectively incorporate desirable traits that would otherwise remain locked away in wild germplasm. Despite the immense potential of wild relatives for improving perennial crops, the first step to exploiting this resource through genomics-assisted breeding is discovering markers linked to useful phenotypes.
While the genetic divergence between cultivated germplasm and wild relatives is precisely why wild relatives offer such unique and diverse traits, it may also cause difficulties for marker discovery and breeding. For example, when relatives differ in ploidy levels or total chromosome number, it may be difficult to produce fertile interspecific hybrids. The domesticated grape, V.
However, progeny from V. Despite occasional sterility, successful pseudo-backcrossing occurred for six subsequent generations, allowing for the introgression of the M. When fertile hybrids are still not possible and a causal locus has been identified, genome editing provides a viable alternative for introgression of valuable traits from wild germplasm. In addition to the difficulties potentially associated with crossing more distant relatives, wild germplasm may have higher levels of diversity, and as such, DNA sequencing and genotyping tools designed for domesticated species may not function as successfully.
For example, SNP arrays are widely used in humans, but do not function as well on organisms with greater genetic diversity because they are designed based on a reference genome. In this case, hybridization intensities were more useful than genotype calls for genetic mapping precisely because of the probe-sequence hybridization issues caused by high levels of genetic divergence across grape species Myles et al. Thus, when mapping in high diversity perennial crops with SNP arrays such as grape Myles et al.
The simultaneous discovery and genotyping of markers eliminates the need for DNA to hybridize to previously designed probes and makes NGS well-suited to high diversity species as well as wild relatives. Despite the proliferation of reference genome sequences, there is a lack of reference genomes for wild relatives.
More than plant genomes were sequenced between and , but only 15 were wild relatives and over half of those were soybean Michael and VanBuren, Thus, there is a clear need for reference genomes in wild relatives in order to map sequence reads allowing for the detection of SNPs for downstream analyses, ultimately allowing for genomics-assisted breeding.
While more genomic resources are still needed for wild species, the number of reference genomes available has continued to increase. Resequencing of several Citrus species including oranges, pummelos, and mandarins enabled researchers to determine the contributions of various wild progenitor species to cultivated citrus Wu et al. Yet, in many cases, resequencing may not be sufficient for the detection of crucial genomic differences between wild and cultivated crops. Within species, a large portion of the genome is present in only a subset of individuals. For example, transcriptome sequencing in maize was used to determine that only The divergence between wild relatives and cultivated plants is likely much greater.
As a result, the genomic region of interest in a wild relative may be a sequence not present in the domesticated crop.
Genome Mapping and Molecular Breeding in Plants - NHBS
DNA sequences present only in wild relatives require de novo assembly rather than resequencing to be mapped. The improvement of genomic resources, such as de novo assembly of wild relative reference genomes, can enable the discovery of markers for MAS and GS. Finally, most sequencing results in some degree of missing data in the final table of genotypes. Missing sequence data can be filled in using imputation. However, imputation generally requires that genomic data be aligned to a reference genome.
Popular imputation softwares, including Beagle Browning and Browning, and fastPhase Scheet and Stephens, , rely on the input of SNPs ordered according to a reference genome, which is not possible for many wild relatives with limited genomic resources. Several methods such as Random Forest and k -nearest neighbors imputation kNNI can be used when a reference genome is not available Nazzicari et al.
LinkImpute is an imputation software based on kNNI, which updates the method to use linkage between markers rather than distance between samples when calculating neighbors. When compared to existing imputation methods, LinkImpute had a similar run time and accuracy to Beagle, despite not requiring positional information for markers Money et al. As the ability to impute missing data without a reference genome improves, reduced representation sequencing techniques with high missing data, such as GBS, will continue to facilitate the discovery of new markers for genomics-assisted breeding in wild relatives.
While there is opportunity for great improvement to elite perennial crops through genomics-assisted introgression of traits from wild relatives, many barriers remain. Genomic tools designed for domesticated species are either not well-suited to more diverse wild relatives, or may be lacking completely. The same genetic divergence that has resulted in wild relatives harboring unique and desirable traits for breeding also results in difficulties in developing markers to introgress these traits into elite germplasm.
However, given that DNA sequencing costs are likely to continue decreasing, it is essential that researchers begin planning for a future where the collection and analysis of DNA sequence data will not be the bottleneck to successful genetic mapping. Especially for perennial breeders used to working on timescales of decades, the focus should be on the collection of high-quality phenotype data that can always be paired later with genotype data as it becomes available.
Now is the time to establish GWAS and linkage mapping populations that will enable powerful genetic mapping in a future where genotyping costs are negligible and the available genomic analysis tools are far superior to those available today. Although the primary focus of this review is the use of genomics, it is worth noting that there are several difficulties unrelated to genomics that may limit the use of improvement using wild relatives.
First, in order to make use of wild relatives for breeding, new germplasm must be collected. While some wild relative collections are well-characterized and actively in use, such as those described in this review, there are likely many benefits of wild germplasm that remain undiscovered. A focus on the collection and characterization of wild germplasm is the first step towards discovering which relatives and traits will be useful for breeding, and thus be exploitable through genomics.
Among the major barriers to improved characterization of wild germplasm are the locations where such germplasm may be found. Often, wild relatives must be collected from locations that are difficult to access, and thus collecting new wild germplasm can be an expensive and time-consuming process. For example, wild cacao is found in the tropical rainforests of South America Lachenaud et al.
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There are also compulsory quarantine requirements when transferring material between political boundaries. Several decades may pass between the collection of wild germplasm and their use by growers Lachenaud et al. Finally, it is important to consider the cultural and financial ramifications of collecting wild relatives.
In the past, germplasm has been collected from farmers and communities without compensation or recognition. In such a scenario, seeds may be taken from one country and used to benefit the private sector in another country. While there is ample opportunity for commercial crops to benefit from wild relatives, it is necessary that farmers and communities which have preserved wild relatives receive adequate credit and compensation for use of such resources Montenegro, The introgression of valuable wild traits into domesticated crops can only occur when breeders have access to these relatives through gene banks.
The collection of new samples for marker discovery poses a major limitation to establishing such collections. Wild relatives are very under-represented in gene bank collections. Future collection of germplasm is also threatened due to habitat destruction and climate change Maxted et al. As the power of genomic tools increases, genomics will become increasingly effective for introgression of wild traits into perennial crops.
However, the ability to exploit wild relatives for breeding requires that this diversity be protected for future use through gene banks and habitat conservation. Preservation of wild relatives will require a complex approach across many environments on a local, national and international scale Montenegro, It is crucial to begin exhaustive sampling and extensive evaluation of wild germplasm for all major perennial crops, an enormously expensive and time-consuming undertaking.
However, such projects are essential to ensuring a safe and secure future food supply as clonally propagated cultivars continue to be threatened by a constantly evolving environment. An essential step toward the adoption of genomic markers from wild relatives will be methods that accelerate the juvenile period in order to increase the efficiency of backcrossing progeny to domesticated germplasm.
While the use of genomics-assisted breeding can increase the efficiency of selecting for traits of interest and decrease the number of plants that must be propagated, the long juvenile period of many perennials still poses a constraint on the rate of crop improvement. A solution to the problem of long juvenile periods has been found in grapes.
In comparison to the 2—5 years of juvenility generally required for grapes, the Vvgai1 mutant produces fruit 2 months after germination. For example, recent work used microvines to aide in QTL identification for traits such as berry acidity Houel et al. In apple, an early flowering transgenic line containing the BpMADS4 gene from silver birch Betula pendula was combined with MAS to pyramid resistance to apple scab, powdery mildew, and fire blight Flachowsky et al.
However, while transgenic lines are incredibly helpful for decreasing the generation time while breeding, it is often desirable to have a final cultivar for release that does not contain the transgene and is not considered a GMO. Thus, once the rapid cycling of generations is completed, a non-GMO tree possessing desirable traits from wild relatives—but not the transgene—can easily be selected Flachowsky et al.
The creation of similar mutants in other species, which reduce the juvenile phase in long-lived perennials, will be essential to the efficient application of MAS. As an alternative to transgenics, virus-induced gene silencing VIGS can also be used to shorten the juvenile phase in perennials. VIGS uses a viral vector to infect a plant with a particular gene, resulting in an RNA-mediated defense which silences expression of the gene within the plant Lu et al.