**2. QTL mapping and backcrossing**

## **2.1 Identification of the molecular basis of technological properties: QTL mapping**

Numerous properties and phenotypes of wine yeasts are quantitative traits. These present continuous variations among individuals, in opposition to qualitative ones showing discrete variations. Those quantitative traits are due to complex genetic mechanisms, often linked to interactions between several loci. It is possible to identify the genetic determinants of such phenotypes using a QTL mapping. A quantitative trait locus is defined as a region of the genome, often scattered, associated with the phenotypic variation of a quantitative trait. The first study using the principles of QTL was done almost 100 years ago [28]. At the time, Sax [28] performed a genetic analysis correlating the size of beans with the color of pigmentation. Shortly after, the concept was applied to agriculture and since then has been widely used in many different organisms such as *Drosophila melanogaster* [29] and *Arabidopsis thaliana* [30], in crops [31], and in yeast [32, 33].

Thanks to those approaches, chromosomic regions, genes, or even mutations, responsible for several wine yeast properties, have been deciphered. These include traits like acetic acid production, sporulation, ethanol tolerance, growth at high temperature, flocculation, wine aroma production, amino acid consumption, nitrogen requirement, fermentative performances, and sulfur compound production [34–45]. These studies have shown some phenotypes to be particularly complex.

The QTL mapping method is divided into three steps. First, a recombinant population is constituted, second, this population is then phenotyped and genotyped, and, lastly, a statistical analysis to link the regions of the genome to the phenotypes is performed (**Figure 1**).

The recombinant population is usually constituted from a hybrid obtained by crossing two parental strains, selected based on their phenotypic diversity. We can note that it is also possible to start directly with a highly heterozygous diploid parental strain, e.g., selected after evolutionary engineering. The hybrid is induced to sporulate to generate a population of meiotic segregants. The meiotic segregants passed through recombination so that each segregant possesses a random distribution of the alleles of the two parents. As the recombination rate is a crucial point in the accuracy of the final mapping of the QTL, it is also possible to generate an F2 segregant population to increase the allelic mixing. In that case, the initial meiotic segregant population, F1, is submitted to random crossing before a second sporulation round to constitute the F2 haploid segregant population [42, 43].

The phenotyping of the segregant population is a crucial step that can be limiting in the QTL approach. Each segregant has to be phenotyped individually for the trait of interest. The higher the number of segregants that are phenotyped, the

**Figure 1.**

*Schematic representation of the QTL mapping strategy divided in three steps: (1) constitution of the meiotic segregants population, (2) phenotypic and genotypic study, and finally (3) linkage analysis.*

better the precision will be in the mapping of the QTL. Some phenotypes can be measured on plates, such as ethanol tolerance; however, numerous phenotypes of interest for wine yeasts require to perform enological fermentations.

The next step is to create a genetic map constituted by molecular markers differentiating the two parental strains. The aim is to obtain the most homogeneous and dense distribution of the markers throughout the genome. The better the coverage is, the more accurate and precise the QTL mapping will be. Then, genotyping of the segregants attributes a parental origin to each marker. Nowadays, the development of sequencing approaches and the reduction of their costs allow to genotype the strains using whole-genome re-sequencing implementing next-generation sequencing technology [46]. This is done for parental strains as well as for the selected segregants.

Different approaches of QTL mapping can be carried out, using individual genotyping or bulk segregant analysis (BSA). For the individual genotyping, each segregant is genotyped, and a linkage analysis identifies the regions that are more likely to be involved in the phenotype. The powerful method of interval mapping is often used [47]. This method is based on the distances between markers. For each marker, the probability that this locus is a true QTL is calculated by a model. A significant threshold can be established by permutation testing. This approach is based on the hypothesis of a single QTL, but it is possible to identify other QTLs by a composite interval mapping that will iteratively scan the genome and add known QTL to the regression model as QTLs are identified.

In the BSA approach, the segregants that present the same phenotype are pooled together [48, 49]. The aim is to identify the regions of the genome that are common to all the segregants presenting the same phenotype. The allelic frequency between the two bulks or with the control is studied and allows detecting gene variants involved in the phenotype.

**137**

**Figure 2.**

*Yeast Strain Optimization for Enological Applications DOI: http://dx.doi.org/10.5772/intechopen.86515*

production [42, 51, 52].

and a strain to improve (**Figure 2**).

A powerful extension of BSA has also been developed: extreme QTL (X-QTL) mapping [50]. This approach is based on the generation of segregating populations of very large size. Those populations composed of large numbers of progeny with extreme trait values can be constituted using selection for drug or stress resistance

The genomic regions identified by QTL mapping strategies can vary from few to 1000 kilobases. Inside those regions, the sequences of the genes are compared, and non-synonymous mutations between the parental strains are searched in the coding region and the promotor/terminator regions. A study of the function of the genes located in this region using databases allows identifying candidate genes. A functional validation of the candidate genes can then be performed. Allelic replacement and reciprocal hemizygosity analysis (RHA) are the common ways to validate the impact of an allele on the phenotype. Allelic replacement consists in deleting the candidate gene in a parental strain and replacing by the allele of the opposite parent. Hemizygotes are constructed using the hybrid of the parental strains and deleting only one copy of the gene. The obtained strains are tested for their phenotypes. Thanks to those approaches, genes, mutations, or even translocations have been validated for diverse wine yeast properties, such as lag phase duration, fermentation capacity under nitrogen starvation, and ester

**2.2 Transferring properties of interest from one strain to another: backcrossing**

Once markers or mutations have been identified thanks to a QTL mapping strategy, it is possible to manage their transfer from one wine yeast strain to another. Introgression, also called backcrossing, or selection assisted by molecular markers, consists in recursive hybridization between a strain possessing the allele of interest

*Schematic representation of backcrossing cycles or recursive hybridization between the receptor strain (parental strain 1, in red) and the donor strain (parental strain 2, in green). The molecular markers (green cross) are followed at each step by PCR. The final strain (in orange) possesses a major part of its genome coming from the* 

*receptor strain and a small part transferred from the donor and containing the region of interest.*

or by cell sorting. Pooled allelic frequencies are then determined.

*Yeast Strain Optimization for Enological Applications DOI: http://dx.doi.org/10.5772/intechopen.86515*

*Advances in Grape and Wine Biotechnology*

better the precision will be in the mapping of the QTL. Some phenotypes can be measured on plates, such as ethanol tolerance; however, numerous phenotypes of

*Schematic representation of the QTL mapping strategy divided in three steps: (1) constitution of the meiotic* 

The next step is to create a genetic map constituted by molecular markers differentiating the two parental strains. The aim is to obtain the most homogeneous and dense distribution of the markers throughout the genome. The better the coverage is, the more accurate and precise the QTL mapping will be. Then, genotyping of the segregants attributes a parental origin to each marker. Nowadays, the development of sequencing approaches and the reduction of their costs allow to genotype the strains using whole-genome re-sequencing implementing next-generation sequencing technology [46]. This is done for parental strains as well as for the selected segregants. Different approaches of QTL mapping can be carried out, using individual genotyping or bulk segregant analysis (BSA). For the individual genotyping, each segregant is genotyped, and a linkage analysis identifies the regions that are more likely to be involved in the phenotype. The powerful method of interval mapping is often used [47]. This method is based on the distances between markers. For each marker, the probability that this locus is a true QTL is calculated by a model. A significant threshold can be established by permutation testing. This approach is based on the hypothesis of a single QTL, but it is possible to identify other QTLs by a composite interval mapping that will iteratively scan the genome and add known

In the BSA approach, the segregants that present the same phenotype are pooled together [48, 49]. The aim is to identify the regions of the genome that are common to all the segregants presenting the same phenotype. The allelic frequency between the two bulks or with the control is studied and allows detecting gene variants

interest for wine yeasts require to perform enological fermentations.

*segregants population, (2) phenotypic and genotypic study, and finally (3) linkage analysis.*

QTL to the regression model as QTLs are identified.

**136**

**Figure 1.**

involved in the phenotype.

A powerful extension of BSA has also been developed: extreme QTL (X-QTL) mapping [50]. This approach is based on the generation of segregating populations of very large size. Those populations composed of large numbers of progeny with extreme trait values can be constituted using selection for drug or stress resistance or by cell sorting. Pooled allelic frequencies are then determined.

The genomic regions identified by QTL mapping strategies can vary from few to 1000 kilobases. Inside those regions, the sequences of the genes are compared, and non-synonymous mutations between the parental strains are searched in the coding region and the promotor/terminator regions. A study of the function of the genes located in this region using databases allows identifying candidate genes. A functional validation of the candidate genes can then be performed. Allelic replacement and reciprocal hemizygosity analysis (RHA) are the common ways to validate the impact of an allele on the phenotype. Allelic replacement consists in deleting the candidate gene in a parental strain and replacing by the allele of the opposite parent. Hemizygotes are constructed using the hybrid of the parental strains and deleting only one copy of the gene. The obtained strains are tested for their phenotypes. Thanks to those approaches, genes, mutations, or even translocations have been validated for diverse wine yeast properties, such as lag phase duration, fermentation capacity under nitrogen starvation, and ester production [42, 51, 52].

#### **2.2 Transferring properties of interest from one strain to another: backcrossing**

Once markers or mutations have been identified thanks to a QTL mapping strategy, it is possible to manage their transfer from one wine yeast strain to another. Introgression, also called backcrossing, or selection assisted by molecular markers, consists in recursive hybridization between a strain possessing the allele of interest and a strain to improve (**Figure 2**).

#### **Figure 2.**

*Schematic representation of backcrossing cycles or recursive hybridization between the receptor strain (parental strain 1, in red) and the donor strain (parental strain 2, in green). The molecular markers (green cross) are followed at each step by PCR. The final strain (in orange) possesses a major part of its genome coming from the receptor strain and a small part transferred from the donor and containing the region of interest.*

The first step is the selection of a "receptor" strain. This strain possesses a good genetic background and presents numerous properties of interest, except the one aimed to be enhanced. This strain will be crossed with a "donor" strain that possesses the property of interest. A first cross results in a hybrid possessing 50% of the genome of each parental strain. This hybrid is induced to sporulate, and a population of meiotic segregants is constituted. A segregant with the right marker or allele of interest is selected using a simple identification by PCR. This segregant is crossed again with the receptor strain. The second hybrid possesses 75% of the genome of the receptor strain and 25% of the donor strain. Several cycles of breeding/sporulation are performed to regenerate the genome of the receptor strain and to recover its good properties. Generally, four cycles are sufficient and lead to a strain possessing more than 93% of the genome of the receptor strain and less than 7% from the donor, including the genes of interest.

This approach has been implemented in plants for many years [53, 54]. Its application to the improvement of wine yeasts has started more than 10 years ago [14] and since then it has been applied to generate numerous wine yeast strains The production of H2S, lag phase, and POF character [14], volatile thiol release [55], or SO2, H2S, and acetaldehyde [56] have been improved using this approach.
