Molecular breeding (MB) may be defined in a broad-sense as the use of genetic manipulation
performed at DNA molecular levels to improve characters of interest in plants and animals,
including genetic engineering or gene manipulation, molecular marker-assisted selection, genomic selection, etc. More often, however, molecular breeding implies molecular marker-assisted breeding (MAB) and is defined as the application of molecular biotechnologies,
specifically molecular markers, in combination with linkage maps and genomics, to alter and improve plant or animal traits on the basis of genotypic assays. This term is used to describe several modern breeding strategies, including marker-assisted selection (MAS), marker-assisted backcrossing (MABC), marker-assisted recurrent selection (MARS), and genome-wide selection (GWS) or genomic selection (GS) (Ribaut et al., 2010).
MAB is a well-known procedure for the introgression of a target gene (or genes) from a donor line into the genomic background of a recipient line. Critical factors for the success of MAB include the number of target genes, the distance between the flanking markers and the target gene (2-20 cM), and the number of genotypes selected in each BC generation (Ribaut, 2002).
Marker-assisted breeding involves the following activities :-
a. Planting the breeding populations with potential segregation for traits of interest or
polymorphism for the markers used.
b. Sampling plant tissues, usually at early stages of growth, e.g. emergence to young seedling
c. Extracting DNA from tissue sample of each individual or family in the populations, and
preparing DNA samples for PCR and marker screening.
d. Running PCR or other amplifying operation for the molecular markers associated with
or linked to the trait of interest.
e. Separating and scoring PCR/amplified products, by means of appropriate separation
and detection techniques, e.g. PAGE, AGE, etc.
f. Identifying individuals/families carrying the desired marker alleles.
g. Selecting the best individuals/families with both desired marker alleles for target traits
and desirable performance/phenotypes of other traits, by jointly using marker results
and other selection criteria.
h. Repeating the above activities for several generations, depending upon the association
between the markers and the traits as well as the status of marker alleles (homozygous
or heterozygous), and advancing the individuals selected in breeding program until stable
superior or elite lines that have improved traits are developed.
Marker-assisted selection (MAS) refers to such a breeding procedure in which DNA marker
detection and selection are integrated into a traditional breeding program. Taking a single
cross as an example, the general procedure can be described as follow:
a. Select parents and make the cross, at least one (or both) possesses the DNA marker allele(
s) for the desired trait of interest.
b. Plant F1 population and detect the presence of the marker alleles to eliminate false hybrids.
c. Plant segregating F2 population, screen individuals for the marker(s), and harvest the
individuals carrying the desired marker allele(s).
d. Plant F2:3 plant rows, and screen individual plants with the marker(s). A bulk of F3 individuals
within a plant row may be used for the marker screening for further confirmation
in case needed if the preceding F2 plant is homozygous for the markers. Select and
harvest the individuals with required marker alleles and other desirable traits.
56 Plant Breeding from Laboratories to Fields
e. In the subsequent generations (F4 and F5), conduct marker screening and make selection
similarly as for F2:3s, but more attention is given to superior individuals within homozygous
lines/rows of markers.
f. In F5:6 or F4:5 generations, bulk the best lines according to the phenotypic evaluation of
target trait and the performance of other traits, in addition to marker data.
g. Plant yield trials and comprehensively evaluate the selected lines for yield, quality, resistance and other characters of interest.
MAS for major genes or improvement of qualitative traits
In crop plants, many economically important characteristics are controlled by major genes/
QTLs. Such characteristics include resistance to diseases/pests, male sterility, self-incompatibility and others related to shape, color and architecture of whole plants and/or plant parts. These traits are often of mono- or oligogenic inheritance in nature. Even for some quality traits, one or a few major QTLs or genes can account for a very high proportion of the phenotypic variation of the trait (Bilyeu et al., 2006; Pham et al., 2012). Transfer of such a gene to a specific line can lead to tremendous improvement of the trait in the cultivar under development. The marker loci which are tightly linked to major genes can be used for selection and are sometimes more efficient than direct selection for the target genes. In some cases, such advantages in efficiency may be due to higher expression of the marker mRNA in such cases that the marker is actually within a gene. Alternatively, in such cases that the target gene of interest differs between two alleles by a difficult-to-detect SNP, an external marker of which polymorphism is easier to detect, may present as the most realistic option.
Soybean cyst nematode (SCN) (Heterodera glycines Inchinoe) may be taken as an example of MAS for major genes.
Marker-assisted or marker-based backcrossing (MABC) is regarded as the simplest form of
marker-assisted selection, and at the present it is the most widely and successfully used
method in practical molecular breeding. MABC aims to transfer one or a few genes/QTLs of
interest from one genetic source (serving as the donor parent and maybe inferior agronomically or not good enough in comprehensive performance in many cases) into a superior cultivar or elite breeding line (serving as the recurrent parent) to improve the targeted trait. Unlike traditional backcrossing, MABC is based on the alleles of markers associated with or linked to gene(s)/QTL(s) of interest instead of phenotypic performance of target trait. The general procedure of MABC is as follow, regardless of dominant or recessive nature of the target trait in inheritance:
a. Select parents and make the cross, one parent is superior in comprehensive performance
and serves as recurrent parent (RP), and the other one used as donor parent (DP)
should possess the desired trait and the DNA markers allele(s) associated with or
linked to the gene for the trait.
b. Plant F1 population and detect the presence of the marker allele(s) at early stages of
growth to eliminate false hybrids, and cross the true F1 plants back to the RP.
c. Plant BCF1 population, screen individuals for the marker(s) at early growth stages, and
cross the individuals carrying the desired marker allele(s) (in heterozygous status) back
to the RP. Repeat this step in subsequent seasons for two to four generations, depending
upon the practical requirements and operation situations as discussed below.
d. Plant the final backcrossing population (e.g. BC4F1), and screen individual plants with
the marker(s) for the target trait and discard the individuals carrying homozygous
markers alleles from the RP. Have the individuals with required marker allele(s) selfed
and harvest them.
e. Plant the progenies of backcrossing-selfing (e.g. BC4F2), detect the markers and harvest
individuals carrying homozygous DP marker allele(s) of target trait for further evaluation
Applications of MABC
Success in integrating MABC as a breeding approach lies in identifying situations in which
markers offer noticeable advantages over conventional backcrossing or valuable complements to conventional breeding effort. MABC is essential and advantageous when:
1. Phenotyping is difficult and/or expensive or impossible;
2. Heritability of the target trait is low;
3. The trait is expressed in late stages of plant development and growth, such as flowers,
fruits, seeds, etc.
4. The traits are controlled by genes that require special conditions to express;
5. The traits are controlled by recessive genes; and
6. Gene pyramiding is needed for one or more traits.
Among the molecular breeding methods, MABC has been most widely and successfully used
in plant breeding up to date. It has been applied to different types of traits (e.g. disease/pest resistance, drought tolerance and quality) in many species, e.g. rice, wheat, maize, barley, pear millet, soybean, tomato, etc. (Collard et al., 2005; Dwivedi et al., 2007; Xu, 2010).
Marker-assisted gene pyramiding and marker-assisted recurrent selection
Marker-assisted gene pyramiding (MAGP) is one of the most important applications of
DNA markers to plant breeding. Gene pyramiding has been proposed and applied to enhance
resistance to disease and insects by selecting for two or more than two genes at a time.
For example in rice such pyramids have been developed against bacterial blight and blast
(Huang et al., 1997; Singh et al., 2001; Luo et al., 2012). Castro et al. (2003) reported a success in pyramiding qualitative gene and QTLs for resistance to stripe rust in barley. The advantage of using markers in this case allows selecting for QTL-allele-linked markers that have the same phenotypic effect. To enhance or improve a quantitatively inherited trait in plant breeding, pyramiding of multiple genes or QTLs is recommended as a potential strategy
(Richardson et al., 2006). The cumulative effects of multiple-QTL pyramiding have been proven in crop species like wheat, barley and soybean (Richardson et al., 2006; Jiang et al.,
2007a, 2007b; Li et al., 2010; Wang et al., 2012).
Marker-based breeding and conventional breeding: Challenges and perspectives
Marker-assisted breeding became a new member in the family of plant breeding as various
types of molecular markers in crop plants were developed during the 1980s and 1990s. The
extensive use of molecular markers in various fields of plant science, e.g. germplasm evaluation, genetic mapping, map-based gene discovery, characterization of traits and crop improvement, has proven that molecular technology is a powerful and reliable tool in genetic
manipulation of agronomically important traits in crop plants. Compared with conventional
breeding methods, MAB has significant advantages:
a. MAB can allow selection for all kinds of traits to be carried out at seedling stage and
thus reduce the time required before the phenotype of an individual plant is known.
For the traits that are expressed at later developmental stages, undesirable genotypes
can be quickly eliminated by MAS. This feature is particularly important and useful for
some breeding schemes such as backcrossing and recurrent selection, in which crossing
with or between selected individuals is required.
b. MAB can be not affected by environment, thus allowing the selection to be performed
under any environmental conditions (e.g. greenhouse and off-season nurseries). This is
very helpful for improvement of some traits (e.g. disease/pest resistance and stress tolerance)
that are expressed only when favorable environmental conditions present. For
low-heritability traits that are easily affected by environments, MAS based on reliable
markers tightly linked to the QTLs for traits of interest can be more effective and produce
greater progress than phenotypic selection.
c. MAB using co-dominance markers (e.g. SSR and SNP) can allow effective selection of
recessive alleles of desired traits in the heterozygous status. No selfing or test crossing
is needed to detect the traits controlled by recessive alleles, thus saving time and accelerating
d. For the traits controlled by multiple genes/QTLs, individual genes/QTLs can be identified
and selected in MAB at the same time and in the same individuals, and thus MAB
is particularly suitable for gene pyramiding. In traditional phenotypic selection, however,
to distinguish individual genes/loci is problematic as one gene may mask the effect
of additional genes.
e. Genotypic assays based on molecular markers may be faster, cheaper and more accurate
than conventional phenotypic assays, depending on the traits and conditions, and
thus MAB may result in higher effectiveness and higher efficiency in terms of time, resources
and efforts saved.