**5. Marker-assisted backcrossing**

lines that were homozygous for all eight marker alleles linked to the genes/loci of resistance to soybean mosaic virus (SMV). These lines exhibited resistance to SMV strains G1 and G7 and presumably carried all three resistance genes (*Rsv1*, *Rsv3* and *Rsv4*) that would poten‐

Most of the important agronomic traits are polygenic or controlled by multiple QTLs. MAS for the improvement of such traits is a complex and difficult task because it is related to many genes or QTLs involved, QTL x E interaction and epistasis. Usually, each of these genes has a small effect on the phenotypic expression of the trait and expression is affected by environmental conditions. Phenotyping of quantitative traits becomes a complex endeav‐ or consequently, and determining marker-phenotype association becomes difficult as well. Therefore, repeated field tests are required to accurately characterize the effects of the QTLs and to evaluate the stability across environments. The QTL x E interaction reduces the effi‐

Despite a tremendous amount of QTL mapping experiments over the past decade, applica‐ tion and utilization of the QTL mapping information in plant breeding has been constrained

**1.** Strong QTL-environmental interaction which make phenotyping difficult since expres‐

**2.** Lack of universally valid QTL-marker associations applicable across populations. The notion that QTL mapping to identify new QTL markers whenever a new germplasm is

**3.** Deficiencies in QTL statistical analysis which lead to either overestimation or underesti‐

**4.** Often times, there are no QTLs with major effects on the trait and this means a large number of QTLs have to be identified and in many cases this becomes a tough goal to

In order to improve the efficiency of MAS for quantitative traits, appropriate field experi‐ mental designs and approaches have to be employed. Attention should be given to replica‐ tions both over time and space, consistency in experimental techniques, samplings and evaluations, robust data processing and statistical analysis. For example, composite interval mapping (CIM) allows the integration of data from different locations for joint analysis to estimate QTL-environment interaction so that stable QTLs across environments can be iden‐ tified. A saturated linkage map enables accurate identification of both targeted QTLs as well as linked QTLs in coupling and repulsion linkage phases. In practical breeding for improve‐ ment of a quantitative trait, usually not many minor QTLs are considered but only a few major QTLs are used in MAS. In case many QTLs especially minor-effect QTLs are involved, a breeder would prefer to consider the strategy of gene pyramiding (see the later section).

ciency of MAS and epistasis can result in a skewed QTL effect on the trait.

tially provide broad and durable resistance to SMV.

**4.3. MAS for improvement of quantitative traits**

60 Plant Breeding from Laboratories to Fields

by a number of factors (Collard and Mackill, 2008):

sion may vary from one location/year to another;

used, puts some people off and they lose interest in MAS;

mation of the number of QTLs involved and their effect on the trait;

achieve and further complicates identification of marker-QTL association.

#### **5.1. MABC procedure and theoretical and practical considerations**

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 agronom‐ ically or not good enough in comprehensive performance in many cases) into a superior cul‐ tivar 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:


Theoretically, the proportion of the RP genome after n generations of backcrossing is given by 1 – (1/2)n+1 for a single locus and [1 – (1/2)n+1] k for k loci, respectively, for a population large enough in size (or with adequate individuals) and no selection being made during backcrossing (i.e. "blind" backcrossing only). The percentage of the RP genome is the aver‐ age of the population, with some individuals possessing more of the RP genome than oth‐ ers. To fully recover the genome of the RP, 6-8 generations of backcrossing is needed typically in case no selection is made for the RP. However, this process is usually slower than expected for the target gene-carrier chromosome, i.e. linkage drag, especially in case a linkage exists between the target gene and other undesirable traits. On the other hand, the process of introgression of QTLs/genes and recovery of the RP genome may be accelerated by selection using markers flanking QTLs and evenly spaced markers from other chromo‐ somes (i.e. unlinked to QTLs) of the RP (Collard et al., 2005) or selection for the performance of the RP conducted simultaneously. For MABC program, therefore, there are two types of selection recognized: Foreground selection and background selection (Hospital, 2003).

In foreground selection, the selection is made only for the marker allele(s) of donor pa‐ rent at the target locus to maintain the target locus in heterozygous state until the final backcrossing is completed. Then the selected plants are selfed and the progeny plants with homozygous DP allele(s) of selected markers are harvested for further evaluation and release. As described above, this is the general procedure of MABC. The effective‐ ness of foreground selection depends on the number of genes/loci involved in the selec‐ tion, the marker-gene/QTL association or linkage distance and the undesirable linkage to the target gene/QTL.

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 agronom‐ ically or not good enough in comprehensive performance in many cases) into a superior cul‐ tivar 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

**a.** Select parents and make the cross, one parent is superior in comprehensive perform‐ ance 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

**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, depend‐

ing 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

**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 evalua‐

Theoretically, the proportion of the RP genome after n generations of backcrossing is given

large enough in size (or with adequate individuals) and no selection being made during backcrossing (i.e. "blind" backcrossing only). The percentage of the RP genome is the aver‐ age of the population, with some individuals possessing more of the RP genome than oth‐ ers. To fully recover the genome of the RP, 6-8 generations of backcrossing is needed typically in case no selection is made for the RP. However, this process is usually slower than expected for the target gene-carrier chromosome, i.e. linkage drag, especially in case a linkage exists between the target gene and other undesirable traits. On the other hand, the process of introgression of QTLs/genes and recovery of the RP genome may be accelerated by selection using markers flanking QTLs and evenly spaced markers from other chromo‐ somes (i.e. unlinked to QTLs) of the RP (Collard et al., 2005) or selection for the performance of the RP conducted simultaneously. For MABC program, therefore, there are two types of selection recognized: Foreground selection and background selection (Hospital, 2003).

k for k loci, respectively, for a population

target trait in inheritance:

62 Plant Breeding from Laboratories to Fields

and harvest them.

tion and release.

by 1 – (1/2)n+1 for a single locus and [1 – (1/2)n+1]

linked to the gene for the trait.

In background selection, the selection is made for the marker alleles of recurrent parent in all genomic regions of desirable traits except the target locus, or selection against the unde‐ sirable genome of donor parent. The objective is to hasten the restoration of the RP genome and eliminate undesirable genes introduced from the DP. The progress in recovery of the RP genome depends on the number of markers used in background selection. The more mark‐ ers evenly located on all the chromosomes are selected for the RP alleles, the faster recovery of the RP genome will be achieved but larger population size and more genotyping will be required as well. In addition, the linkage drag also can be efficiently addressed by back‐ ground selection using DNA markers, although it is difficult to overcome in a traditional backcrossing program.

Foreground selection and background selection are two respective aspects of MABC with different foci of selection. In practice, however, both foreground and background selection are usually conducted in the same program, either simultaneously or successively. In many cases, they can be performed alternatively even in the same generation. The individuals that have the desired marker alleles for target trait are selected first (foreground selection). Then the selected individuals are screened for other marker alleles again for the RP genome (back‐ ground selection). It is understandable to do so because selection of the target gene/QTL is the essential and only critical point for backcrossing program, and the individuals that do not have the allele of target gene will be discarded and thus it is not necessary to genotype them for other traits.

The efficiency of MABC depends upon several factors, such as the population size for each generation of backcrossing, marker-gene association or the distance of markers from the target locus, number of markers used for target trait and RP background, and undesirable linkage drag. Based on simulations of 1000 replicates, Hospital (2003) pre‐ sented the expected results of a typical MABC program, in which heterozygotes were selected at the target locus in each generation, and RP alleles were selected for two flanking markers on target chromosome each located 2 cM apart from the target locus and for three markers on non-target chromosomes. As shown in Table 2, a faster recov‐ ery of the RP genome could be achieved by MABC with combined foreground and background selection, compared to traditional backcrossing. Therefore, using markers can lead to considerable time savings compared to conventional backcrossing (Frisch et al., 1999; Collard et al., 2005).


**Table 2.** Expected results of a MABC program with combined foreground and background selection used; Adapted from Hospital (2003).

In a MABC program, the population to be analyzed should contain at least one genotype that has all favorable alleles for a particular QTL. Later, the number of QTLs may be in‐ creased progressively, but not beyond six QTLs in most cases because of prohibitive difficul‐ ty in handling all QTLs (Hospital, 2003). In addition, the more QTLs/genes are transferred, the larger the proportion of unwanted genes would be due to linkage drag. In general, most of the unwanted genes are located on non-target chromosomes in early BC generations, and are rapidly removed in subsequent BC generations. On the contrary, the quantity of DP genes on the target chromosome decreases much more slowly, and even after generation BC6 many of the unwanted donor genes are still located on the target chromosome in segre‐ gating state (Newbury, 2003). Given a total genome length is 3000 cM, 1% donor DNA frag‐ ments after six backcrosses represents a 30 cM chromosomal segment or region, which may host many unwanted genes, especially if the DP is a wild genetic resource. Young and Tanksley (1989) genotyped a collection of tomato varieties in which the resistance gene was previously transferred at the *Tm*-2 locus with RLFP markers. Their data indicated that the size of chromosomal segment retained around the *Tm*-2 locus during backcross breeding was very variable, with one line exhibiting a donor segment of 50 cM after 11 backcrosses and other one possessing 36 cM donor segment after 21 backcrosses. This clearly demon‐ strates the need for background selection.

As discussed above, linkage drag can be reduced by performing background selection. Typi‐ cally, two markers flanking the target gene are used, and the individuals (or double re‐ combinants) that are heterozygous at the target locus and homozygous for the recipient (RP) alleles at both flanking markers are selected. Use of closer flanking markers leads to more effective and faster reduction of linkage drag compared to distant markers. However, less distance between two flanking markers implies less probability of double recombination, and thus larger populations and more genotyping are needed. In order to optimize genotyp‐ ing effort (i.e. the cost of the program), therefore, it is important to determine the minimal population sizes necessary to ensure the desired genotypes can be obtained. Hospital and Decoux (2002) developed a statistical software for determining the minimum population size required in BC program to identify at least one individual that is double-recombinant with heterozygosity at target locus and homozygosity for recurrent parent alleles at flanking marker loci. In addition, for closely-linked flanking markers, it is unlikely to obtain double recombinant genotypes through only one generation of backcrossing. Therefore, additional backcrossing should be conducted. For instance, in one BC generation (e.g. BC1) single re‐ combination on one side of the target gene is selection, and single recombination on the oth‐ er side may be selected in another BC generation (e.g. BC2) (Young and Tanksley 1989). In this way, individuals with desired RP alleles at two flanking markers and donor allele at tar‐ get locus can be finally obtained.

To accelerate the recovery of RP genome on non-target chromosomes, scientists suggested using markers in backcrossing and discussed how many makers should be used (Tanksley et al., 1989; Hospital et al. 1992; Visscher et al. 1996). In background selection, the ap‐ proaches involve selecting individuals that are of homozygous recipient type at a collection of markers located on non-carrier chromosomes. From a point of both effectiveness and effi‐ ciency, it is important to determine an appropriate number of markers to be used. More markers do not necessarily mean better benefits in practice. Generally, several markers are involved and MABC should be performed over two or more generations. It is unlikely that the selection objective can be realized in a single BC generation.

Dense marker coverage of non-target chromosomes is not mandatory to increase the overall proportion of recurrent parent genome, unless fine-mapping of specific chromosome re‐ gions is highly important. An appropriate number of markers and optimal position on chro‐ mosomes are important. Computer simulation suggested that for a chromosome of 100 cM, two to four markers are sufficient, and selection based on markers would be most efficient if the markers are optimally positioned along the chromosomes (Servin and Hospital, 2002). In practice, at least two or three markers per chromosome are needed, and every chromosome should be involved. In such a MABC scheme, three to four generations of backcrossing is generally enough to achieve more than 99% of the recurrent parent genome. With respect to the time necessary to release new varieties, the gain due to background selection can be eco‐ nomically valuable. In addition, background selection is more efficient in late BC genera‐ tions than in early BC generations. For example, if a BC breeding scheme is conducted over three successive BC generations and yet the preference is to genotype individuals only once, then it is more efficient to genotype and select the individuals in BC3 generation rather than in the BC1 generation (Hospital et al. 1992, Ribaut et al. 2002).

#### **5.2. Application of MABC**

**Backcross generation**

from Hospital (2003).

**Number of individuals**

64 Plant Breeding from Laboratories to Fields

strates the need for background selection.

**% homozygosity of recurrent parent**

BC1 70 38.4 60.6 79.0 75.0 BC2 100 73.6 87.4 92.2 87.5 BC3 150 93.0 98.8 98.0 93.7 BC4 300 100.0 100.0 99.0 96.9

**All other chromosomes**

**Table 2.** Expected results of a MABC program with combined foreground and background selection used; Adapted

In a MABC program, the population to be analyzed should contain at least one genotype that has all favorable alleles for a particular QTL. Later, the number of QTLs may be in‐ creased progressively, but not beyond six QTLs in most cases because of prohibitive difficul‐ ty in handling all QTLs (Hospital, 2003). In addition, the more QTLs/genes are transferred, the larger the proportion of unwanted genes would be due to linkage drag. In general, most of the unwanted genes are located on non-target chromosomes in early BC generations, and are rapidly removed in subsequent BC generations. On the contrary, the quantity of DP genes on the target chromosome decreases much more slowly, and even after generation BC6 many of the unwanted donor genes are still located on the target chromosome in segre‐ gating state (Newbury, 2003). Given a total genome length is 3000 cM, 1% donor DNA frag‐ ments after six backcrosses represents a 30 cM chromosomal segment or region, which may host many unwanted genes, especially if the DP is a wild genetic resource. Young and Tanksley (1989) genotyped a collection of tomato varieties in which the resistance gene was previously transferred at the *Tm*-2 locus with RLFP markers. Their data indicated that the size of chromosomal segment retained around the *Tm*-2 locus during backcross breeding was very variable, with one line exhibiting a donor segment of 50 cM after 11 backcrosses and other one possessing 36 cM donor segment after 21 backcrosses. This clearly demon‐

As discussed above, linkage drag can be reduced by performing background selection. Typi‐ cally, two markers flanking the target gene are used, and the individuals (or double re‐ combinants) that are heterozygous at the target locus and homozygous for the recipient (RP) alleles at both flanking markers are selected. Use of closer flanking markers leads to more effective and faster reduction of linkage drag compared to distant markers. However, less distance between two flanking markers implies less probability of double recombination, and thus larger populations and more genotyping are needed. In order to optimize genotyp‐ ing effort (i.e. the cost of the program), therefore, it is important to determine the minimal population sizes necessary to ensure the desired genotypes can be obtained. Hospital and Decoux (2002) developed a statistical software for determining the minimum population size required in BC program to identify at least one individual that is double-recombinant with heterozygosity at target locus and homozygosity for recurrent parent alleles at flanking

**% recurrent parent genome**

**Conventional backcross**

**Marker-assisted backcross**

**alleles at selected markers**

**Chromosome with target locus**

> Success in integrating MABC as a breeding approach lies in identifying situations in which markers offer noticeable advantages over conventional backcrossing or valuable comple‐ ments to conventional breeding effort. MABC is essential and advantageous when:


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 re‐ sistance, 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). In maize, for example, *Bacillus thuringiens* is a bacterium that produces insecticidal toxins, which can kill corn borer larvae when they ingest the toxins in corn cells (Ragot et al. 1995). The integration of the *Bt* transgene into various corn genetic backgrounds has been achieved by using MABC. Ar‐ oma in rice is controlled by a recessive gene which is due to an eight base-pair deletion and three single nucleotide polymorphism in a gene that codes for betaine aldehyde dehydrge‐ nase 2 (Bradbury et al., 2005a). This discovery allows identification of the aromatic and non-ar‐ omatic rice varieties and discriminates homozygous recessive and dominant as well as heterozygous individuals in segregating population for the trait. MABC has been used to se‐ lect for aroma in rice (Bradbury et al. 2005b). High lysine *opaque2* gene in corn was incorporat‐ ed using MABC (Babu et al. 2005). However, the rate of success decreases when large numbers of QTLs are targeted for introgression. Sebolt et al. (2000) used MABC for two QTL for seed pro‐ tein content in soybeans. However, only one QTL was confirmed in BC3F4:5. When that QTL was introduced in three different genetic backgrounds, it had no effect in one background. In tomato, Tanksley and Nelson (1996) proposed a MABC strategy, called advanced backcross-QTL (AB-QTL), to transfer resistance genes from wild relative/unadapted genotype into elite germplasm. The strategy has proven effective for various agronomically important traits in to‐ mato, including fruit quality and black mold resistance (Tanksley and Nelson, 1996; Bernacchi et al., 1998; Fulton et al., 2002). In addition, AB-QTL has been used in other crop species, such as rice, barley, wheat, maize, cotton and soybean, collectively demonstrating that this strategy is effective in transferring favorable alleles from the wild/unadapted germplasm to elite germ‐ plasm (Wang and Chee, 2010; Concibido et al., 2003).

In barley, a marker linked (0.7 cM) to the *Yd2* gene for resistance to barley yellow dwarf virus was successfully used to select for resistance in a backcrossing scheme (Jefferies et al., 2003). Compared to lines without the marker, the BC2F2-derived lines carrying the linked marker had lighter leaf symptoms and higher yield when infected by the virus. In maize, marker-facilitat‐ ed backcrossing was also successfully employed to improve complex traits such as grain yield. Using MABC, six chromosomal segments each in two elite lines, Tx303 and Oh43, were trans‐ ferred into two widely used inbred lines, B73 and Mo17, through three generations of back‐ crossing followed by two selfing generations. Then the enhanced lines with better performance were selected based on initial evaluations of testcross hybrids. The single-cross hybrids of enhanced B73 x enhanced Mo17 out-yielded the check hybrids by 12-15% (Stuber et al., 1999). Zhao et al. (2012) reported that a major quantitative trait locus (named *qHSR1*) for re‐ sistance to head smut in maize was successfully integrated into ten high-yielding inbred lines (susceptible to head smut). Each of the ten high-yielding lines was crossed with a donor pa‐ rent Ji 1037 that contains *qHSR1* and is completely resistant to head smut, followed by five gen‐ erations of backcrossing to the respective recurrent parents. In BC1 through BC3 only phenotypic selection was conducted to identify highly resistant individuals after artificial in‐ oculation. In BC4phenotypic selection, foreground selection and recombinant selection were conducted to screen for resistant individuals with the shortest *qHSR1* donor regions. In BC5, phenotypic selection, foreground selection and background selection were performed to iden‐ tify resistant individuals with the highest proportion of the recurrent parent genome, fol‐ lowed by one generation of self-pollination to obtain homozygous genotypes at the *qHSR1* locus. The ten improved inbred lines all showed substantial resistance to head smut, and the hybrids derived from these lines also showed a significant increase in the resistance. Semagn et al. (2006b) provided a detail review on the progress and prospects of MABC in crop breeding.

**4.** The traits are controlled by genes that require special conditions to express;

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 re‐ sistance, 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). In maize, for example, *Bacillus thuringiens* is a bacterium that produces insecticidal toxins, which can kill corn borer larvae when they ingest the toxins in corn cells (Ragot et al. 1995). The integration of the *Bt* transgene into various corn genetic backgrounds has been achieved by using MABC. Ar‐ oma in rice is controlled by a recessive gene which is due to an eight base-pair deletion and three single nucleotide polymorphism in a gene that codes for betaine aldehyde dehydrge‐ nase 2 (Bradbury et al., 2005a). This discovery allows identification of the aromatic and non-ar‐ omatic rice varieties and discriminates homozygous recessive and dominant as well as heterozygous individuals in segregating population for the trait. MABC has been used to se‐ lect for aroma in rice (Bradbury et al. 2005b). High lysine *opaque2* gene in corn was incorporat‐ ed using MABC (Babu et al. 2005). However, the rate of success decreases when large numbers of QTLs are targeted for introgression. Sebolt et al. (2000) used MABC for two QTL for seed pro‐ tein content in soybeans. However, only one QTL was confirmed in BC3F4:5. When that QTL was introduced in three different genetic backgrounds, it had no effect in one background. In tomato, Tanksley and Nelson (1996) proposed a MABC strategy, called advanced backcross-QTL (AB-QTL), to transfer resistance genes from wild relative/unadapted genotype into elite germplasm. The strategy has proven effective for various agronomically important traits in to‐ mato, including fruit quality and black mold resistance (Tanksley and Nelson, 1996; Bernacchi et al., 1998; Fulton et al., 2002). In addition, AB-QTL has been used in other crop species, such as rice, barley, wheat, maize, cotton and soybean, collectively demonstrating that this strategy is effective in transferring favorable alleles from the wild/unadapted germplasm to elite germ‐

In barley, a marker linked (0.7 cM) to the *Yd2* gene for resistance to barley yellow dwarf virus was successfully used to select for resistance in a backcrossing scheme (Jefferies et al., 2003). Compared to lines without the marker, the BC2F2-derived lines carrying the linked marker had lighter leaf symptoms and higher yield when infected by the virus. In maize, marker-facilitat‐ ed backcrossing was also successfully employed to improve complex traits such as grain yield. Using MABC, six chromosomal segments each in two elite lines, Tx303 and Oh43, were trans‐ ferred into two widely used inbred lines, B73 and Mo17, through three generations of back‐ crossing followed by two selfing generations. Then the enhanced lines with better performance were selected based on initial evaluations of testcross hybrids. The single-cross hybrids of enhanced B73 x enhanced Mo17 out-yielded the check hybrids by 12-15% (Stuber et al., 1999). Zhao et al. (2012) reported that a major quantitative trait locus (named *qHSR1*) for re‐ sistance to head smut in maize was successfully integrated into ten high-yielding inbred lines (susceptible to head smut). Each of the ten high-yielding lines was crossed with a donor pa‐ rent Ji 1037 that contains *qHSR1* and is completely resistant to head smut, followed by five gen‐

**5.** The traits are controlled by recessive genes; and **6.** Gene pyramiding is needed for one or more traits.

66 Plant Breeding from Laboratories to Fields

plasm (Wang and Chee, 2010; Concibido et al., 2003).

Currently, a cooperative marker-based backcrossing project for high-oleic acid in soybean has been initiated among multiple U.S. land-grant universities and USDA-ARS. Backcross‐ ing and selection will be performed using the markers tightly linked to the high-oleic genes/ loci. Hopefully, the high-oleic (80% or higher) traits will be successfully transferred from mutant lines or derived lines into other locally superior cultivars/lines, or combined with other unique traits like low linolenic acid (Pham et al., 2012).
