GENETIC IMPROVEMENT

- 21 Genomic Tools for Improvement Malay C. Saha, Andrew A. Hopkins, and Zeng-Yu Wang

ABSTRACT Genomics provides insight into the genetic basis for traits of interest in plants. Molecular (i.e., DNA) markers have been used for the genetic improvement of crop species. Several molecular marker systems have been developed, of which microsatellite or simple sequence repeat (SSR) has become the marker class of choice owing to its many advantages over other marker systems. A comprehensive molecular marker system has been developed for tall fescue (Lolium arundinaceum) and used for the construction of genetic linkage maps, genetic diversity analysis, and marker-assisted breeding. Use of genomic knowledge and molecular markers is expected to contribute greatly to the development of improved tall fescue cultivars in the coming decades. Keywords: genome, molecular marker, gene sequence, comparative genomics, polymorphism, genetic linkage map, quantitative trait loci. Abbreviations: A, adenine; AFLP, amplified fragment length polymorphism; C, cytosine; cDNA, complementary DNA; cM, centimorgan; DNA, deoxyribonucleic acid; EST, expressed sequence tag sequences; FaGI, Festuca Gene Index; G, guanine; ITS, internal transcribed spacer region; IVDMD, in vitro dry matter digestibility; LG, linkage group; mRNA, messenger RNA; NCBI, National Center for Bioinformatics; PCR, polymerase chain reaction; PP, primer pair; QTL, quantitative trait loci; RAPD, randomly amplified polymorphic DNA; rDNA, ribosomal

DNA; RFLP, restriction fragment length polymorphism; SSR, simple sequence repeat (microsatellite); T, thymine; TIGR, The Institute for Genetic Resources.

INTRODUCTION A genome contains the genetic material that is passed from parent to offspring. The genome embodies the genetic blueprint that determines or influences various characteristics, such as height, seed yield, grazing tolerance, etc., of an individual plant. The nuclear genome is packaged in chromosomes found in the nucleus of the cell. Chromosomes contain deoxyribonucleic acid, better known as DNA, which is composed, in part, of the nucleotideforming bases adenine (A), thymine (T), guanine (G), and cytosine (C). The arrangement (i.e., sequence) of these bases on the DNA molecule determines which enzymes the plant produces; in turn, these enzymes provide the biochemical basis for the expression of traits by the plant. Thus, variation in DNA sequence can lead to trait differences passed on to offspring (see Chapter 19).

TALL FESCUE GENOME STRUCTURE Tall fescue contains three genomes (PPG1G1G2G2) and is an allohexaploid (2n = 6x = 42), meaning that the six sets of chromosomes present in tall fescue have been derived from different ancestor species (see Chapter 2). The P genome is derived from the diploid progenitor meadow fescue (L. pratensis Huds.; 2n = 2x = 14) while the G1G2 genome is from tetraploid fescue (F. arundinacea var glaucescens Boiss; 2n = 4x = 28) (Xu et al., 1991). The tall fescue genome contains more than 5 billion pairs of bases, or approximately 5.27-5.83 x 106 kilobases (kb) (Seal, 1983). Only a portion of this DNA contributes to gene expression. The large genome size and the presence of a large number of non-expressed DNA sequences makes identifying and

selecting genes of interest very challenging. Molecular markers and maps have been developed to facilitate this selection process in tall fescue.

GENOMIC TOOLS AND TECHNIQUES Conventional plant breeding is time-consuming and largely dependent on crop phenotype and environmental conditions. The development of molecular techniques for crop improvement has led to a great increase in our knowledge and understanding of the structure and behavior of crop genomes. In the past three decades, great advances have been made and several molecular tools have been developed. Most of these tools have been used for the genetic improvement of major cereal species, but only a few of those have been used for tall fescue improvement. A brief summary of those tools and techniques is discussed in this chapter. Molecular markers A molecular marker is a fragment of DNA that is associated with a part of the genome and can be identified by a simple assay. Molecular markers are based on differences in DNA sequence and, as such, are not subject to environmental influence; they are abundant throughout the entire genome, and tests can be carried out at any time during plant development. Molecular markers are detected as differences in DNA fragment size, which arise from differences in DNA sequence. Molecular markers are used widely in cultivar identification, assessment of genetic variability among and within population(s), molecular mapping (i.e., determining the linear order of molecular markers in a genome), and marker-assisted breeding (tagging important trait(s) in a breeding program). Thus, they are considered the basic tools of genomic research. The effectiveness of molecular markers depends on their ability to identify variation in the DNA of a population, often known as marker polymorphism. They are genetic components and highly

heritable. The genome size of most plant species ranges between 108 to 1010 base pairs, so even a small proportion of variation in DNA can yield a large number of potential markers (Paterson et al., 1991). The ideal marker class should be abundant, stable, easily detectable, and have a high degree of polymorphism. The marker classes commonly used for germplasm characterization and genetic improvement of tall fescue are described below. Restriction fragment length polymorphisms (RFLPs). Restriction fragment length polymorphisms (Tanksley et al., 1989) were the first set of molecular markers developed that were linked to genes controlling important traits. The RFLPs are co-dominant and all three morphs (i.e., forms) can be identified, so these are highly informative because homozygotes, in which all copies of a specific gene have the same form, do not provide any information. Major disadvantages of RFLPs are the robust, laborious, and costly protocols needed to generate these markers. In the early 1990s, attempts were undertaken to generate RFLP markers for tall fescue. These were used for the construction of genetic linkage maps (Xu et al., 1995), comparative mapping (Chen et al., 1998), genetic diversity analysis (Xu et al., 1994), phylogenetic analysis (Xu and Sleper, 1994), analyses of Festuca Lolium hybrids (Chen et al., 1995), cultivar identification, and differentiation of monosomic lines (Eizenga et al., 1998). Randomly amplified polymorphic DNA (RAPD). The polymerase chain reaction (PCR) amplifies discrete fragments of DNA and can be used to detect polymorphisms quickly. Randomly amplified polymorphic DNA was the first marker of this kind (Williams et al., 1990). However, imperfect reproducibility of RAPD markers was a major problem. Phylogenetic analysis of the Festuca-Lolium complex (Charmet et al., 1997) and detection of Festuca genome introgression into a Lolium background (Wang et al., 2003) have been examined using RAPD markers.

Amplified fragment length polymorphisms (AFLPs). Amplified fragment length polymorphism markers provide an efficient system for molecular mapping, because a large number of markers can be generated within a short time (Vos et al., 1995). However, developing AFLP markers is costly and requires skilled personnel. In addition, an AFLP marker linked to a specific trait in one population will rarely be useful for marking the same trait in a different population. As a result, AFLPs are not used commonly in framework mapping (Jones et al., 2002). Hundreds of tall fescue AFLP markers were developed and used for the construction of a genetic linkage map (Saha et al., 2005) and genetic diversity analysis of forage grass species (Mian et al., 2002; Mian et al., 2005). Microsatellites. Microsatellites, also known as simple sequence repeats (SSRs), have become highly useful molecular markers for plant improvement. Microsatellites have the potential advantages of reliability, reproducibility, discrimination, standardization, and cost effectiveness over RFLPs. In addition, SSR markers can be used across a wider range of populations and species than more restrictive markers such as AFLPs. High-throughput detection systems make SSR genotyping much faster, easier, and cost efficient than many other types of markers. Development of SSR markers is costly but, once developed, they can be used for many different purposes. Microsatellites are composed of short stretches of DNA with a repetitive sequence, for example, CTGCTGCTGCTGCTGCTGCTG. Primers are short stretches of DNA that are designed from the start (forward primer) and end (reverse primer) of sequences that flank such repeat regions. Using PCR, primers can amplify fragments of DNA that vary in length of the repetitive sequences and/or adjacent sequences, which in turn can serve as molecular markers for tall fescue and other grasses (Mian et al., 2005). Thus, sequence information is essential to

generate SSR markers. Microsatellites can be identified from genomic DNA, which refers to segments of DNA derived from either expressed or non-expressed regions. Expressed sequence tags also are considered to be important resources for the development of SSR markers. Microsatellite markers have been developed from tall fescue sequences (Saha et al., 2004, 2006) and used for the construction of genetic linkage maps of tall fescue (Saha et al., 2005), annual ryegrass (L. multiflorum) perennial ryegrass (L. perenne) (Warnke et al., 2004), and bentgrass (Agrostis stolonifera) (Zhao et al., 2006), and to conduct genetic diversity and phylogenetic analysis of grass species (Saha et al., 2004; Zwonitzer et al., 2004; Hopkins and Saha, 2005; Mian et al., 2005). Cross-species applicability of these microsatellite markers was also evaluated (Saha et al., 2004; Mian et al., 2005). However, the development of molecular markers for these complex polyploid species lag far behind those for major cereal crops [e.g., rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum)]. DNA sequences A genome is all the DNA in an organism. DNA is made up of four nucleotide-forming bases A, T, C, and G. The exact order of nucleotides in a genome is called its DNA sequence. Attempts were undertaken to develop genome sequences of tall fescue. Expressed sequence tag (EST) sequences. Expressed Sequence Tags are short stretches of DNA sequence (usually less than 500 nucleotides) derived from a gene expressed in the cell or tissue being studied. Transcribed regions give rise to messenger RNA (mRNA), which can be used to make complementary DNA (cDNA). A set of genes is expressed as mRNAs from a cell or tissue at very different levels at any given time. Both the gene identity and the expression levels provide valuable clues to the biology of a cell or tissue. Thus, ESTs are key molecular tools used for genomic research. In addition, ESTs provide a valuable source of potential

molecular markers; these allow for the construction of comparative maps of expressed genes in related species (Cato et al., 2001; Kantety et al., 2002). The ESTs are only a tag of the cDNA (2,000 or more nucleotides) and often are sufficient to identify a specific mRNA and its corresponding gene. Several EST databases have grown exponentially because of their potential use in plant and animal genetic improvement programs (Messing and Llaca, 1998). Each of four major plant species (Arabidopsis thaliana, rice, maize, and wheat) has more than one million ESTs in the dbEST of the National Center for Bioinformatics (NCBI) database. A total of 44,512 Festuca ESTs has been published in dbEST (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide; verified 3 Jan. 2009). Most of the sequences were derived from tall fescue and a few from F. mairei and F. rubra. Most of these ESTs were contributed from the research programs of the Noble Foundation where high-quality ESTs were generated from nine cDNA libraries of tall fescue representing tissues from different plant organs, developmental stages, and abiotic stress conditions (Table 21-1). About 5,000 ESTs from each of eight libraries were developed. A ninth cDNA library constructed from field-stressed plants was sequenced and 1,211 ESTs were developed. Heat responsive gene transcripts were cloned by using differential expression between a heat-tolerant and a heat-sensitive fescue genotype. Of the 2,495 sequences generated, 656 were singlets and the remaining 1,839 ESTs were grouped into 434 clusters (Zhang et al., 2005). All these EST sequences are available at the dbEST.

Table 21-1. Development of tall fescue expressed sequence tags (ESTs) from cDNA libraries constructed from different plant tissues and growing conditions.
cDNA libraries Stem Root Leaf Young seedling Floral meristem Drought stress shoot Drought stress root Heat stress shoot Field stress shoot Number of ESTs 5,035 5,689 4,939 4,784 5,014 5,051 4,992 5,119 1,211 Library description Shoots from field grown KY31(E-) plants at E1 -E3 and R1-R3 growth stages Roots from 25 KY31(E-) plants collected after 3-10 weeks of growth All leaves of primary tiller of KY31(E-) plants grown in greenhouse 3-7 days old seedlings of KY31(E-) plants from germinator Floral meristems (3-15mm) of field grown KY31plants collected in spring Whole shoots from drought stressed and re-stressed KY31(E-) plants Roots from drought stressed and re-stressed KY31(E-) plants in greenhouse Four weeks old KY31(E-) plants heat stressed (44C) in growth chamber Shoots from 2 tillers of 3 KY31 plants grown at 35.2 - 38.7C

SSH library constructed between heat tolerant and sensitive tall Heat stress SSH 2,495 fescue accessions Young seedling, floral meristem, root, leaf, stem, and stress tolerant libraries Total 44,329 KY31(E-) = Kentucky 31 endophyte free genotypes SSH = suppression subtractive hybridization

Genomic sequences. A total of 5,320 genomic sequences developed from a (GA-CT)n enriched tall fescue genomic library is available at the NCBI. Genomic DNA from a pool of 31 KY-31 plants was isolated and used for the library construction. A biotinylated (GA)n oligonucleotide was used to capture and enrich for DNA fragments containing (GA/CT)n repeats. A protocol suggested by Hamilton et al. (1999) was used for the library construction, with minor

modifications. The detailed protocol can be viewed at http://bioserver.georgetown.edu/faculty/hamilton (verified 27 April 2007). A CoreNucleotide collection of 930 sequences from Festuca and related species is available also at the NCBI and a Festuca Gene Index (FaGI) has been established at The Institute for Genetic Resources (TIGR) (http://compbio.dfci.harvard.edu/tgi/plant.html; verified 3 Jan. 2009). Sequence characteristics and marker development. Publicly available ESTs have become a cost effective, time efficient, and unique source of SSR markers (Scott et al., 2000; Cordeiro et al., 2001; Eujayl et al., 2002; Thiel et al., 2003; Eujayl et al., 2004; Saha et al., 2004). Vast EST databases are available, and a large number of SSR markers has been developed for species such as rice, wheat, barley (Hordeum vulgare), and maize. Because EST-SSRs are derived from transcribed regions of DNA, they are more conserved and show less marker polymorphism than genomic SSRs (Cho et al., 2000; Thiel et al., 2003). However, EST-SSRs are associated with expressed genes and usually are concentrated in the gene-rich regions of the genome. Thus, many microsatellite markers are needed to get good genome-wide coverage. In five cereal species, it was found that 1.5 to 4.7% of the ESTs contained SSRs suitable for marker development. The percent of tall fescue ESTs containing SSRs is lower (1.3%) than those for barley (3.4%), wheat (3.2%), rice (4.7%), and sorghum (Sorghum bicolor) (3.6%) (Kantety et al., 2002; Saha et al., 2004). It is similar to that found in maize, 1.5% (Kantety et al., 2002). Analysis of 20,000 tall fescue ESTs resulted in the development of 157 primer pairs (PPs), approximately 0.8% of the total sequences (Saha et al., 2004). Subsequently, 43,000 tall fescue ESTs developed at the Noble Foundation were searched for SSRs and a total of 780 PPs developed. The first group of 157 PPs was released to the public domain (Saha et al., 2004). An

additional 348 primers have been developed and are being assessed for their transferability across 16 different grass species. The tall fescue genome appears rich in GC rather than AT nucleotides, as was reported for some other plant genomes, for example, soybean (Glycine max) and other legumes (BrownGuedira et al., 2000). The distribution of di-, tri-, tetra-, and pentanucleotide repeats in tall fescue was similar to that reported by earlier investigators (Eujayl et al., 2004; Thiel et al., 2003; Kantety et al., 2002). Usually, di-nucleotide repeats are the most abundant in genomic SSRs, whereas tri-nucleotide motifs are the most abundant in EST-SSRs (La Rota et al., 2005). Among 20,000 tall fescue ESTs, trinucleotide motifs were the most abundant type of SSRs (70%), followed by di- (20%), tetra- (5%) and pentanucleotide (5%) motifs. The CCG/GGC motif was the most abundant trinucleotide repeat, while GA/CT was the most abundant dinucleotide repeat (Saha et al., 2004). Detailed information on 145 tall fescue EST-SSR primers, including primer sequences, marker name, EST ID, expected band size, annealing temperature, SSR repeat motif and number, observed band size range, amplification results on 11 genotypes of seven species, and gene functional annotation, is available as electronic supplementary material (Saha et al., 2004). In species where EST databases are not well established, genomic libraries are considered an important source for SSR marker development. In tall fescue, EST sequences are few. Furthermore, EST-SSR loci have a tendency to be clustered in gene-rich regions of the genome, and this clustering limits the potential for genome-wide coverage (Warnke et al., 2004; Yu et al., 2004; La Rota et al., 2005; Saha et al., 2005). Genomic SSRs are highly polymorphic. They tend to be distributed widely throughout the genome, giving better map coverage than EST-SSRs (Taramino et al., 1997; Warnke et al., 2004; La Rota et al., 2005; Saha et al., 2005). Construction

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and screening of partial genomic libraries, and sequencing of SSR-positive clones are considered effective methods for microsatellite development (Rafalski et al., 1996). Microsatellite markers can be developed either from non enriched or highly enriched genomic libraries (e.g., normal genomic libraries vs. genomic libraries enriched for a particular repeat motif) (Edwards et al., 1996). Highly-enriched genomic libraries significantly reduced the cost and effort for microsatellite development compared to non enriched libraries (Kijas et al., 1994; Edwards et al., 1996). A (GA-CT)n--enriched genomic library of tall fescue was developed. A total of 5,712 sequences was characterized, and after deleting questionable and short sequences, 5,320 high quality sequences were screened for SSR motifs. About 20% of the sequences were found to be SSR positive. Nine hundred six of these were candidate SSR sequences that had repeat motifs >18 bases, and at least 25 bases of flanking DNA sequences at both ends (Saha et al., 2006). Five hundred eleven PPs were developed. Four hundred twenty-five of these PPs were singleton SSRs. Thus, genomic SSR primers were developed from 56% of the SSR containing sequences. The sequences of the 511 PPs, along with the marker name, sequence ID, primer sequences, Tm, and expected sizes are available at Saha et al. (2006). Cross-species amplification of molecular markers Conservation of grass genomes has been documented in distantly related grass species (Ahn and Tanksley, 1993; Kurata et al., 1994; Gale and Devos, 1998). Comparative genetics revealed the conservation of gene and marker orders within the Poaceae family (Ahn and Tanksley, 1993; Van Deynze et al., 1995; Jones et al., 2002; Alm et al., 2003; Kuleung et al., 2004). Comparative genomics facilitates identification of putative orthologous loci (genes that encode proteins with the same function in different species) controlling agronomic traits within

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the Poaceae (Paterson et al., 1995; Devos and Gale, 1997). It also assists extending genetic information from model species to more complicated species (Gale and Devos, 1998). As the grass genomes are highly conserved, molecular markers developed in one species can be used for genetic analysis of other species. In the past, comparative genomics efforts relied primarily on the hybridization-based RFLP technique. For example, three chromosome I specific maize clones were detected on the chromosomes of homeologous group 3 (3a, 3b, and 3c) in a tall fescue map (Xu et al., 1995). Attempts were undertaken to assess the evolutionary relationship of meadow fescue with tall fescue through comparative RFLP mapping (Chen et al., 1998). A group of 33 RFLP markers that was mapped in meadow fescue and tall fescue detected highly conserved linkage groups between the two species. Eight of the nine markers that mapped to linkage group I of meadow fescue also were present in linkage group I of tall fescue. However, changes in marker sequences, map distances, and homeologous linkage groups were detected between the two species. This indicated that the P genome diverged substantially during evolution from the diploid meadow fescue to the hexaploid tall fescue (Chen et al., 1998). A major drawback of RFLP-based maps was low resolution, which often failed to determine the preserved order of genes between related species at micro levels. The application of a PCR-based codominant marker system for comparative genomics would be highly desirable. These markers have been found to be efficient in transferring genetic information across species (Kantety et al., 2002; Thiel et al., 2003; Eujayl et al., 2004; Saha et al., 2004). Studies by Jones et al. (2002) and Alm et al. (2003) revealed that forage species (ryegrass and meadow fescue) were highly orthologous, that is, they had a common ancestry and were colinear with the ancestries of rice, oat (Avena sativa), maize, and sorghum. High rates of transferability of SSR

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loci across species (>50%) within a genus have been reported (Peakall et al., 1998; Gaitn-Sols et al., 2002; Dirlewanger et al., 2002; Thiel et al., 2003; Eujayl et al., 2004; Saha et al., 2004). However, the transferability of SSR loci across genera and beyond appears to be low (White and Powell, 1997; Peakall et al., 1998; Roa et al., 2000; Thiel et al., 2003). The EST-SSR markers are expected to be more conserved and have a higher rate of transferability across species than genomic SSR markers (Scott et al., 2000). Thanks to this transferability, the EST-SSR markers have good potential for application in cross species genetic studies (Kantety et al., 2002; Thiel et al., 2003; Eujayl et al., 2004; Saha et al., 2004). Tall fescue EST-SSR markers are fairly polymorphic across species and can be used to discriminate genotypes with wider genetic bases (Mian et al., 2005). One genotype from each of 12 forage and cereal grass species was screened using tall fescue EST-SSR primer pairs. Nearly 43% of the primer pairs produced PCR bands in at least 10 species. Ten percent of primer pairs were species specific (Mian et al., 2005). From this initial screening, 71 primer pairs with clear banding patterns were selected for screening 54 genotypes. Forty-six of the 71 selected primer pairs worked in all 12 species. In a similar study, tall fescue EST-SSRs were found highly conserved (Saha et al., 2004). A set of tall fescue EST-SSR markers was identified which can amplify characteristic SSR type products across a wide range of species in the Poaceae family (Mian et al., 2005; Saha et al., 2004). In many forage grass species, molecular markers have yet to be developed. Thus, highly conserved tall fescue microsatellite markers are of great value for species in which molecular marker information is very limited or not developed at all. Tall fescue EST-SSRs were found most effective in closely related species, and the complexity of banding pattern increased with the increasing genetic distance from tall fescue. A total of 511 tall fescue genomic SSRs was tested for functionality in 10 genotypes representing six different

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grass species (Saha et al., 2006). The proportion of PPs that produced clean SSR products ranged from 66% (tall fescue) to 48% (rice). Polymorphism rates were tested in the parents of mapping populations and rates varied from 68% (tall fescue) to 34% (rice). Breeders often are interested in incorporating ryegrass genes into tall fescue, or vice versa. Molecular markers have proven useful in monitoring introgression between species. A repetitive DNA sequence specific to the Festuca genome was used as a probe to monitor chromatin introgression in Festuca/Lolium hybrids (Cao and Sleper, 2001). Mapping of tall fescue EST-SSRs in ryegrass linkage groups (Wranke et al., 2004) indicated the applicability of these markers for genome introgression studies in the Festuca Lolium complex. Of 27 polymorphic EST-SSR loci, 15 were mapped to ryegrass linkage groups (LGs) (Warnke et al., 2004). Seven of these marker loci were uniquely mapped on both male and female maps (NFFA031 and NFFA75 on LG 1; NFFA015, 036, and 048 on LG 6; NFFA019 and NFFA069 on LG 7). Repetitive DNA sequences contribute significantly to the understanding of genomes of higher plants. Repetitive DNA sequences tend to be genome specific and represent 20 to 90% of a whole genome and, in most cases, have no known function. Genome-specific repetitive sequences were developed and characterized in Festuca species (Cao and Sleper, 2001). Two repetitive sequences (TF436 and TF521) were identified from a hexaploid tall fescue PstI genomic library (Xu and Sleper, 1994). The sequence of TF521 was P genome specific and hybridized only with meadow fescue and tall fescue, but not in tetraploid fescue (F. arundinacea var. glaucescens) or Lolium. The sequence of TF436 was tetraploid fescue specific and did not hybridize to diploid meadow fescue or Lolium. Further study suggested meadow fescue and tetraploid fescue as the probable progenitors of tall fescue. Hybridization results with repetitive

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DNA and non repetitive RFLP probes implied no significant DNA structural differentiation in the genomes of meadow and tetraploid fescue. Both genomic in situ hybridization (GISH) (Humphreys et al., 1995) and RFLP hybridization (Cao and Sleper, 2001) experiments successfully differentiated meadow fescue from F. arundinacea var. glaucescens. Xu and Sleper (1994) also successfully used TF436 clone to detect alien chromatin introgression from F. mairei in the progenies of the hybrid F. mairei L. perenne. Molecular linkage maps Genetic linkage maps correspond to the linear order of molecular markers in a specific genome. Linkage maps are constructed by following the segregation of molecular markers in a population and placing them in linear order based on pair-wise recombination frequencies. The occurrence of many polymorphisms over the total genome is a highly desirable characteristic for a mapping population. Backcross, F2, and recombinant inbred lines (RIL) are the most commonly used populations for molecular mapping. In outcrossing and self-incompatible species, pseudo F1 populations are used to construct genetic linkage maps. Constructing genetic linkage maps of tall fescue is impaired by genome complexity, heterozygosity within clones, a high level of self-incompatibility, and lack of morphological genetic markers (Xu et al., 1991). The two-way pseudo-testcross procedure is considered an efficient way to construct genetic linkage maps in outcrossing species like tall fescue (Ritter et al., 1990; Hemmat et al., 1994). In the pseudo-testcross procedure, a mapping population is developed by hybridizing two unrelated highly heterozygous parents to produce a set of F1 progeny. High levels of marker segregation are observed in most of these populations. However, construction of linkage maps is complicated because one or both parents may be heterozygous at a certain locus, markers may be dominant or codominant, and the linkage phase of marker alleles

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usually is unknown (Maliepaard et al., 1997). Most of the forage grass species are polyploids and outcrossing, thus genomic information for many forage species is quite limited when compared to other major crop species. RFLP map. The first genetic linkage map of tall fescue was generated from an F2 population using 108 RFLP markers (Xu et al., 1995). The mapping population was constructed by crossing HD28-56, a plant with high forage quality, to a selected plant of Kentucky-31, the most widely grown cultivar in the United States. A high level of marker polymorphism was observed in the population. Polymorphic probes detected multiple segregating fragments. Two- and three-point linkage analysis was performed using MAPMAKER software. The RFLP marker data in the population indicated clear and strong evidence of disomic inheritance, meaning that tall fescue behaved genetically like a diploid organism that contained two sets of chromosomes. Earlier cytogenetic (Sleper, 1985) and isozyme studies (Lewis et al., 1980) also suggested disomic inheritance in tall fescue. All these results supported the conclusion that tall fescue is an allohexaploid. Ninety-five markers were used to construct a tall fescue genetic linkage map (Xu et al., 1995). The map covered 1274 centimorgan (cM) on 19 LGs with an average of five loci per LG, and a marker density of 17.9 cM per marker (map distances are calculated on the basis of recombination events; one cM equals 1% recombinant offspring). Thirteen markers remained unlinked. Markers that segregated at more than one locus allowed identification of linkage groups that belonged to the same homoeologous (that is, base chromosome) group. Five out of seven homoeologous groups were identified. Linkage groups 15 to 19 were not placed in any of the homoeologous groups. Several LGs were assigned to the three genomes (PG1G2) using

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genome-specific probes. The genome-specific probes hybridized with one or occasionally two of the three genomes in tall fescue. AFLP and SSR map. The first PCR-based genetic linkage map of tall fescue was constructed with AFLP and SSR markers (Saha et al., 2005). Conserved grass EST-SSR markers were developed from the EST sequences conserved across different cereal grass species (Kantety et al., 2002). Tall fescue and conserved grass EST-SSRs, genomic SSRs from Festuca Lolium hybrids, and AFLP markers were used to construct the parental maps followed by biparental consensus maps (Fig. 21-1). A two-way pseudo-testcross mapping strategy was followed for map construction. Seven hundred seventy-three AFLP and 343 microsatellite markers were used to construct the linkage maps. The AFLP markers contributed considerably to genome coverage and to providing links to associate distantly located SSR loci. The majority of markers segregated from either parent and showed a 1:1 Mendelian segregation ratio. However, 12% of the markers segregated from both parents and showed a 3:1 segregation pattern. Markers present in both parents and showing a 3:1 segregation ratio were useful for identifying homologous groups between maps (Maliepaard et al., 1997). The female (HD28-56) map included 558 loci placed in 22 LGs and covered 2,013 cM of the genome. The male (R43-64) map comprised 579 loci grouped in 22 LGs with a total map length of 1,722 cM. The marker density in the two maps varied from 3.61 cM (female parent) to 2.97 (male parent) cM per marker. A biparental consensus map was constructed based on common markers in parental maps. The consensus map covered 1,841 cM on 17 LGs, with an average of 54 loci per LG, and had an average marker density of 2.0 cM per marker. Six of the seven predicted homeologous groups were identified. The female parental map length was greater than the male parental map. A distinctly reduced

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HD-8
N FF A 0 1 7 _ 2 0 8 B3B7_300 P G T _ G C T2 6 7 N FF A 1 1 2 _ 2 5 0 B 4D7_216 P G T _ G C T5 6 P G T_ T G 2 0 1 N FF A 1 3 8 _ 2 1 8 N FF A 1 5 5 _ 1 8 5 N FF A 1 5 5 _ 2 4 5 P G T_ T G 2 5 7 PG T _G G A251 P G T _T G C257 B3B1_250 PG G _AG 420 P G T_ A G C2 7 1 N FF A 0 2 1 _ 2 3 3 N FF A 1 3 4 _ 2 6 0 N FF A 0 7 5 _ 2 0 5 P CC _T CG 231 N FF A 1 2 9 _ 2 2 0 P C C_T G 231 P G T _ G C T1 9 0 B1B6_308 N FF A 1 3 4 _ 2 5 2 N FF A 1 2 9 _ 2 3 2 P G G _ TG 7 5 N FF A 1 3 4 _ 3 9 0 N FF A 0 7 3 _ 2 4 8 N FF A 0 7 5 _ 3 1 5 N FF A 1 2 0 _ 2 3 0 P G T _T G C365 P C C_C G C55 P G T_ A G C3 6 5 P G G _ G CG 179 N FF A 1 2 9 _ 1 6 0 B5E1_252 N FF A 1 3 4 _ 5 6 0 N FF A 1 3 4 _ 4 6 0 P G G _ G CG 124 P C C_C G C226 N FF A 1 1 2 _ 2 3 0 P C C_C G C83

2-C

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145

R-7
N F FA 1 2 9 _ 2 4 0 N F FA 0 2 1 _ 2 3 3 E G G _G CG 209 P G G _ TC G 1 6 5 B 3B1_260 N F FA 1 2 9 _ 2 3 2 N F FA 1 2 0 _ 2 5 2 P G G _G CG 96 P G T_ T G 1 5 4 N F FA 1 2 9 _ 1 6 0 P G T_ G G A 3 3 2 N F FA 1 2 0 _ 2 4 4 P G T_ A G C 2 7 3 E CC _A G 188 B 3B1_264 P CC _G G A 173 N F FA 1 5 5 _ 2 5 8 N F FA 1 5 5 _ 2 4 1 N F FA 1 4 0 _ 2 3 0 B 3B7_290 N F FA 1 3 8 _ 2 0 4 E A G _ TG 1 8 5 P G T_ G C T1 0 8 P G G _G CG 123 P G T_ G C T3 5 8 N F FA 1 1 2 _ 2 4 2

Parental maps

Integrated map

B3B7_300 NF FA 0 1 7 _ 2 0 8 P G T_ G C T2 6 7 NF FA 1 1 2 _ 2 5 0 B 4D7_216 P G T_ G C T5 6 P G T_ T G 2 0 1 NF FA 1 3 8 _ 2 1 8 NF FA 1 5 5 _ 1 8 5 NF FA 1 5 5 _ 2 4 5 P G T_ T G 2 5 7 P G T_ T G C2 5 7 P G T_ G G A 2 5 1 B3B1_250 N F F A 1 2 9 _ 2 4 0 * ** PG G _AG 420 P G T_ A G C 2 7 1 NF FA 0 2 1 _ 2 3 3 NF FA 1 3 4 _ 2 6 0 NF FA 0 7 5 _ 2 0 5 P CC _ TC G 2 3 1 NF FA 1 2 0 _ 2 4 4 * * NF FA 1 2 9 _ 2 2 0 P CC _ TG 2 3 1 P G G _ TC G 1 6 5 P G T_ G C T1 9 0 B1B6_308 NF FA 1 2 0 _ 2 5 2 NF FA 1 3 4 _ 2 5 2 NF FA 1 2 9 _ 2 3 2 P G T_ G G A 3 3 2 P G G _ TG 7 5 NF FA 1 3 4 _ 3 9 0 NF FA 0 7 3 _ 2 4 8 P G T_ T G 1 5 4 NF FA 0 7 5 _ 3 1 5 P G G _G CG 96 NF FA 1 2 0 _ 2 3 0 E G G _G CG 209 P CC _CG C 55 P G T_ A G C 3 6 5 NF FA 1 2 9 _ 1 6 0 P G T_ A G C 2 7 3 B5E1_252 N F F A 1 3 4 _ 5 6 0 * ** E CC _A G 188 B3B1_264 N F F A 1 3 4 _ 4 6 0 * ** NF FA 1 5 5 _ 2 5 8 P G G _ G C G 1 2 4 * ** NF FA 1 5 5 _ 2 4 1 NF FA 1 4 0 _ 2 3 0 * B3B7_290 P C C _ G G A 1 7 3 ** P C C _ C G C 2 2 6 ** E A G _ TG 1 8 5 * * * P G T_ G C T1 0 8 * N F F A 1 3 8 _ 2 0 4 * ** NF FA 1 1 2 _ 2 3 0 P G G _G CG 123 P G T_ G C T3 5 8 NF FA 1 1 2 _ 2 4 2 P C C _ C G C 8 3 ** B 3 B 1 _ 2 6 0 ** *

Figure 21-1. Parental linkage groups (LG) of HD-8 (HD28-56 map) and R-7 (R43-64 map) along with the integrated group 2-C of a tall fescue genetic linkage map (Saha et al., 2005). The cM distance scale is at the extreme left. Bars indicate length of each LG. Marker names are placed at the right side of each bar. The AFLP markers are prefixed with a P or E. Tall fescue and conserved grass EST-SSRs are indicated as NFFA and CNL, respectively. The Festuca Lolium genomic SSRs begin with a B. The last three digits of each marker indicate the size in base pairs.

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level of recombination was found in the male parent compared to the female parent. Markers in general were evenly distributed throughout the genome. However, clustering of markers in some regions and gaps of 10 to 21 cM in some LGs were also evident. These results indicated that recombination events were not evenly distributed throughout the genome. High levels of segregation distortion (23% of total markers) were observed in the cross between two distantly related, heterozygous tall fescue genotypes. Segregation distortion has been reported frequently in tall fescue (Xu et al., 1995; Saha et al., 2005) and other grass species (Warnke et al., 2004; Brummer et al., 1993; Wang et al., 1994). Distorted markers segregated from both parents of the HD 28-56 x R43 - 64 mapping population and were present in most of the linkage groups. The distribution of skewed markers in both parental maps indicated that both the male and female gametophytes and/or sporophytes were involved in segregation distortion. Clustering of markers showing significant segregation distortion was observed in four of the LGs of the consensus map. The AFLP and microsatellite based maps (Saha et al., 2005) were a substantial improvement over the RFLP-based map (Xu et al., 1995) in both map coverage and marker density. Microsatellite map. Recently, a substantial number of microsatellite and sequence tagged site (STS) primers (>1800 primer pairs) has been developed for tall fescue. The same mapping population used for the construction of the AFLP-SSR map has been used to construct the biparental and consensus maps. This map has been used to identify molecular markers associated with traits of interest and to map quantitative trait loci (QTL), regions of the genome that contain a cluster of genes influencing a quantitative trait. This is helpful since it indicates that these traits are conditioned by a large number of genes.

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GENETIC DIVERSITY AND MOLECULAR PHYLOGENY Only a few RFLP based studies have been conducted to evaluate genetic relationships among forage grass species. Xu and Sleper (1994) used RFLPs to study the phylogenetic relationship of tall fescue with six related grass species. Charmet et al. (1997) used RFLP and chloroplast DNA (cpDNA) for phylogenetic analysis of the Festuca-Lolium complex. The RFLP probes generated from turf grass species were employed successfully for establishment of phylogenetic relationship in turf grasses (Yaneshita et al., 1993). Genetic variation within forage and turf grass species has been investigated by several groups using RAPD markers (Huff et al., 1993; Valls et al., 1993; Wu and Lin, 1994; Sun et al., 1999; Diaby and Casler, 2003; Casler et al., 2003) and AFLP markers (Roldan-Ruiz et al., 2000; Zhang et al., 1999; Mian et al., 2002; Larson et al., 2003; Vergara and Bughrara, 2003). However, the anonymous and dominant nature of RAPD and AFLP markers limit their utility in genetic studies, particularly across species. The PCR based codominant and portable SSR markers are more useful than dominant markers for such studies (Wang et al., 2001). The SSRs were found highly effective for differentiating cultivars of perennial ryegrass (Kubik et al., 2001) and discriminating between Lolium and Festuca grasses (Paakinskien et al., 2000). Variability among grass species has been assessed using tall fescue EST-SSR markers (Mian et al., 2005). A dendrogram produced from the DICE similarity coefficients based on 1,666 microsatellite bands clearly separated each species and clustered all genotypes of each species together. Two of the three Festuca species (tall fescue and meadow fescue, now classified as Lolium species, as described in Chapter 2) were grouped together while the third species, F. rubra, was grouped with Dactylis glomerata (orchardgrass). All three genera (Lolium, Festuca, and Dactylis) of the tribe Poeae formed a loose cluster (Mian et al., 2005). The

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phylogenetic tree based on the nucleotide substitution data of DNA sequences (obtained from EST-SSR PP NFFA150) revealed results similar to those obtained from SSR marker analysis. The genetic tree placed the three genera of the tribe Poeae and three genera of tribe Triticeae in exactly the same classes, as depicted by the SSR analysis. The tree obtained from the analysis of sequences from another SSR PP (NFFA036) was quite similar except it pushed meadow fescue close to Lolium and red fescue toward tall fescue. Clustering of two Festuca (now considered Lolium) species (formerly F. arundinacea and F. pratensis) from a third (F. rubra) indicated separation of broad leaf fescues (subg. Schedonorus) from fine leaf fescues (subg. Festuca). According to the rate of nucleotide substitution in chloroplast DNA (cpDNA) spacers, the split of broad-leaved and fine-leaved fescues occurred some nine million years ago (Charmet et al., 1997). Internal transcribed spacer regions (ITSs) are well suited for intrageneric studies and used for the sequence analysis in grasses (Hsiao et al., 1995). Trees of the Festuca/Lolium complex obtained from cpDNA, ribosomal DNA (rDNA), and ITSs analyses showed the same differentiation of the three major groups i) fine-leaved fescues, ii) broad-leaved fescues and, iii) ryegrasses (Charmet et al., 1997). Clustering of F. rubra with D. glomerata rather than with tall fescue and meadow fescue was detected in several independent studies (Charmet et al., 1997; Levslaiho et al., 1987; Mian et al., 2005). Tetraploid fescue was more closely related to hexaploid tall fescue than was meadow fescue; this may be because tetraploid fescue shares two genomes (G1G2) whereas meadow fescue shares only one (P) genome with tall fescue (PG1G2).

QUANTITATIVE TRAIT LOCI (QTL) ANALYSIS Quantitative traits show continuous phenotypic variation in a population due to combined effects of genes and environments. Examples of quantitative traits in tall fescue include

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digestibility, forage yield, and drought stress tolerance. Improvement of quantitative traits is an important goal of many breeding programs. Analysis of QTL has been a major area of genetic study for many decades. Traditionally, study of quantitative traits largely depended on statistical approaches based on means, variances, and covariances of relatives. Thus the biological nature of the quantitative traits in terms of number of genes involved and their chromosomal location was largely unknown. With the advent of molecular markers and specific statistical methods, it became possible to follow the segregation of quantitative traits via linked markers (Tanksley, 1993). Mapping markers linked to QTLs identifies regions of the genome that contain gene(s) involved in the expression of quantitative traits. Efforts are underway to apply molecular markers for the genetic improvement of quantitative traits of tall fescue. Two examples include increased digestibility and improved drought stress tolerance. Phenotypic data, including in vitro dry matter digestibility (IVDMD), have been collected from the mapping population described by Saha et al. (2005) for three consecutive years. These data are being evaluated in conjunction with SSR marker data to identify possible QTLs associated with IVDMD. These QTL will be evaluated subsequently for their effectiveness in a selection scheme aimed at improving IVDMD of tall fescue. Database and literature searches have identified a number of genes that appear to be important for osmotic stress (drought, cold, salt, abscisic acid, and oxidative stress). The majority of these genes originate from the model plant Arabidopsis thaliana. Sequences from these genes are being screened to identify corresponding molecular markers in tall fescue. These markers then will be evaluated for their association with drought stress tolerance in tall fescue. Those markers that are strongly correlated with stress tolerance can be used subsequently in breeding improved tall fescue cultivars.

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Marker assisted breeding A prerequisite for marker-assisted breeding is a robust set of informative markers for the species of interest. Location of QTLs can be identified on genetic linkage maps and associated markers can be used in marker-assisted selection to improve economically important traits of tall fescue (Xu et al., 1995). An efficient way to accelerate development of disease resistant cultivars is marker-assisted selection (Frisch et al., 1999). Identification of markers that are linked to the genes of interest is very important. Marker-assisted selection was used to pyramid disease resistance genes in rice (Huang et al., 1997). With appropriate markers, a large number of plants can be screened quickly at an early stage, thereby reducing experimental size, labor requirements, and time. Microsatellite markers associated with forage digestibility in tall fescue were identified. Seven markers associated with high digestibility and six others linked to low digestibility were used to initiate marker-assisted breeding. These markers contributed 2.3 to 10.1% effect on forage digestibility as estimated using IVDMD. It would be useful to select for alleles leading to increased digestibility while at the same time eliminating alleles contributing to decreased digestibility. It is estimated that a 1% increase in IVDMD can lead up to a 3.2% increase in daily animal live-weight gains (ADG) (Casler and Vogel 1999). Thus, use of these markers might increase the forage digestibility in resulting populations. The population developed through MAS will be evaluated with populations based on phenotypic selection (IVDMD analysis) and combination of marker and phenotypic selection, to determine the effectiveness of MAS.

CONCLUSIONS

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Genomic tools have considerable utility for crop improvement. Substantial genomic information has been developed in major crop species. Genomic tools for most of the forage and turf grass species have yet to be developed. Molecular markers (RFLPs, RAPDs, AFLPs, and SSRs) have been developed for tall fescue, the most important cool-season perennial forage and turf grass species. These markers have been used for phylogenetic analysis, cross species amplification, construction of genetic linkage maps, cultivar fingerprinting, and tagging of QTLs for traits of interest. Use of these resources in cultivar development programs will expedite greatly the cultivar improvement process.

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