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Genetic linkage maps of centipedegrass [Eremochloa ophiuroides (Munro) Hack] based on sequence-related amplified polymorphism and expressed sequence tag-simple sequence repeat markers
Scientia Horticulturae 156 (2013) 86–92
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Genetic linkage maps of centipedegrass [Eremochloa ophiuroides (Munro) Hack] based on sequence-related amplified polymorphism and expressed sequence tag-simple sequence repeat markers Yiqi Zheng a,b , Hailin Guo a , Guozhang Zang b , Jianxiu Liu a,∗ a b
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, PR China College of Forestry, Henan University of Science and Technology, Luoyang 471003, PR China
a r t i c l e
i n f o
Article history: Received 24 July 2012 Received in revised form 27 March 2013 Accepted 29 March 2013 Keywords: Centipedegrass Sequence-related amplified polymorphism (SRAP) Expressed sequence tag-simple sequence repeat (EST-SSR) Genetic mapping
a b s t r a c t Centipedegrass [Eremochloa ophiuroides (Munro) Hack] is a warm-season turfgrass native to the southeast of China. In this study, the first genetic linkage maps of centipedegrass were constructed using PCR-based markers based on a segregation population consisting of 89 F1 progeny derived from a cross between two ecotypes (E102 and E092(1)). A combination of sequence-related amplified polymorphisms (SRAPs) and expressed sequence tag-simple sequence repeats (EST-SSRs) from wheat was used for map construction. Eighty nine loci, including 85 SRAP and 4 EST-SSR loci, in the female (E102) linkage map were placed in five major and five minor (two triplet and three doublets) linkage maps, covering 623.6 cM with an average map distance of 7.1 cM between adjacent markers. In the male (E092(1)) linkage map, 71 loci including 71 SRAP and 0 EST-SSR loci were assigned to seven major and five triplets linkage maps, with genome coverage of 673.1 cM, and a mean inter-marker separation of 9.6 cM. The information presented in this study establishes a foundation for extending genetic mapping in this species, serves as a framework for mapping quantitative trait loci, and provides basic information for future molecular breeding. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Centipedegrass [Eremochloa ophiuroides (Munro) Hack] is one of the most important warm-season turfgrass native to Southeast Asia, with China being the probable area of origin (Hanson et al., 1969). Commonly referred to as “lazy man’s grass”, the management requirements of centipedegrass are generally considered lower than most warm-season turfgrass. It can tolerate a wide range of soil types, and is relatively resistant to insect and disease infestations. These low input requirements and high stress tolerances make centipedegrass popular for home lawns, landscaping, roadsides, and recreational fields in tropical and subtropical areas around the world (Hanna, 1995). This species reproduces sexually (2n = 2x = 18; Brown, 1950) and is commonly cross-pollinated because of a self-incompatibility mechanism (Hanna and Burton, 1978). Genetic linkage mapping is useful for determining the locations of genes on a chromosome, with order and position based on recombination frequencies observed in progeny populations. Plant genome maps may include important linkage relationships among molecular markers and trait genes that breeders wish to
∗ Corresponding author. Tel.: +86 025 84347012; fax: +86 025 84347057. E-mail address: [email protected] (J. Liu). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.03.030
manipulate for cultivar improvement to increase the efficiency of breeding programs. The genetic improvement of centipedegrass is impaired by its high heterozygosity and self-incompatibility. The latter makes linkage mapping of centipedegrass more complicated than that of other species that can readily be inbred. The double pseudo-testcross mapping strategy (Grattapaglia and Sederoff, 1994) is considered to be an efficient way to construct molecular genetic maps in plant species like centipedegrass. This method has been applied to a number of perennial fruit and forest trees (Beedanagari et al., 2005; Lowe and Walker, 2006), as well as to ornamental plants such as lily (Abe et al., 2002), rose (Dugo et al., 2005; Oyant et al., 2008), and chrysanthemum (Zhang et al., 2010, 2011). At present, a number of linkage maps have been constructed in turf and forage grasses, including tall fescue (Festuca arundinacea Schreb.; Xu et al., 1995; Saha et al., 2005), ryegrass (Lolium perenne L.; Bert et al., 1999; Warnke et al., 2004), zoysiagrass (Zoysia japonica Steud.; Cai et al., 2004; Li et al., 2009), bermudagrass (Cynodon dactylon × transvaalensis; Bethel et al., 2006), Kentucky bluegrass (Poa pratensis L.; Porceddu et al., 2002), and orchardgrass (Dactylis glomerata L.; Xie et al., 2011). However, there have been no reports of genetic linkage studies conducted in centipedegrass. SRAP (sequence-related amplified polymorphism) is a new PCRbased marker (Li and Quiros, 2001). This method aims to amplify open reading frames with particular primer pairs. It combines simplicity, reliability, moderate throughout ratio, and facile sequencing
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of selected bands. As a novel molecular marker, SRAP has been applied in map construction, locating genes, quantitative trait loci (QTL) mapping (Lin et al., 2005; Okazaki et al., 2007; Sun et al., 2007; Xie et al., 2011; Zhang et al., 2011), comparative genetics (Li et al., 2003), and genetic diversity assessment (Ferrio et al., 2003; Budak et al., 2004; Milla-Lewis et al., 2011). Furthermore, SRAP does not require gene sequence information. This is particularly useful for plant species such as centipedegrass with poorly researched genomes. Microsatellites or simple sequence repeats (SSRs) are a ubiquitous class of repetitive DNA sequences consisting of tandemrepeated short motifs (one to six nucleotides) (Morgante et al., 2002). SSR markers developed from genomic libraries (gSSR) have been widely used for mapping and population genetic analysis because of their high level of polymorphism, abundance and dispersion throughout the genome, codominant nature of inheritance, and reproducibility (Gupta and Varshney, 2000; Squirrell et al., 2003). Nevertheless, there are several disadvantages to gSSR markers, including high initial cost of development and low transferability across genera and beyond (Roa et al., 2000; Kindiger, 2006). However, with the increase in cDNA sequence information in public databases, exploitation of microsatellites from expressed sequence tags (ESTs) has become an attractive alternative strategy (Gupta and Varshney, 2000). Additionally, EST-SSRs are derived from transcripts, wherein flanking regions are expected to be more conserved, and thus tend to show higher levels of transferability in cross-amplification, and the possibility of tagging and mapping of genes and QTLs (Varshney et al., 2005). The map location of such SSRs may provide functionally associated genetic markers for direct characterization of the QTLs for putatively correlated traits. This would be particularly advantageous for mapping the genome of species with relatively little available genomic information, such as centipedegrass. A number of studies on the transferability of ESTSSRs in turf and forage grasses have been performed (Saha et al., 2005; Wang et al., 2005; Sim et al., 2009; Zeid et al., 2010; Xie et al., 2011). However, the identification and use of PCR-based markers in centipedegrass lags behind other major turf and forage grasses. To date there are only three types of molecular markers used in centipedegrass, DNA amplification fingerprinting (DAF) (Weaver et al., 1995), random amplification of polymorphic DNA (RAPD) (Xuan et al., 2005), and SRAP (Milla-Lewis et al., 2011), all of which were used only to evaluate the genetic diversity. In the present study, a genetic linkage map of centipedegrass based on a double pseudo-testcross mapping strategy was constructed using SRAP and EST-SSR markers from wheat. To our knowledge, this is the first report of linkage maps for centipedegrass. These maps will be valuable for studies to identify markers associated with traits of interest and for comparative analysis with other grass species.
2. Materials and methods 2.1. Plant material and DNA isolation Two E. ophiuroides ecotypes (E102 and E092(1)) were used as parents of the mapping population. E102 originated from Georgia in the USA, whereas E092(1) originated from Chongqing City of southwest China. A controlled cross between the two parents was performed in the nursery at the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, China. An F1 mapping population consisting of 89 individuals was derived by crossing E102 with E092(1). The true F1 plants, confirmed using six SRAP markers with different heterozygous alleles in both parents, were used as the mapping population. Genomic DNA from the F1 individuals and the two parents was
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extracted using the cetyltrimethylammonium bromide (CTAB) protocol (Murray and Thompson, 1980). DNA concentration was estimated by the intensity of fluorescence of ethidium bromidestained samples on 0.8% agarose gels. 2.2. SRAP profiling A set of 40 SRAP primers (20 forward and 20 reverse primers, see Table 1) was designed following Li and Quiros (2001), giving a total of 400 primer combinations to search for polymorphism between the parents. Informative primer combinations were used to genotype the mapping population. The PCR amplifications were carried out in a 20 L reaction mixture containing 60 ng genomic DNA, 260 mol/L dNTPs (Invitrogen), 1.5 mmol/L MgCl2 , 0.25 mol/L primer (Invitrogen), 2 L Taq buffer, and 0.5 U Taq DNA polymerase (Promega). The procedure for PCR was conducted according to Li and Quiros (2001) as follows: an initial denaturing step was performed at 94 ◦ C for 5 min followed by 5 cycles of 94 ◦ C for 1 min, 35 ◦ C for 1 min, and 72 ◦ C for 1 min, followed by 35 cycles of 94 ◦ C for 1 min, 50 ◦ C for 1 min, and 72 ◦ C for 1 min, with a final extension step at 72 ◦ C for 7 min. PCR products were electrophoresed through 10% non-denaturing polyacrylamide gels (acrylamide-bisacrylamide (19:1), 1× TBE). Following electrophoresis, the gel was stained with AgNO3 solution. 2.3. EST-SSR profiling A total of 301 EST-SSR primer pairs from wheat were selected for genetic mapping (provided by Xiu E Wang, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Cytogenetics Institute, Nanjing Agricultural University), of which 85 pairs were from homoeologous group 1, 100 pairs were from homoeologous group 4, and 116 pairs were from homoeologous group 6 of wheat (Yu et al., 2004; Xue et al., 2008). PCR amplifications were performed in a volume of 10 L, each containing 1× PCR buffer (10 mmol/L Tris–HCl pH 8.3, 50 mmol/L KCl), 1 mmol/L MgCl2 , 200 mol/L dNTPs (Invitrogen), 4 ng SSR primer, 1 U Taq polymerase (Promega), and 10 ng template DNA. The following PCR profile was used: initial denaturation at 94 ◦ C for 3 min, 35 cycles of 94 ◦ C for 50 s, X ◦ C (X ◦ C was determined empirically for each primer pair) for 1 min, 72 ◦ C for 1 min, and a final extension at 72 ◦ C for 10 min (Wang et al., 2007). Amplification fragments were separated on 8% non-denaturing polyacrylamide gels (acrylamide-bis-acrylamide (19:1), 1× TBE). Following electrophoresis, the gel was stained with AgNO3 solution. 2.4. Marker nomenclature and segregation analysis The value of each marker was assigned according to its presence or absence in each progeny, i.e. if the fragment was present in a progeny, it was designated as an “1”, and if the fragment was absent from the corresponding position of a progeny, the value was an “0”. Ambiguous and lost fragments were designated as “–”. Each fragment was identified by the primer pair combination used to amplify it, along with a sequential suffix, numbered in descending order of molecular weight. The segregation of markers heterozygous in one parent only (testcross markers) was tested against a Mendelian segregation ratio of 1:1 using a 2 test (P < 0.05), while those heterozygous in both parents (intercross markers) were tested against a 3:1 ratio (P < 0.05). Segregation of markers that did not fit either ratio stated above was treated as distorted. 2.5. Map construction According to the double pseudo-testcross mapping strategy (Grattapaglia and Sederoff, 1994), the markers were separated
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Table 1 SRAP primer sequences used for polymorphism analysis in the mapping population. Forward primer
Sequence (5 –3 )
Reverse primer
Sequence (5 –3 )
Me1 Me2 Me3 Me4 Me5 Me6 Me7 Me8 Me9 Me10 Me11 Me12 Me13 Me14 Me15 Me16 Me17 Me18 Me19 Me20
TGAGTCCAAACCGGATA TGAGTCCAAACCGGAGC TGAGTCCAAACCGGACC TGAGTCCAAACCGGACA TGAGTCCAAACCGGTGC TGAGTCCAAACCGGAGA TGAGTCCAAACCGGACG TGAGTCCAAACCGGAAA TGAGTCCAAACCGGAAC TGAGTCCAAACCGGAAT TGAGTCCAAACCGGAAG TGAGTCCAAACCGGTAG TGAGTCCAAACCGGTTG TGAGTCCAAACCGGTGT TGAGTCCAAACCGGTCA TGAGTCCAAACCGGGAC TGAGTCCAAACCGGGTA TGAGTCCAAACCGGGGT TGAGTCCAAACCGGCAG TGAGTCCAAACCGGCAT
Em1 Em2 Em3 Em4 Em5 Em6 Em7 Em8 Em9 Em10 Em11 Em12 Em13 Em14 Em15 Em16 Em17 Em18 Em19 Em20
GACTGCGTACGAATTCAA GACTGCGTACGAATTCTG GACTGCGTACGAATTGAC GACTGCGTACGAATTTGA GACTGCGTACGAATTAAC GACTGCGTACGAATTGCA GACTGCGTACGAATTGAG GACTGCGTACGAATTGCC GACTGCGTACGAATTTCA GACTGCGTACGAATTCAT GACTGCGTACGAATTAAT GACTGCGTACGAATTTGC GACTGCGTACGAATTCGA GACTGCGTACGAATTATG GACTGCGTACGAATTAGC GACTGCGTACGAATTACG GACTGCGTACGAATTTAG GACTGCGTACGAATTTCG GACTGCGTACGAATTGTC GACTGCGTACGAATTGGT
into two parental segregation patterns. Each pattern contained the testcross markers segregation from the respective parent and the intercross presenting in both parents. Linkage maps were generated for each parent independently. Map construction in each parent was carried out using Joinmap v3.0 (Van Ooijen and Voorrips, 2001) by applying a LOD threshold of 4.0. The calculation of the linkage maps utilized all pair-wise recombination estimation of <0.45 and a LOD score >0.01. The Kosambi (1944) mapping function was used to convert recombination fractions into centiMorgand (cM). Homologous linkage groups between the parental maps were attempted using the intercross markers shared by the linkage groups of both parents. The resulting linkage maps were drawn by using MapChart 2.1 software (Voorrips, 2002). 2.6. Estimation of genome length The expected size of the centipedegrass genome was estimated by Li [(ki + 1)/(ki − 1)] as described by Chakravarti et al. (1991), where L is the length of linkage group (cM), k is the number of marker loci, ki is the number of marker loci on the ith linkage group, where i = 1, 2, . . ., t, and t is the number of linkage groups. Genome coverage was estimated by the ratio between the cumulative map length and the expected genome size. 3. Results 3.1. Polymorphism and segregation of molecular markers Of the 400 SRAP primer combinations applied to the two parental DNA samples, 162 (40.5%) were informative and used to genotype the mapping population. In total, 288 polymorphic bands were generated. The number of polymorphic bands produced per
primer combination ranged from one to four (mean 1.8). Of these polymorphic bands, 191 (66.3%) fit a 1:1 segregation ratio, 16 (5.6%) fit a 3:1 ratio, and the remaining 81 (28.1%) showed segregation distortion. Among the markers segregating only in E102, 103 (72.0%) segregated in a 1:1 ratio, and 40 (28.0%) were distorted, while in E092(1), the respective numbers were 88 (72.1%) and 34 (27.9%). Of the 23 intercross markers, 16 (69.6%) fit the expected 3:1 ratio, with the remaining seven (30.4%) showing segregation distortion (Table 2). Among the 301 EST-SSR primers screened from wheat, 183 (60.8%) generated clear and scored bands, and only 12 (6.6%) displayed a polymorphic banding pattern and were used to screen the mapping population generating 18 polymorphic fragments (1.5 polymorphic fragments per primer). Of the 18 EST-SSR loci, 14 (77.8%) were testcross markers, and the remaining four (22.2%) showed segregation distortion. Six of the markers present in E102 (66.7%) fit a 1:1 segregation ratio, and three (33.3%) were distorted, while in E092(1), the respective numbers were eight (88.9%) and one (11.1%). No EST-SSR markers were present in both parents (Table 2).
3.2. Construction of parental genetic maps The parental linkage maps were developed independently following the double pseudo-testcross mapping strategy. The SRAP and EST-SSR markers were used to construct the genetic linkage maps based on segregation data obtained from 89 F1 progeny. Two parental maps were constructed with markers segregating in a 1:1 and 3:1 ratio. Those markers present in both parents that segregated in a 3:1 ratio were used to find the homologous groups in the two parental maps.
Table 2 Segregation analysis of markers in the mapping population. Parameters SRAP Polymorphic markers Expected segregation ratio Segregation distortion (P < 0.05) EST-SSR Polymorphic markers Expected segregation ratio Segregation distortion (P < 0.05)
Present only in E102
Present only in E092(1)
Present in both parents
Total
143 103 (72.0%) 40 (28.0%)
122 88 (72.1%) 34 (27.9%)
23 16 (69.6%) 7 (30.4%)
288 207 (71.9%) 81 (28.1%)
9 6 (66.7%) 3 (33.3%)
9 8 (88.9%) 1 (11.1%)
The numbers in parentheses indicate the percentages.
0 0 0
18 14 (77.8%) 4 (22.2%)
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Fig. 1. E102 genetic linkage map of centipedegrass based on SRAP and EST-SSR markers at a LOD 4.0. *Markers showing segregation distortion at P = 0.05.
The maternal map was constructed with 85 SRAP and four ESTSSR loci, leaving 81 SRAP and five EST-SSR loci unlinked. A total of 89 marker loci were distributed in ten LGs covering a length of 623.6 cM, with the SRAP loci predominating in all LGs except LG 8. Wheat EST-SSRs were only present in LG 3 and LG 7. Five LGs linked four or more (maximum 30) markers, two triplet and three doublets, giving a mean of 8.9 markers per LG. The LGs varied in genetic length from 8.1–130.5 cM (mean 62.4 cM). The intermarker distance ranged from 0.02–38.3 cM (mean 7.1 cM), with 9 gaps of >20 cM. LG 1 was the longest, with 30 loci spanning 130.5 cM, whereas LG 10 was the shortest, with two loci spanning 8.1 cM (Table 3, Fig. 1). For the paternal genetic linkage map, 71 markers (all SRAP loci) were assignable to 12 LGs at a LOD score of 4.0 and covered 673.1 cM, leaving 74 SRAP and nine EST-SSR loci unlinked. Seven LGs contained at least four markers, five were triplets, giving a mean of 5.9 markers per LG. The LGs varied in genetic length from 11.7–119.1 cM, with an average LG length of 56.1 cM. The intermarker distance ranged from 0.5–37.5 cM (mean 9.6 cM), with 12 gaps of >20 cM. LG 1 was the longest, with 18 loci spanning 119.1 cM, whereas LG 12 was the shortest, with three loci spanning 11.7 cM (Table 3, Fig. 2). The comparison between male and female maps was based on common markers present in both parents (Figs. 1 and 2). In all, there were two common markers (Me2Em1-2 and Me2Em4-2). The Me2Em1-2 appeared in LG 3 of female map and LG 1 of male map. The Me2Em4-2 appeared in LG 2 of female map and LG 9 of male map.
3.3. Estimating genome length and map coverage Using method no. 4 of Chakravarti et al. (1991), we estimated a total genome size of 911.9 cM for the female parent and 1023.0 cM for the male parent. Based on these estimates for genome length, the coverage of the female and male maps was 68.5% and 65.8%, respectively. 4. Discussion 4.1. Map construction and marker distribution Centipedegrass is a perennial outbreeding turfgrass species with high heterozygosity and a self-incompatibility system, which hampers its breeding and complicates linkage mapping. The double pseudo-testcross mapping strategy (Grattapaglia and Sederoff, 1994) is an efficient way to construct molecular marker-based genetic maps in plant species like centipedegrass. In this study, we constructed the first PCR marker-based genetic linkage map of centipedegrass. Two comprehensive maps were developed, a maternal map consisting of 89 markers arranged on 10 LGs, with a total length of 623.6 cM and a mean inter-marker distance of 7.1 cM (Table 3, Fig. 1), and a paternal map that included 71 markers ordered into 12 LGs covering 673.1 cM, with a marker density of 9.6 cM (Table 3, Fig. 2). The maps represent 68.5% and 65.8% coverage of the estimated genome length, respectively. The estimated genome size and the length of the paternal map were both higher than those of the maternal map. Differences in map length
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Fig. 2. E092(1) genetic linkage map of centipedegrass based on SRAP and EST-SSR markers at a LOD 4.0. *Markers showing segregation distortion at P = 0.05.
may indicate a reduced level of recombination in the female parent. Differences in recombination frequency between male and female parents have been reported in other species (Warnke et al., 2004; Saha et al., 2005). In theory, the number of LGs should be consistent with the number of haploid chromosomes. As a diploid species, the number of chromosomes for centipedegrass is 2n = 18. The number of LGs in parental map was more than the number of haploid chromosomes, which has been reported in many other species (Xie et al., 2011; Zhang et al., 2011; Lu et al., 2012). The LGs would become fused if additional markers were included. Adding more individuals and using different types of mapping populations would also integrate the linkage groups. Future in situ hybridization studies using centipedegrass-specific genome probes may be useful for determining the relationships between LGs and chromosomes. The marker density in the parental maps varied from 7.1 cM (the maternal map) to 9.6 cM (the paternal map) per marker. Markers were highly clustered in some regions, but there were gaps of >20 cM in some LGs (9 gaps in the maternal map and 12 gaps in the paternal map). To fill the gaps, additional markers of different types and a larger population size are needed. In an out-breeding population, however, linkage maps can be constructed for each parent separately, but these can only be aligned if sufficient intercross markers are available. If most of the markers are dominant, the only possibility is to establish a bridge by means of a 3:1 markers present in both parents linked to a 1:1 marker present in one parents, which is a configuration with low information (Brennan et al., 2008). If enough markers are in common between maps, the maps can be combined into a single integrated linkage map (Peach et al., 2003; Saha et al., 2005). In our research, 16 out of 306 polymorphic loci (5.2%) displayed
a 3:1 segregation ratio. We attempted to find the homologues between the genetic LGs of both parents using intercross markers after developing their frameworks. However, this approach failed, as there were few (only two) common markers between the LGs of the two parents. To align and compare the homology of both maps effectively, either more intercross markers need to be generated and mapped in a larger progeny set, or, preferably, more informative codominant marker types such as SSRs need to be placed on the maps. 4.2. Segregation distortion High levels of segregation distortion (29.5%) were observed in this study. Segregation distortion has been reported in tall fescue (Xu et al., 1995; Saha et al., 2005), ryegrass (Warnke et al., 2004), orchardgrass (Xie et al., 2011), and zoysiagrass (Cai et al., 2004). Various processes can cause segregation distortion, such as genetic isolation mechanisms, chromosome loss, structural rearrangement, genetic load, and viability genes (Kuang et al., 1999). Non-biological factors such as scoring errors and sampling errors can also lead to distortion in segregation ratios (Nikaido et al., 1999; Echt and Nelson, 1997). Centipedegrass, as a perennial outbreeding turfgrass, has a self-incompatibility system, and the genes linked to self-incompatibility could cause segregation distortion. However, the mapping population was developed by crossing two unrelated heterozygous parents. Thus, the involvement of selfincompatibility genes in segregation distortion is doubtful. Clustering of distorted markers was also observed in this study. In total, 75.7% of the skewed markers were distributed on linkage groups LG 1 and 3 in the female map, and LG 1 and 9 in the male map (Table 3, Figs. 1 and 2). This has also been reported in other species,
8
0 1 1 0 2 1 0 1 0 1 1 0
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such as rice (Xu et al., 1998), tomato (Foolad et al., 1998), ryegrass (Bert et al., 1999), and tall fescue (Saha et al., 2005). Such clustering reflects sites of reduced recombination (Beedanagari et al., 2005) or linkage with self-incompatibility genes (Warnke et al., 2004). Further study is needed to determine the main factor causing segregation distortion in centipedegrass.
12 9.6 673.1 71 0 71 9 25
18 7 8 10 4 3 4 3 5 3 3 3 0 2 0 1 2 2 2 0 0 0 – – 11 0 10 0 1 0 1 0 2 0 – –
SRAP
0 0 0 0 0 0 0 0 0 0 0 0
18 7 8 10 4 3 4 3 5 3 3 3
119.1 100.9 68.7 60.7 58.7 46.9 46.7 44.6 38.8 38.3 38.0 11.7
7.0 16.8 9.8 6.7 19.6 23.5 15.6 22.3 9.7 19.2 19.0 5.9
3 1 0 0 1 0 0 1 4 0 2 0
4.3. Transferability of wheat EST-SSR markers in centipedegrass
No. of gaps (>20 cM)
Genetic studies of centipedegrass are hindered because of the scarcity of molecular markers available. It has become widely accepted to screen EST-based markers derived from one species with other species in the same genus and even across genera within the same family. The transferability of EST-SSR markers from the crop family Poaceae has been examined in tall fescue (Saha et al., 2005), ryegrass (Sim et al., 2009), bermudagrass, creeping bentgrass, Kentucky bluegrass (Wang et al., 2005; Zeid et al., 2010), and orchardgrass (Xie et al., 2011). The results of these studies showed that EST-SSR markers from major cereal crops can be useful for application in minor grass species with few or no available molecular markers. In present study, a few EST-SSR markers of wheat were used to screen for their transferability and to construct the centipedegrass linkage map. Of the 301 EST-SSR primers screened from wheat, 183 (60.8%) generated clear and scored bands. The transfer rate of EST-SSR markers from wheat to centipedegrass was similar to that from wheat to bermudagrass (58.33%) and seashore paspalum (65.10%) (Wang et al., 2005). However, among these 183 EST-SSR primers, only 12 (6.6%) displayed a polymorphic banding pattern between parents, generating 18 polymorphic fragments. Ultimately, only four EST-SSR markers were linked in the genetic maps (all in the maternal maps). Although the EST-SSR markers from wheat used to construct the linkage map will be useful for the synteny studies of centipedegrass. However, the number of relevant markers was too low (only four) to study the detailed synteny between the two species. In the future, EST-SSRs from other homoeologous groups of wheat and other species could be used in centipedegrass. Acknowledgement This project was supported by the National Nature Science Foundation of China (no. 30670200 and no. 30972017).
7.1 623.6 4 85 Total/mean
89
0 0 1 0 0 0 3 0 0 0 – – 30 10 25 7 4 3 0 2 2 2 – –
30 10 26 7 4 3 3 2 2 2 – –
130.5 100.5 93.3 90.3 72.4 52.6 49.4 16.4 10.1 8.1 – –
4.5 11.2 3.7 15.1 24.1 26.3 24.7 16.4 10.1 8.1 – –
References
1 2 3 4 5 6 7 8 9 10 11 12
Marker density (cM/marker) Map length (cM) Total EST-SSR SRAP
E102
Parental map Linkage group
Table 3 Distribution of SRAP and EST-SSR marker loci in different groups of parental maps.
Markers segregation distortion
E092(1)
EST-SSR
Total
Map length (cM)
Marker density (cM/marker)
Markers segregation distortion
No. of gaps (>20 cM)
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Genetic linkage maps of centipedegrass [Eremochloa ophiuroides (Munro) Hack] based on sequence-related amplified polymorphism and expressed sequence tag-simple sequence repeat markers Yiqi Zheng a,b , Hailin Guo a , Guozhang Zang b , Jianxiu Liu a,∗ a b
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, PR China College of Forestry, Henan University of Science and Technology, Luoyang 471003, PR China
a r t i c l e
i n f o
Article history: Received 24 July 2012 Received in revised form 27 March 2013 Accepted 29 March 2013 Keywords: Centipedegrass Sequence-related amplified polymorphism (SRAP) Expressed sequence tag-simple sequence repeat (EST-SSR) Genetic mapping
a b s t r a c t Centipedegrass [Eremochloa ophiuroides (Munro) Hack] is a warm-season turfgrass native to the southeast of China. In this study, the first genetic linkage maps of centipedegrass were constructed using PCR-based markers based on a segregation population consisting of 89 F1 progeny derived from a cross between two ecotypes (E102 and E092(1)). A combination of sequence-related amplified polymorphisms (SRAPs) and expressed sequence tag-simple sequence repeats (EST-SSRs) from wheat was used for map construction. Eighty nine loci, including 85 SRAP and 4 EST-SSR loci, in the female (E102) linkage map were placed in five major and five minor (two triplet and three doublets) linkage maps, covering 623.6 cM with an average map distance of 7.1 cM between adjacent markers. In the male (E092(1)) linkage map, 71 loci including 71 SRAP and 0 EST-SSR loci were assigned to seven major and five triplets linkage maps, with genome coverage of 673.1 cM, and a mean inter-marker separation of 9.6 cM. The information presented in this study establishes a foundation for extending genetic mapping in this species, serves as a framework for mapping quantitative trait loci, and provides basic information for future molecular breeding. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Centipedegrass [Eremochloa ophiuroides (Munro) Hack] is one of the most important warm-season turfgrass native to Southeast Asia, with China being the probable area of origin (Hanson et al., 1969). Commonly referred to as “lazy man’s grass”, the management requirements of centipedegrass are generally considered lower than most warm-season turfgrass. It can tolerate a wide range of soil types, and is relatively resistant to insect and disease infestations. These low input requirements and high stress tolerances make centipedegrass popular for home lawns, landscaping, roadsides, and recreational fields in tropical and subtropical areas around the world (Hanna, 1995). This species reproduces sexually (2n = 2x = 18; Brown, 1950) and is commonly cross-pollinated because of a self-incompatibility mechanism (Hanna and Burton, 1978). Genetic linkage mapping is useful for determining the locations of genes on a chromosome, with order and position based on recombination frequencies observed in progeny populations. Plant genome maps may include important linkage relationships among molecular markers and trait genes that breeders wish to
∗ Corresponding author. Tel.: +86 025 84347012; fax: +86 025 84347057. E-mail address: [email protected] (J. Liu). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.03.030
manipulate for cultivar improvement to increase the efficiency of breeding programs. The genetic improvement of centipedegrass is impaired by its high heterozygosity and self-incompatibility. The latter makes linkage mapping of centipedegrass more complicated than that of other species that can readily be inbred. The double pseudo-testcross mapping strategy (Grattapaglia and Sederoff, 1994) is considered to be an efficient way to construct molecular genetic maps in plant species like centipedegrass. This method has been applied to a number of perennial fruit and forest trees (Beedanagari et al., 2005; Lowe and Walker, 2006), as well as to ornamental plants such as lily (Abe et al., 2002), rose (Dugo et al., 2005; Oyant et al., 2008), and chrysanthemum (Zhang et al., 2010, 2011). At present, a number of linkage maps have been constructed in turf and forage grasses, including tall fescue (Festuca arundinacea Schreb.; Xu et al., 1995; Saha et al., 2005), ryegrass (Lolium perenne L.; Bert et al., 1999; Warnke et al., 2004), zoysiagrass (Zoysia japonica Steud.; Cai et al., 2004; Li et al., 2009), bermudagrass (Cynodon dactylon × transvaalensis; Bethel et al., 2006), Kentucky bluegrass (Poa pratensis L.; Porceddu et al., 2002), and orchardgrass (Dactylis glomerata L.; Xie et al., 2011). However, there have been no reports of genetic linkage studies conducted in centipedegrass. SRAP (sequence-related amplified polymorphism) is a new PCRbased marker (Li and Quiros, 2001). This method aims to amplify open reading frames with particular primer pairs. It combines simplicity, reliability, moderate throughout ratio, and facile sequencing
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of selected bands. As a novel molecular marker, SRAP has been applied in map construction, locating genes, quantitative trait loci (QTL) mapping (Lin et al., 2005; Okazaki et al., 2007; Sun et al., 2007; Xie et al., 2011; Zhang et al., 2011), comparative genetics (Li et al., 2003), and genetic diversity assessment (Ferrio et al., 2003; Budak et al., 2004; Milla-Lewis et al., 2011). Furthermore, SRAP does not require gene sequence information. This is particularly useful for plant species such as centipedegrass with poorly researched genomes. Microsatellites or simple sequence repeats (SSRs) are a ubiquitous class of repetitive DNA sequences consisting of tandemrepeated short motifs (one to six nucleotides) (Morgante et al., 2002). SSR markers developed from genomic libraries (gSSR) have been widely used for mapping and population genetic analysis because of their high level of polymorphism, abundance and dispersion throughout the genome, codominant nature of inheritance, and reproducibility (Gupta and Varshney, 2000; Squirrell et al., 2003). Nevertheless, there are several disadvantages to gSSR markers, including high initial cost of development and low transferability across genera and beyond (Roa et al., 2000; Kindiger, 2006). However, with the increase in cDNA sequence information in public databases, exploitation of microsatellites from expressed sequence tags (ESTs) has become an attractive alternative strategy (Gupta and Varshney, 2000). Additionally, EST-SSRs are derived from transcripts, wherein flanking regions are expected to be more conserved, and thus tend to show higher levels of transferability in cross-amplification, and the possibility of tagging and mapping of genes and QTLs (Varshney et al., 2005). The map location of such SSRs may provide functionally associated genetic markers for direct characterization of the QTLs for putatively correlated traits. This would be particularly advantageous for mapping the genome of species with relatively little available genomic information, such as centipedegrass. A number of studies on the transferability of ESTSSRs in turf and forage grasses have been performed (Saha et al., 2005; Wang et al., 2005; Sim et al., 2009; Zeid et al., 2010; Xie et al., 2011). However, the identification and use of PCR-based markers in centipedegrass lags behind other major turf and forage grasses. To date there are only three types of molecular markers used in centipedegrass, DNA amplification fingerprinting (DAF) (Weaver et al., 1995), random amplification of polymorphic DNA (RAPD) (Xuan et al., 2005), and SRAP (Milla-Lewis et al., 2011), all of which were used only to evaluate the genetic diversity. In the present study, a genetic linkage map of centipedegrass based on a double pseudo-testcross mapping strategy was constructed using SRAP and EST-SSR markers from wheat. To our knowledge, this is the first report of linkage maps for centipedegrass. These maps will be valuable for studies to identify markers associated with traits of interest and for comparative analysis with other grass species.
2. Materials and methods 2.1. Plant material and DNA isolation Two E. ophiuroides ecotypes (E102 and E092(1)) were used as parents of the mapping population. E102 originated from Georgia in the USA, whereas E092(1) originated from Chongqing City of southwest China. A controlled cross between the two parents was performed in the nursery at the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, China. An F1 mapping population consisting of 89 individuals was derived by crossing E102 with E092(1). The true F1 plants, confirmed using six SRAP markers with different heterozygous alleles in both parents, were used as the mapping population. Genomic DNA from the F1 individuals and the two parents was
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extracted using the cetyltrimethylammonium bromide (CTAB) protocol (Murray and Thompson, 1980). DNA concentration was estimated by the intensity of fluorescence of ethidium bromidestained samples on 0.8% agarose gels. 2.2. SRAP profiling A set of 40 SRAP primers (20 forward and 20 reverse primers, see Table 1) was designed following Li and Quiros (2001), giving a total of 400 primer combinations to search for polymorphism between the parents. Informative primer combinations were used to genotype the mapping population. The PCR amplifications were carried out in a 20 L reaction mixture containing 60 ng genomic DNA, 260 mol/L dNTPs (Invitrogen), 1.5 mmol/L MgCl2 , 0.25 mol/L primer (Invitrogen), 2 L Taq buffer, and 0.5 U Taq DNA polymerase (Promega). The procedure for PCR was conducted according to Li and Quiros (2001) as follows: an initial denaturing step was performed at 94 ◦ C for 5 min followed by 5 cycles of 94 ◦ C for 1 min, 35 ◦ C for 1 min, and 72 ◦ C for 1 min, followed by 35 cycles of 94 ◦ C for 1 min, 50 ◦ C for 1 min, and 72 ◦ C for 1 min, with a final extension step at 72 ◦ C for 7 min. PCR products were electrophoresed through 10% non-denaturing polyacrylamide gels (acrylamide-bisacrylamide (19:1), 1× TBE). Following electrophoresis, the gel was stained with AgNO3 solution. 2.3. EST-SSR profiling A total of 301 EST-SSR primer pairs from wheat were selected for genetic mapping (provided by Xiu E Wang, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Cytogenetics Institute, Nanjing Agricultural University), of which 85 pairs were from homoeologous group 1, 100 pairs were from homoeologous group 4, and 116 pairs were from homoeologous group 6 of wheat (Yu et al., 2004; Xue et al., 2008). PCR amplifications were performed in a volume of 10 L, each containing 1× PCR buffer (10 mmol/L Tris–HCl pH 8.3, 50 mmol/L KCl), 1 mmol/L MgCl2 , 200 mol/L dNTPs (Invitrogen), 4 ng SSR primer, 1 U Taq polymerase (Promega), and 10 ng template DNA. The following PCR profile was used: initial denaturation at 94 ◦ C for 3 min, 35 cycles of 94 ◦ C for 50 s, X ◦ C (X ◦ C was determined empirically for each primer pair) for 1 min, 72 ◦ C for 1 min, and a final extension at 72 ◦ C for 10 min (Wang et al., 2007). Amplification fragments were separated on 8% non-denaturing polyacrylamide gels (acrylamide-bis-acrylamide (19:1), 1× TBE). Following electrophoresis, the gel was stained with AgNO3 solution. 2.4. Marker nomenclature and segregation analysis The value of each marker was assigned according to its presence or absence in each progeny, i.e. if the fragment was present in a progeny, it was designated as an “1”, and if the fragment was absent from the corresponding position of a progeny, the value was an “0”. Ambiguous and lost fragments were designated as “–”. Each fragment was identified by the primer pair combination used to amplify it, along with a sequential suffix, numbered in descending order of molecular weight. The segregation of markers heterozygous in one parent only (testcross markers) was tested against a Mendelian segregation ratio of 1:1 using a 2 test (P < 0.05), while those heterozygous in both parents (intercross markers) were tested against a 3:1 ratio (P < 0.05). Segregation of markers that did not fit either ratio stated above was treated as distorted. 2.5. Map construction According to the double pseudo-testcross mapping strategy (Grattapaglia and Sederoff, 1994), the markers were separated
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Table 1 SRAP primer sequences used for polymorphism analysis in the mapping population. Forward primer
Sequence (5 –3 )
Reverse primer
Sequence (5 –3 )
Me1 Me2 Me3 Me4 Me5 Me6 Me7 Me8 Me9 Me10 Me11 Me12 Me13 Me14 Me15 Me16 Me17 Me18 Me19 Me20
TGAGTCCAAACCGGATA TGAGTCCAAACCGGAGC TGAGTCCAAACCGGACC TGAGTCCAAACCGGACA TGAGTCCAAACCGGTGC TGAGTCCAAACCGGAGA TGAGTCCAAACCGGACG TGAGTCCAAACCGGAAA TGAGTCCAAACCGGAAC TGAGTCCAAACCGGAAT TGAGTCCAAACCGGAAG TGAGTCCAAACCGGTAG TGAGTCCAAACCGGTTG TGAGTCCAAACCGGTGT TGAGTCCAAACCGGTCA TGAGTCCAAACCGGGAC TGAGTCCAAACCGGGTA TGAGTCCAAACCGGGGT TGAGTCCAAACCGGCAG TGAGTCCAAACCGGCAT
Em1 Em2 Em3 Em4 Em5 Em6 Em7 Em8 Em9 Em10 Em11 Em12 Em13 Em14 Em15 Em16 Em17 Em18 Em19 Em20
GACTGCGTACGAATTCAA GACTGCGTACGAATTCTG GACTGCGTACGAATTGAC GACTGCGTACGAATTTGA GACTGCGTACGAATTAAC GACTGCGTACGAATTGCA GACTGCGTACGAATTGAG GACTGCGTACGAATTGCC GACTGCGTACGAATTTCA GACTGCGTACGAATTCAT GACTGCGTACGAATTAAT GACTGCGTACGAATTTGC GACTGCGTACGAATTCGA GACTGCGTACGAATTATG GACTGCGTACGAATTAGC GACTGCGTACGAATTACG GACTGCGTACGAATTTAG GACTGCGTACGAATTTCG GACTGCGTACGAATTGTC GACTGCGTACGAATTGGT
into two parental segregation patterns. Each pattern contained the testcross markers segregation from the respective parent and the intercross presenting in both parents. Linkage maps were generated for each parent independently. Map construction in each parent was carried out using Joinmap v3.0 (Van Ooijen and Voorrips, 2001) by applying a LOD threshold of 4.0. The calculation of the linkage maps utilized all pair-wise recombination estimation of <0.45 and a LOD score >0.01. The Kosambi (1944) mapping function was used to convert recombination fractions into centiMorgand (cM). Homologous linkage groups between the parental maps were attempted using the intercross markers shared by the linkage groups of both parents. The resulting linkage maps were drawn by using MapChart 2.1 software (Voorrips, 2002). 2.6. Estimation of genome length The expected size of the centipedegrass genome was estimated by Li [(ki + 1)/(ki − 1)] as described by Chakravarti et al. (1991), where L is the length of linkage group (cM), k is the number of marker loci, ki is the number of marker loci on the ith linkage group, where i = 1, 2, . . ., t, and t is the number of linkage groups. Genome coverage was estimated by the ratio between the cumulative map length and the expected genome size. 3. Results 3.1. Polymorphism and segregation of molecular markers Of the 400 SRAP primer combinations applied to the two parental DNA samples, 162 (40.5%) were informative and used to genotype the mapping population. In total, 288 polymorphic bands were generated. The number of polymorphic bands produced per
primer combination ranged from one to four (mean 1.8). Of these polymorphic bands, 191 (66.3%) fit a 1:1 segregation ratio, 16 (5.6%) fit a 3:1 ratio, and the remaining 81 (28.1%) showed segregation distortion. Among the markers segregating only in E102, 103 (72.0%) segregated in a 1:1 ratio, and 40 (28.0%) were distorted, while in E092(1), the respective numbers were 88 (72.1%) and 34 (27.9%). Of the 23 intercross markers, 16 (69.6%) fit the expected 3:1 ratio, with the remaining seven (30.4%) showing segregation distortion (Table 2). Among the 301 EST-SSR primers screened from wheat, 183 (60.8%) generated clear and scored bands, and only 12 (6.6%) displayed a polymorphic banding pattern and were used to screen the mapping population generating 18 polymorphic fragments (1.5 polymorphic fragments per primer). Of the 18 EST-SSR loci, 14 (77.8%) were testcross markers, and the remaining four (22.2%) showed segregation distortion. Six of the markers present in E102 (66.7%) fit a 1:1 segregation ratio, and three (33.3%) were distorted, while in E092(1), the respective numbers were eight (88.9%) and one (11.1%). No EST-SSR markers were present in both parents (Table 2).
3.2. Construction of parental genetic maps The parental linkage maps were developed independently following the double pseudo-testcross mapping strategy. The SRAP and EST-SSR markers were used to construct the genetic linkage maps based on segregation data obtained from 89 F1 progeny. Two parental maps were constructed with markers segregating in a 1:1 and 3:1 ratio. Those markers present in both parents that segregated in a 3:1 ratio were used to find the homologous groups in the two parental maps.
Table 2 Segregation analysis of markers in the mapping population. Parameters SRAP Polymorphic markers Expected segregation ratio Segregation distortion (P < 0.05) EST-SSR Polymorphic markers Expected segregation ratio Segregation distortion (P < 0.05)
Present only in E102
Present only in E092(1)
Present in both parents
Total
143 103 (72.0%) 40 (28.0%)
122 88 (72.1%) 34 (27.9%)
23 16 (69.6%) 7 (30.4%)
288 207 (71.9%) 81 (28.1%)
9 6 (66.7%) 3 (33.3%)
9 8 (88.9%) 1 (11.1%)
The numbers in parentheses indicate the percentages.
0 0 0
18 14 (77.8%) 4 (22.2%)
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Fig. 1. E102 genetic linkage map of centipedegrass based on SRAP and EST-SSR markers at a LOD 4.0. *Markers showing segregation distortion at P = 0.05.
The maternal map was constructed with 85 SRAP and four ESTSSR loci, leaving 81 SRAP and five EST-SSR loci unlinked. A total of 89 marker loci were distributed in ten LGs covering a length of 623.6 cM, with the SRAP loci predominating in all LGs except LG 8. Wheat EST-SSRs were only present in LG 3 and LG 7. Five LGs linked four or more (maximum 30) markers, two triplet and three doublets, giving a mean of 8.9 markers per LG. The LGs varied in genetic length from 8.1–130.5 cM (mean 62.4 cM). The intermarker distance ranged from 0.02–38.3 cM (mean 7.1 cM), with 9 gaps of >20 cM. LG 1 was the longest, with 30 loci spanning 130.5 cM, whereas LG 10 was the shortest, with two loci spanning 8.1 cM (Table 3, Fig. 1). For the paternal genetic linkage map, 71 markers (all SRAP loci) were assignable to 12 LGs at a LOD score of 4.0 and covered 673.1 cM, leaving 74 SRAP and nine EST-SSR loci unlinked. Seven LGs contained at least four markers, five were triplets, giving a mean of 5.9 markers per LG. The LGs varied in genetic length from 11.7–119.1 cM, with an average LG length of 56.1 cM. The intermarker distance ranged from 0.5–37.5 cM (mean 9.6 cM), with 12 gaps of >20 cM. LG 1 was the longest, with 18 loci spanning 119.1 cM, whereas LG 12 was the shortest, with three loci spanning 11.7 cM (Table 3, Fig. 2). The comparison between male and female maps was based on common markers present in both parents (Figs. 1 and 2). In all, there were two common markers (Me2Em1-2 and Me2Em4-2). The Me2Em1-2 appeared in LG 3 of female map and LG 1 of male map. The Me2Em4-2 appeared in LG 2 of female map and LG 9 of male map.
3.3. Estimating genome length and map coverage Using method no. 4 of Chakravarti et al. (1991), we estimated a total genome size of 911.9 cM for the female parent and 1023.0 cM for the male parent. Based on these estimates for genome length, the coverage of the female and male maps was 68.5% and 65.8%, respectively. 4. Discussion 4.1. Map construction and marker distribution Centipedegrass is a perennial outbreeding turfgrass species with high heterozygosity and a self-incompatibility system, which hampers its breeding and complicates linkage mapping. The double pseudo-testcross mapping strategy (Grattapaglia and Sederoff, 1994) is an efficient way to construct molecular marker-based genetic maps in plant species like centipedegrass. In this study, we constructed the first PCR marker-based genetic linkage map of centipedegrass. Two comprehensive maps were developed, a maternal map consisting of 89 markers arranged on 10 LGs, with a total length of 623.6 cM and a mean inter-marker distance of 7.1 cM (Table 3, Fig. 1), and a paternal map that included 71 markers ordered into 12 LGs covering 673.1 cM, with a marker density of 9.6 cM (Table 3, Fig. 2). The maps represent 68.5% and 65.8% coverage of the estimated genome length, respectively. The estimated genome size and the length of the paternal map were both higher than those of the maternal map. Differences in map length
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Fig. 2. E092(1) genetic linkage map of centipedegrass based on SRAP and EST-SSR markers at a LOD 4.0. *Markers showing segregation distortion at P = 0.05.
may indicate a reduced level of recombination in the female parent. Differences in recombination frequency between male and female parents have been reported in other species (Warnke et al., 2004; Saha et al., 2005). In theory, the number of LGs should be consistent with the number of haploid chromosomes. As a diploid species, the number of chromosomes for centipedegrass is 2n = 18. The number of LGs in parental map was more than the number of haploid chromosomes, which has been reported in many other species (Xie et al., 2011; Zhang et al., 2011; Lu et al., 2012). The LGs would become fused if additional markers were included. Adding more individuals and using different types of mapping populations would also integrate the linkage groups. Future in situ hybridization studies using centipedegrass-specific genome probes may be useful for determining the relationships between LGs and chromosomes. The marker density in the parental maps varied from 7.1 cM (the maternal map) to 9.6 cM (the paternal map) per marker. Markers were highly clustered in some regions, but there were gaps of >20 cM in some LGs (9 gaps in the maternal map and 12 gaps in the paternal map). To fill the gaps, additional markers of different types and a larger population size are needed. In an out-breeding population, however, linkage maps can be constructed for each parent separately, but these can only be aligned if sufficient intercross markers are available. If most of the markers are dominant, the only possibility is to establish a bridge by means of a 3:1 markers present in both parents linked to a 1:1 marker present in one parents, which is a configuration with low information (Brennan et al., 2008). If enough markers are in common between maps, the maps can be combined into a single integrated linkage map (Peach et al., 2003; Saha et al., 2005). In our research, 16 out of 306 polymorphic loci (5.2%) displayed
a 3:1 segregation ratio. We attempted to find the homologues between the genetic LGs of both parents using intercross markers after developing their frameworks. However, this approach failed, as there were few (only two) common markers between the LGs of the two parents. To align and compare the homology of both maps effectively, either more intercross markers need to be generated and mapped in a larger progeny set, or, preferably, more informative codominant marker types such as SSRs need to be placed on the maps. 4.2. Segregation distortion High levels of segregation distortion (29.5%) were observed in this study. Segregation distortion has been reported in tall fescue (Xu et al., 1995; Saha et al., 2005), ryegrass (Warnke et al., 2004), orchardgrass (Xie et al., 2011), and zoysiagrass (Cai et al., 2004). Various processes can cause segregation distortion, such as genetic isolation mechanisms, chromosome loss, structural rearrangement, genetic load, and viability genes (Kuang et al., 1999). Non-biological factors such as scoring errors and sampling errors can also lead to distortion in segregation ratios (Nikaido et al., 1999; Echt and Nelson, 1997). Centipedegrass, as a perennial outbreeding turfgrass, has a self-incompatibility system, and the genes linked to self-incompatibility could cause segregation distortion. However, the mapping population was developed by crossing two unrelated heterozygous parents. Thus, the involvement of selfincompatibility genes in segregation distortion is doubtful. Clustering of distorted markers was also observed in this study. In total, 75.7% of the skewed markers were distributed on linkage groups LG 1 and 3 in the female map, and LG 1 and 9 in the male map (Table 3, Figs. 1 and 2). This has also been reported in other species,
8
0 1 1 0 2 1 0 1 0 1 1 0
91
such as rice (Xu et al., 1998), tomato (Foolad et al., 1998), ryegrass (Bert et al., 1999), and tall fescue (Saha et al., 2005). Such clustering reflects sites of reduced recombination (Beedanagari et al., 2005) or linkage with self-incompatibility genes (Warnke et al., 2004). Further study is needed to determine the main factor causing segregation distortion in centipedegrass.
12 9.6 673.1 71 0 71 9 25
18 7 8 10 4 3 4 3 5 3 3 3 0 2 0 1 2 2 2 0 0 0 – – 11 0 10 0 1 0 1 0 2 0 – –
SRAP
0 0 0 0 0 0 0 0 0 0 0 0
18 7 8 10 4 3 4 3 5 3 3 3
119.1 100.9 68.7 60.7 58.7 46.9 46.7 44.6 38.8 38.3 38.0 11.7
7.0 16.8 9.8 6.7 19.6 23.5 15.6 22.3 9.7 19.2 19.0 5.9
3 1 0 0 1 0 0 1 4 0 2 0
4.3. Transferability of wheat EST-SSR markers in centipedegrass
No. of gaps (>20 cM)
Genetic studies of centipedegrass are hindered because of the scarcity of molecular markers available. It has become widely accepted to screen EST-based markers derived from one species with other species in the same genus and even across genera within the same family. The transferability of EST-SSR markers from the crop family Poaceae has been examined in tall fescue (Saha et al., 2005), ryegrass (Sim et al., 2009), bermudagrass, creeping bentgrass, Kentucky bluegrass (Wang et al., 2005; Zeid et al., 2010), and orchardgrass (Xie et al., 2011). The results of these studies showed that EST-SSR markers from major cereal crops can be useful for application in minor grass species with few or no available molecular markers. In present study, a few EST-SSR markers of wheat were used to screen for their transferability and to construct the centipedegrass linkage map. Of the 301 EST-SSR primers screened from wheat, 183 (60.8%) generated clear and scored bands. The transfer rate of EST-SSR markers from wheat to centipedegrass was similar to that from wheat to bermudagrass (58.33%) and seashore paspalum (65.10%) (Wang et al., 2005). However, among these 183 EST-SSR primers, only 12 (6.6%) displayed a polymorphic banding pattern between parents, generating 18 polymorphic fragments. Ultimately, only four EST-SSR markers were linked in the genetic maps (all in the maternal maps). Although the EST-SSR markers from wheat used to construct the linkage map will be useful for the synteny studies of centipedegrass. However, the number of relevant markers was too low (only four) to study the detailed synteny between the two species. In the future, EST-SSRs from other homoeologous groups of wheat and other species could be used in centipedegrass. Acknowledgement This project was supported by the National Nature Science Foundation of China (no. 30670200 and no. 30972017).
7.1 623.6 4 85 Total/mean
89
0 0 1 0 0 0 3 0 0 0 – – 30 10 25 7 4 3 0 2 2 2 – –
30 10 26 7 4 3 3 2 2 2 – –
130.5 100.5 93.3 90.3 72.4 52.6 49.4 16.4 10.1 8.1 – –
4.5 11.2 3.7 15.1 24.1 26.3 24.7 16.4 10.1 8.1 – –
References
1 2 3 4 5 6 7 8 9 10 11 12
Marker density (cM/marker) Map length (cM) Total EST-SSR SRAP
E102
Parental map Linkage group
Table 3 Distribution of SRAP and EST-SSR marker loci in different groups of parental maps.
Markers segregation distortion
E092(1)
EST-SSR
Total
Map length (cM)
Marker density (cM/marker)
Markers segregation distortion
No. of gaps (>20 cM)
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