A Genetic Linkage Map of Diploid Paspalum notatum.
Paspalum notatum Flugge is a rhizomatous species widely distributed in native grasslands from central Mexico to Argentina and throughout the Caribbean Islands (Chase, 1929). The diploid form, Pensacola bahiagrass (P. notatum var. saurae Parodi, 2n = 2x = 20), is one of the most popular perennial pasture grasses in the southern USA (Burton, 1974). Although it was named after the Pensacola region of Florida, it probably originated from a relatively small area of central-eastern and northeastern Argentina (Burton, 1967). The species is sexual and self-incompatible (Burton, 1955).
Molecular marker technologies have led to the rapid development of detailed genetic maps for many crop plants (Martin, 1998). Genetic linkage maps provide a framework for studying simple and complex traits. Moreover, the possibility of using common RFLP probes across several taxa provides a means of comparing maps between both related and unrelated species (Ahn and Tanksley, 1993). Comparative studies in the Gramineae have led to the observation of an extensive similarity between genomes of several species (Devos and Gale, 1997; Glaszmann et al., 1997; Moore et al., 1995). With regard to the forage grasses, genetic linkage maps are available for Lolium, where a number of important quantitative trait loci (QTLs) have been identified (Bert et al., 1999; Hayward et al., 1994, 1998). Likewise, a map of Setaria italica (L.) P. Beauv. and its comparative analysis with rice has been reported (Devos et al., 1998; Wang et al., 1998). Among the apomictic grasses, several markers linked to the genomic region controlling this reproductive mode have been identified in Pennisetum and Brachiaria (Gustine et al., 1997; Ozias-Akins et al., 1993; Pessino et al., 1997; 1998). Moreover, an apospory-specific genomic region (ASGR) with restricted genetic recombination has been identified in Pennisetum hybrids (Ozias-Akins et al., 1998).
The objective of this work was the construction of a genetic linkage map of diploid Paspalum notatum to be used in genetic studies as well as in programs involving the breeding of the species. The strategy of map construction was based on a comparative approach that employed heterologous RFLP clones evenly distributed over the maize and rice genetic maps to cover the Paspalum genome uniformly and together with random markers (RAPD and AFLP) to condense the linkage groups. In addition, several markers reported to be associated with the apomictic mode of reproduction in the Gramineae were located on the map.
MATERIALS AND METHODS
The mapping population was generated at IBONE, Corrientes, Argentina, in the summer of 1995-1996. Crosses were made between two diploid genotypes (2n = 2x = 20) of Paspalum notatum. The female parent ([Q4084.sub.10]) was collected from a natural population occurring at Cayasta, Santa Fe province, Argentina. The pollen donor plant ([Tift.sub.9]) originated from the Coastal Plain Experimental Station, Tifton, GA, USA. Although diploid races are highly self-sterile (Burton, 1955), emasculation of the female parent was carried out to prevent the generation of self-pollinated progenies. Emasculation was performed in an artificial fog chamber as described by Burton (1948). Briefly, culms of plant [Q4084.sub.10] bearing a piece of rhizome and a panicle due to flower on the following day were brought into a fog chamber in which the humidifier system was turned on. At anthesis, anthers protruded but dehiscence was delayed due to the high relative humidity. In this condition, anthers were eliminated with sharp pointed tweezers. The non-flowered spikelets were eliminated. Afterward, pollen from a plant of cultivar Tifton ([Tift.sub.9]) was dusted onto the emasculated spikelets. Inflorescences were enclosed in a glassine bag to prevent further pollination and culms were placed in a shaded part of the greenhouse until seed maturity. Seeds were germinated in petri dishes at 35 [degrees] C and seedlings were transferred to pots with soil. A segregating [F.sub.1] population was developed and grown under greenhouse conditions. Genomic DNA was extracted according to Dellaporta et al. (1983) from 3 to 5 g of leaf tissue. RFLP marker analysis was carried out as described in Ortiz et al. (1997) using EcoRI, HindIII, BamHI, or DraI restriction enzymes. One hundred five maize clones from the University of Missouri-Columbia (UMC and CSU) (Table 1) and 44 rice and four oat clones from Cornell University (RZ and CDO, respectively) (Table 2), were used as probes to identify polymorphism between parents and segregate markers in the progeny. Small filters containing DNA from both parental genotypes and five offspring were used in this step. Clones that revealed a polymorphic hybridization pattern were selected for the linkage analysis. RAPD markers were identified by means of arbitrary decamer oligonucleotides from Operon Technologies Inc. (Alameda, CA, USA) series L and J, and from British Columbia University, Set 1, No. 1 to 80. Amplifications were performed according to the procedure previously described by Ortiz et al. (1997). AFLP marker analysis was carried out as described in Vos et al. (1995), except for the following modifications: the ligation mixture included 50 pmol of MseI adapters, 5 pmol of EcoRI adapters, 0.5 mM ATP, 10 units of T4 DNA ligase, 1 x R/L buffer, and sterile distilled water to a final volume of 25 [micro]L. Preamplifications included 2 [micro]L of the ligation mix, 1 x Taq activity buffer (Promega Corp., Madison, WI), 0.2 mM dNTPs, 30 ng of EcoRI and MseI primers (Keygene), and 1U of Taq polymerase enzyme (Promega) in a final volume reaction of 25 [micro]L. Thermocycler parameters were: 94 [degrees] C for 30 s, 60 [degrees] C for 30 s and 72 [degrees] C for 60 s, 30 times. Following the PCR reaction, each tube was supplemented with 475 [micro]L of TE. Selective PCR reactions included 4 [micro]L of the preamplification, 1x Taq activity buffer, 0.2 mM of each dNTP, 30 ng of each oligonucleotide, 0.75 U of Taq polymerase (Promega), and sterile distilled water to a final volume of 25 [micro]L. Cycles for selective amplification were carried out at 94 [degrees] C for 30 s, 65 [degrees] C for 30 s, and 72 [degrees] C for 60 s 1 time, then 12 cycles in which the annealing temperature was reduced by 0.7 [degrees] C and 22 cycles of 94 [degrees] C for 30 s, 56 [degrees] C for 30 s, and 72 [degrees] C for 60 s. Five microliters of the amplification reactions were mixed with an equal volume of formamide, heated to 90 [degrees] C for 3 min and loaded in 4.5% (w/v) acrylamide/1x TBE denaturing gels. Gel electrophoresis was carried out at 60 W for 2 h. Gels were fixed in 10% (v/v) acetic acid for 20 min, silver-stained by the DNA Silver Staining System (Promega), and air dried overnight. RFLP and RAPD markers generated by the same clone or primer were identified with the corresponding name followed by a letter (a, b, c, etc.). RAPD markers were named according to their primer's name designated at the University of British Columbia or Operon Technology, Inc. AFLP markers were labeled with a prefix MxEx (x = 1 - n) designating the specific MseI and EcoRI primers (Keygene N.V., Wageningen, the Netherlands) used during selective amplification, respectively. A final number indicated the relative position in the gel.
Table 1. Maize RFLP probes used to detect polymorphic loci in the Paspalum mapping population. Clones were hybridized against DNA from both parental genotypes of the mapping cross and five progenies. Maize chromosome Probes detecting loci in a given maize([dagger]) chromosome No. 1 ([double dagger]) UMC13, UMC14, UMC58, UMC67, UMC83, UMC128, UMC140, UMC157, UMC161, UMC164, CSU61, CSU134, CSU164 13 2 UMC2, UMC6, UMC34, UMC36, UMC49, UMC53, UMC55, UMC61, UMC122, UMC125, UMC131, UMC139, CSU109, CSU133, CSU148, CSU184 16 3 UMC16, UMC32, UMC63, UMC92, UMC96, UMC121, UMC157, CSU21, CSU32, CSU38, CSU56, CSU58 12 4 UMC31, UMC42, UMC52, UMC156, CSU36, CSU91, CSU100 7 5 UMC40, UMC72, UMC90, UMC104, UMC147, CSU33, CSU137, CSU149 8 6 UMC28, UMC38, UMC65, UMC85, CSU146 5 7 UMC56, UMC80, UMC116, UMC125, CSU5, CSU8, CSU11, CSU13, CSU27, CSU81, CSU129 11 8 UMC48, UMC89, UMC103, UMC120, UMC124, UMC150, CSU110, CSU142, CSU165 9 9 UMC20, UMC70, UMC81, UMC94, UMC95, UMC109, UMC113, CSU31, CSU54, CSU93, CSU94, CSU95, CSU147, CSU158 14 10 UMC44, UMC57, UMC64, UMC130, UMC163, CSU46, CSU48, CSU86, CSU103, CSU136 10 Total 105 ([dagger]) According to Heredia-Diaz et al. 1994. ([double dagger]) Italics indicates clones detecting polymorphism between parents of the mapping population and at least one segregating fragment. Table 2. Rice and oat RFLP probes used to detect polymorphic loci in Paspalum mapping population. Clones were hybridized against DNA from both parental genotypes of the mapping cross and five progenies. Rice Rice (rz) and oat (cdo) probes detecting loci chromosome in a given rice([dagger]) chromosome No. 1 RZ382,([double dagger]) RZ413, RZ444, RZ543, RZ995, RZ358 6 2 RZ87, RZ166, RZ273, RZ446, RZ567, RZ599 6 3 RZ329, RZ630, RZ574 3 4 RZ86, RZ69, RZ569, RZ590 4 5 RZ244, RZ296, RZ455 3 6 RZ2, RZ144, RZ242, RZ338, RZ588, RZ612 6 7 RZ395, RZ509, RZ753 3 8 RZ143, RZ323 2 9 RZ206, RZ698 2 10 CDO98, RZ421, RZ583, RZ892 4 11 CDO520, RZ141, RZ525, RZ537, RZ797 5 12 CDO459, CDO127, RZ397, RZ670 4 Total 48 ([dagger]) According to Causse et al. 1994. ([double dagger]) Italics indicates clones detecting polymorphism between parents of the mapping population and at least one segregating fragment.
RFLP mapping data were collected from filters containing DNA from both parental genotypes and 126 individuals of the progeny. Data from RAPD and AFLP experiments were obtained from both parents and 65 progenies. Segregation information was collected from all informative fragments, regardless of whether segregation originated in one or both parents. As a result of using an [F.sub.1] population derived from non-inbred parents, five types of single loci segregating ratios (1:1m, 1:1 [Male], 1:2:1, 3:1 or 1:1:1:1) were expected for the codominant markers (RFLP), while two types of segregation (3:1 or 1:1) were anticipated for the dominant markers (RAPD and AFLP) (Ritter et al. 1990). A Chi-square test was used to determine the goodness of fit between the observed and the expected number of genotypes for each class of segregation ratio. Segregation ratios that differed from the expected value (at P = 0.05 or less) were classified as distorted.
A combined genetic linkage map was constructed with the segregation information derived form both parental genotypes and the computational program JoinMap 1.4 (Stam, 1993). JoinMap allows the use of raw data of various types of segregation and the combination of linkage information from different populations to construct integrated linkage maps. The program estimates map distances for a given group of markers by using essentially a least square procedure (Stam, 1993). JoinMap permits a choice of "linklod" and "maplod." Linklod is for finding linkage groups. Given a set of markers within a linkage group, only those additional markers which are linked with at least one marker of the group with a LOD score not less than a linklod are included. This procedure leads to a unique grouping of markers depending on the supplied LOD value. Maplod defines the minimum LOD score for recombination rates to be used for map construction. Two separate parental data files were created with the segregating markers generated from each progenitor. Each parental data file included those markers segregating in 1:1, 3:1, or 1:2:1 ratio for the corresponding parent. Markers segregating in both parents, showing a 1:1:1:1 segregation ratio, were partitioned according to the alleles contributed by the relevant parent and included in the correspondent parental data file with an expected segregation ratio of 1:1 (Jacobs et al., 1995). The two parental data files were combined into one according to the JoinMap software's instructions and used as an input file for estimation of linkage between markers. Linkage groups were first established by groups of markers linked at LOD (linklod) = 4. Afterward, a second set of markers was added to the formed groups at linklod = 3.0. Finally, some markers were assigned to the groups at linklod = 2.0 and recombination frequencies [is less than] 0.3. Since JoinMap uses a Chi-square test for the independence of segregation, a LOD score of 2 is reliable for assigning markers to linkage groups. Marker orders within the linkage groups were initially determined in each parental map, in which markers segregating in 1:1, 3:1, and 1:2:1 can be used directly (Stam, 1993). The combined map was constructed maintaining the marker order established for each female and male linkage group by using the JoinMap option "fixed sequence" only if resulted in a minimal increase in the Chi-square value for the overall goodness-of-fit of the combined map ([chi square] [is less than] 6.0). Kosambi's mapping function (Kosambi, 1944) was used to convert recombination values to map units. The computer program Drawmap (Van Ooijen, 1994) was used for a graphic representation of the map.
Marker Selection and Level Of Polymorphism
Considering that neither genomic nor cDNA libraries of P. notatum are still available, we decided to use heterologous maize, rice, and oat clones for revealing RFLP loci in this study. Single or low copy clones of these species have been used as a reference for comparative mapping in the Gramineae family (Ahn and Tanksley, 1993; Van-Deynze et al., 1998). On the basis of the colinearity observed between the grass genomes (Moore et al., 1995), dispersed RFLP clones selected from both the maize and rice genetic maps (Heredia-Diaz et al., 1994; Causse et al., 1994) were tested to detect polymorphisms between the parents revealed as segregating fragments in the progeny. The clones selected were assumed to detect loci uniformly distributed over the Paspalum genome. Of the initial 153 probes used, 57 (37.2%) cross hybridized with the Paspalum DNA and showed differences between parents with at least one of the four enzymes employed in the digestion of the DNA. Each clone revealed one to several segregating fragments, producing 71 RFLP loci. The rest of the probes did not generate informative patterns because of a lack of hybridization, no polymorphism, or smearing. All maize and rice clones tested and selected for mapping, with the corresponding chromosomes where they map to, are indicated in Tables 1 and 2, respectively. Screening of RAPD's oligonucleotides showed an overall efficiency for detecting polymorphism of about 28%. Informative primers produced on to four bands in the amplification patterns, with one to two segregating fragments. Sixteen RAPD loci were selected for mapping. Likewise, five AFLP primer combinations (M32E31, M32E33, M33E31, M33E32, and M34E42) (62.5% of the total assayed) that generated 62 polymorphic bands were selected for mapping purposes.
Although diploid races of P. notatum are highly self-sterile and the female parent of the mapping cross was emasculated (see Material and Methods), each progeny plant was examined for the presence of bands segregating from the male parent. This test was performed to prevent the inclusion in the analysis of any undesirable individual derived from self-pollination. All 126 progenies selected for the linkage analysis produced specific bands derived from the male parent with several markers, indicating its hybrid origin. As the segregation analysis involved the use of an [F.sub.1] mapping population, i.e., derived from non-inbred parents, different types of segregation were observed at different loci within the population. When codominant markers (RFLP) were used, loci segregating in 1:1, 3:1, 1:2:1, or 1:1:1:1 ratios were detected. Loci with a 1:1 or 3:1 segregation ratios were resolved with the dominant markers (RAPD and AFLP). The majority of the markers (94.6%) segregated according to the expected Mendelian ratios, but about 5% displayed a varying degree of distorted segregation in the progeny (7.5 [is less than] [chi square] [is less than] 55.11). Most markers (64.5%) detected polymorphisms, which originated in only one of the parental genotypes, while the rest (35.5%) displayed polymorphism generated from both parental plants. One hundred forty-nine polymorphic loci were used for constructing the linkage map. The number of markers scored in each class and the origin of the polymorphisms is shown in Table 3.
Table 3. Mode of segregation and number of markers used for constructing the P. notatum linkage map. P1 and P2 = parent [Q4084.sub.10] and parent [Tift.sub.9], respectively. Parental genotype Offspring P1 x P2 genotype Expected ratios Parental map Aa x aa Aa:aa 1:1 P1 aa x Aa aa:Aa 1:1 P2 Aa x Aa A-:aa 3:1 P1 + P2 Aa x Aa AA:Aa:aa 1:2:1 P1 + P2 Aa x Ab AA:Ab:aA:ab 1:1:1:1 P1 + P2 Parental genotype Number of P1 x P2 markers % Aa x aa 55 37.0 aa x Aa 41 27.5 Aa x Aa 32 21.5 Aa x Aa 9 6.0 Aa x Ab 12 8.0
Construction of a Combined Genetic Linkage Map
Linkage analysis was performed employing all segregant fragments regardless of whether they originated from one or both parents. Because the female and male meioses are independent from each other, the information generated by the analysis of the alleles segregating from each parental genotype can be considered as being produced from two independent populations (Jacobs et al., 1995). Thus, a combined map was constructed with the segregation information from both parents, by merging both parental data sets in one file according to the JoinMap1.4 software instructions (Stam, 1993). The combined map was based on the markers shared by both parental genotypes (Table 3). The parental data file corresponding to the female parent ([Q4084.sub.10]) included 108 markers and that corresponding to the male parent ([Tift.sub.9]), 94. An outline map, comprised of 55 markers (49%) on the 10 expected Paspalum linkage groups was first defined by using a LOD = 4.0. Afterwards, a second set of markers was added to the formed groups by using a minimum LOD = 3.0. Finally, some markers were mapped at a minimum LOD = 2.0 (Fig. 1). One hundred twelve markers were assigned to the linkage groups, while 37 remained unlinked. The markers' order within linkage groups was first independently established in each parental map at a maplod = 0.05. Common markers showed a similar order in most of the female and male linkage groups, and no contradictions were detected when the combined linkage groups were constructed by maintaining the markers' order of the parental maps using the JoinMap option "fixed sequence" (see Materials and Methods). However, combined Linkage Group 7 could not be assembled preserving the markers' order of the parental maps and the more likely order of the markers was determined directly from the data at a maplod = 0.5. The relative position of the loci and the distance between them are shown in Fig. 1. According to the map distances, a total of 991 cM was covered, with an average distance between markers of 9 cM. The largest group putatively labeled as Linkage Group 1, according to the map length of the group, comprises 11 markers covering 136 cM. The smallest group (Linkage Group 10) contains eight markers covering 59 cM. Some parts of the map appeared densely populated such as Linkage Groups 2 and 6, where 15 and 24 loci were allocated over 122 and 98 cM, respectively. However, some regions were only sparsely mapped such as Linkage Groups 8 and 9. Three of the RFLP markers that showed distorted segregation ratios (RZ590, UMC44, and UMC120) mapped together on Linkage Group 10 while three distorted AFLP markers (M32E3111, M33E3111, M33E317) mapped to the central region of Linkage Group 2. The rest of the markers showing distorted segregation ratios (UMC13, UMC156, CSU31) were distributed among the different linkage groups (Fig. 1).
RFLP probes used in this study had been previously located on the rice and maize genetic maps (Ahn and Tanksley, 1993; Heredia-Diaz et al., 1994; Van Deynze et al., 1998). According to this information, several genomic regions of Paspalum appear to be syntenic to regions of the maize or rice genomes. For instance, Linkage Group 1 shows three clones of rice chromosome 1 (RZ444, RZ413, RZ543), two of which are duplicated in maize chromosome 3 and 8, plus other clones of maize chromosome 8 (UMC103, RZ244). Linkage Group 3 carries two clones of maize chromosome 7 (CSU5, CSU11). Linkage Group 4 has four clones that map to maize chromosome 5 (UMC104, UMC72, RZ273, CSU137) and 3 (RZ143, RZ206, RZ588) which map to maize chromosome 6. Linkage Group 5 presents three clones of maize chromosome 5 (CSU137, UMC147, RZ273) linked with clones of rice chromosome 10 (RZ421) and 4 (RZ69). Linkage Group 6 carries four clones of maize chromosome 1 (CSU164, UMC13, CSU61, UMC128) and 5 clones of maize chromosome 6 or 9 (RZ144, UMC62, UMC65, RZ206, UMC113). Linkage Group 8 shows two clones of maize chromosome 6 (CSU146, CSU56) and clones of maize chromosome 1 (CSU164) and 9 (CSU54). Linkage Group 10 presented two clones of maize chromosome 7 (CSU129, UMC80) and three from maize chromosome 10 (CSU86, RZ590, UMC44). The location of each RFLP marker in the rice and maize maps is shown in Fig. 1.
Localization of Markers Reported as Linked to Apomixis
Several clones of maize chromosome 5 (including UMC72 and UMC147) and rice chromosome 2 (including RZ273), have been found to belong to the linkage group that includes the factor(s) for apospory in Brachiaria hybrids (Pessino et al., 1997, 1998). To identify a putative genomic region associated with the character apomixis in P. notatum, the three mentioned RFLP clones (UMC72, UMC147, and RZ273) were tested on the mapping population. Clone UMC72 generated two segregating fragments. One of them (UMC72a) remained unlinked, but the other (UMC72b) mapped to Linkage Group 4, close to a marker generated by clone RZ273 (RZ273a). Interestingly, other clones of maize chromosome 5 (CSU137 and UMC104) were also found in this genomic region. Likewise, a fragment produced by UMC147 (UMC147b) was allocated to Linkage Group 5 and was linked to CSU137b of maize chromosome 5, RZ421 of rice chromosome 10, and maize chromosome 1. A second marker produced by clone RZ273 (RZ273b) is present at the end of this group. The other fragment produced by clone UMC147 (UMC147a) mapped to Paspalum Linkage Group 2 close to CSU48 of maize chromosome 10 and CDO127 duplicated in maize chromosome 4 and 10. On the other hand, clone UMC28 of the long arm of maize chromosome 6, which has been found to cosegregate with diplospory in maize-Tripsacum hybrids (Leblanc et al., 1995), mapped at one end of Paspalum Linkage Group 2 linked to marker CSU93 of maize chromosome 9.
There are two main attributes of diploid Pensacola bahiagrass which make it an exceptional material for molecular and genetic studies: first, it has a small DNA content per haploid genome (C value) estimated by Jarret et al. (1995) to be of the order of 0.6 pg, and, second, it has a completely sexual mode of reproduction, but has close tetraploid apomictic relatives. Genetic information generated at the diploid level could be extended to other races of Paspalum, with a more complex genetic structures and reproductive systems. The strategy employed in this work included the use of an [F.sub.1] population derived from non-inbred parents. Since all the diploid citotypes of P. notatum have originated from a relatively small area in the northeastern Argentina (Burton, 1967), the parents of the mapping cross may be more closely related than is apparent. Nevertheless, a significant level of polymorphism between both plants allowed the generation of a useful mapping population. RFLP, RAPD, and AFLP markers were applied to the construction of combined linkage groups that permitted the utilization of all segregating markers regardless of whether they segregate from one or both parents of the mapping cross. The combination of genetic information from different populations is possible when common polymorphic markers exist. Thus, the combined map was constructed on the basis of the markers shared by both parental genotypes. This procedure led to the construction of a denser map and the inclusion of polymorphic markers not simultaneously segregating from both parents. Common marker order in the male and female linkage groups was consistent, with the exception of Linkage Group 7. This might indicate the presence of chromosomal rearrangements of one parental genotype relative to the other in this group. Markers generated by the use of heterologous RFLP clones and RAPD and AFLP markers showed a relatively high level of polymorphism of potential utility for breeding research in this species. The total length of the map appeared to be similar to those reported for others grasses (Hayward et al., 1998; Wang et al., 1998), indicating a good degree of genome coverage. Several single copy clones of rice and maize hybridized with two or more segregating fragments that were located to different linkage groups, apparently at random. This outcome denotes some degree of gene duplication at the diploid level in Paspalum.
The utilization of heterologous clones for generating RFLP loci led to the observation that several genomic regions appeared to be composed of clones located in maize or rice syntenic regions. However, other regions are still lacking a sufficient number of markers for a comprehensive comparative analysis. The generation of more RFLP markers by anchored probes, as described by Van-Deynze et al. (1998), will allow the saturation of these regions and a more detailed study of their genetic composition and syntenic relationships. Regarding the location of markers that have been reported as linked to apomixis in related species, Paspalum Linkage Group 4 and 5, which included clones linked to apospory in Brachiaria (Pessino et al., 1998), appear to be regions worthy of further investigation. Besides, Linkage Group 2, which presented a clone linked to diplospory in maize-Tripsacum hybrids (Leblanc et al., 1995), should also be considered, since diplospory has been observed in some Paspalum species (Bonilla and Quarin, 1997).
The map presented here can be used as a genetic framework for basic and applied studies in Paspalum. It also establishes the first example of a genetic linkage map of a subtropical forage grass with both sexual and apomictic forms. The search for and identification of markers linked to traits of agronomic interest such as apomixis, and the identification of QTL for characters such as cold resistance or dry matter production can be rapidly exploited in marker assisted selection--breeding programs. Furthermore, this map will provide a foundation for extension of mapping to the tetraploid members of the Paspalum complex, many of which are important forage crops.
Authors wish to thank Theresa A. Musket from the University of Missouri-Columbia and Ying Fan from Cornell University for providing some of the RFLP clones used in this study.
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Abbreviations: AFLP, amplified fragment length polymorphism; cM, centimorgan; LOD logarithms of the odds; PCR, polymerase chain reaction; QTL, quantitative trait loci; RAPD, random amplified polymorphic DNA; RFLP, restriction fragment length polymorphism.
Juan Pablo A. Ortiz,(*) Silvina C. Pessino, Vishnu Bhat, Michael D. Hayward, and Camilo L. Quarin
J.P.A. Ortiz, S.C. Pessino, and C.L. Quarin, Instituto de Botanica del Nordeste (IBONE), Facultad de Ciencias Agrarias, UNNE, CC209, (3400) Corrientes, Argentina; V. Bhat, Indian Grassland and Fodder Research Institute, UP 284 003, Jhansi, India; M.D. Hayward, Institute of Grassland and Environmental Research, Aberystwyth, Ceredigion SY23 3EB, UK. This work was partly financed by a grant from the Commission of the European Communities-STD3 program (TS3-CT93-0242). JPAO and SCP were supported by IBONE-Facultad de Ciencias Agrarias, UNNE, Corrientes, Argentina. JPAO received a grant by the International Foundation for Science (C/2870-1), Sweden. SCP was granted by Fundacion Antorchas, Buenos Aires, Argentina. JPAO, SCP and CLQ are members of CONICET, Argentina. Received 25 May 2000. Juan Pablo A. Ortiz, Corresponding author (firstname.lastname@example.org).
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|Author:||Ortiz, Juan Pablo A.; Pessino, Silvina C.; Bhat, Vishnu; Hayward, Michael D.; Quarin, Camilo L.|
|Date:||May 1, 2001|
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