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Apospory-linked molecular markers in buffelgrass.

The ability to interchange at will obligate asexual and sexual reproduction in crop breeding material would allow plant breeders to quickly develop superior varieties and to fix heterosis in a single parent (Hanna and Bashaw, 1987; Jefferson, 1993). Attempts at moving chromosomes with apomixis genes from related species into high value crops with high yield of viable seed has not been accomplished. Even if successful, it would still require classical breeding approaches and multiple generations to create each new "asexual" line. Furthermore, it would be equally difficult to convert such a plant back to a sexually reproducing type. An alternate approach is to clone an apomixis gene and move it by genetic transformation into specific crops. While achieving this is not imminent, the necessary intermediate steps have been accomplished in a variety of plants and crops. Because the expression product of an apomixis gene has not been found, a crucial step in cloning the gene is to map its position on a chromosome. Some preliminary steps have been taken to do this in Pennisetum glaucum (L.) R. Br. (Lubbers et al., 1994; Ozias-Akins et al., 1993).

Production of apomictic seed by buffelgrass is based on formation of meiotically unreduced, aposporous embryo sacs of the Panicum type followed by pseudogamous, parthenogenetic development of the embryo (Fisher et al., 1954; Nogler et al., 1984). The embryo sacs are cytologically different from meiotically reduced Polygonum type sexual embryo sacs of buffelgrass. Crosses of sexual with aposporous genotypes of buffelgrass yielded segregation ratios compatible with an inheritance model that postulates expression of apospory requires the dominant allele (A) of a single autotetrasomically inherited locus, assuming random assortment of chromatids (Sherwood et al., 1994). Within this model, crosses of obligately sexual maternal parents of genotype aaaa with aposporous pollen parents of the simplex (Aaaa) and duplex (AAaa) levels, respectively, give progeny segregating in ratios of 15:13 and 3:11 (sexual: aposporous). The data were not compatible with models for disomic or allotetrasomic inheritance of the apospory locus:

From this system, crosses of sexual and apomictic parents can produce populations for which linked markers will provide linkage estimates to the apoospory gene for mapping. Relatively small populations of 75 progeny suffice for autotetraploids if the markers are linked by coupling (daSilva et al., 1996; Sorrells, 1992; Wu et al., 1992). While RAPD markers are much easier to produce than RFLP markers, they are not always found on homeologous chromosomes as are RFLP markers. However, estimation of maximum likelihood estimators of the recombination fraction (Wu et al., 1992) can indicate whether a selected marker is linked by coupling (the same chromosome) or by repulsion (homologous chromosome). RAPD markers linked by coupling behave like SDRF (single-dose restriction fragment) markers (Wu et al., 1992). Therefore, our approach is to map dominant RAPD markers. In the case of autotetraploid random chromatid assortment, an SDRF (one allele) or a DDRF (double-dose restriction fragment, two alleles) will segregate in a 15:13 or 3:11 (absent/present) ratio, respectively, but in both cases a single RAPD marker will be produced, even if it is in a duplex plant. SDRF markers have been shown to be useful in genetic analysis of phenotypic characters in tetraploid alfalfa (Yu and Pauls, 1993a,b).

A RAPD marker (C04-600) and a STS (sequence-tagged site) marker (UGT197-144) were associated with apomixis in Pennisetum sp. (Lubbers et al., 1994; Ozias-Akins et al., 1993). Both loci were present on an alien chromosome, which had been introduced into a trihybrid with pearl millet background (Dujardin and Hanna, 1989; Hanna et al., 1993) by backcrossing P. squamulatum Fresen to P. glaucum through a bridging species, P. purpureum Schumacher. These two markers were also reported in apomictic P. ciliare (Lubbers et al., 1994). Additional RAPD markers are needed to prepare an initial map of loci linked to the apospory locus in buffelgrass. Our approach to identifying RAPD markers linked to aposporous reproduction in buffelgrass was through bulked segregant analysis (Michelmore et al., 1991) of two F1 populations segregating for apospory. Using MAPMAKER/EXP 3.0 software (Lander et al., 1987; Lincoln et al., 1992), we identified two new markers that could be mapped, and two additional markers that will require larger populations to place on a map. Pairwise linkage analysis of the new markers together with an STS marker previously reported for Pennisetum sp. provided the first preliminary map for the buffelgrass chromosome bearing the apospory gene.



Dr. E.C. Bashaw (USDA-ARS, College Station, TX) provided tillers of obligately sexual buffelgrass plant B-2s and seed of obligately aposporous cultivar Higgins (Bashaw, 1968). Obligately aposporous buffelgrass line B-12-9 was selected at University Park, PA, from segregating seed provided by Dr. Bashaw. The plants were grown in a greenhouse as described by Gustine et al. (1996). Inheritance studies indicated the genotypes were as follows: B-2s, aaaa; B-12-9, Aaaa; Higgins, AAaa (Sherwood et al., 1994).


B-2s was used as the maternal parent in test crosses with B-12-9 and Higgins. Crosses were made in a greenhouse by means of the dialysis bagging technique of Sherwood et al. (1994). Progeny were classified for reproductive mode using 20 to 60 cleared pistils of each progeny (Young et al., 1979).

Molecular Markers

An STS marker and RAPD markers were generated by the polymerase chain reaction (PCR). Genomic DNA was isolated from parents and progeny by the procedure of Gustine et al. (1996). Subsamples of 40 (10 sexual, 30 aposporous) and 30 (10 sexual, 20 aposporous) progeny from the B-2s x B-12-9 and B-2s x Higgins crosses, respectively, were screened for molecular markers by bulk segregant analysis (Michelmore et al., 1991). Each bulked DNA sample contained 10 [micro]g of genomic DNA from each of 10 sexual or 10 apomictic progeny (100 [micro]g total) in 500 [micro]L of deoxyribonuclease-free water. DNA samples from parents and individual progeny contained 10 [micro]g of genomic DNA in 500 [micro]L of deoxyribonuclease-free water. PCR reaction concentrations, amplification conditions, and agarose gel separation of RAPD markers were described in Gustine et al. (1996). PCRs were conducted in thin-walled GeneAmp reaction tubes (Perkin Elmer Cetus, Norwalk, CT) in a final volume of 30 [micro]L. Reaction components consisted of genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 MM [MgCl.sub.2], 0-01 g gelatin [kg.sup.-1] water, 0.1 mM each of dATP, dTTP, dGTP, and dCTP, 0.2 mM primer or primer pair, and 1 unit of Taq polymerase. Each reaction contained 10 ng of DNA from a single individual, or 100 ng of bulked DNA (10 individuals x 10 ng). Ten nanograms of bulked DNA was insufficient for amplification of markers if only one individual of the bulk contained a specific marker, and therefore 100 ng of bulked DNA was needed. Reaction components other than primer or template were supplied by Perkin Elmer Cetus or Boehringer Mannheim Corp. (Indianapolis, IN). PCRs were done in a Perkin Elmer Cetus 480 thermo cycler: 1 cycle of 1 min 45 s at 94 [degrees] C, 50 s at 36 [degrees] C, and 1 min 45 s at 72 [degrees] C; 42 cycles of 50 s at 94 [degrees] C, 50 s at 36 4[degrees] C, and 1 min 45 s at 72 [degrees] C; 1 cycle of 50 s at 94 [degrees] C, 50 s at 36 [degrees] C, and 2 min 30 s at 72 [degrees] C; and hold at 4 [degrees] C. Amplified DNA sequences were separated in agarose gels (14 g agarose [kg.sup.-1] water) in 0.5 x TBE buffer, stained in ethidium bromide, and photographed under UV illumination (Sambrook et al., 1989).

Bulked DNA and parental samples were screened with 500 decanucleotide primers to identify markers amplified in apomictic parents and bulked apomictic progeny, but not detected in B-2s and bulked sexual progeny. Oligonucleotide primers were from kits OPA-OPC and OPE-OPZ (Operon Technologies, Inc., Alameda, CA). We screened 120 primers from kits OPA, OPB, OPC, OPE, OPF, and OPZ. Biogenetic Services, Inc. (Brookings, SD) screened the 20 OPF primers and the remaining 380 primers (kits OPG - OPY). Gel data were recorded on Polaroid 665 prints and negatives. Presence or absence of gel bands was independently analyzed by two observers at different times to reduce bias error.

A total of 82 individual progeny was analyzed for markers generated from decamer primers OPA20, OPB14, OPC04, OPJ 16, OPM02, OPN15, and the forward and reverse primers UGT197 (Ozias-Akins et al., 1993). The 70 individuals in seven bulks and 12 individuals from unbulked progeny of the two crosses were analyzed. UGT197 forward and reverse octadecanoic nucleic acid primers were synthesized by the Nucleic Acid Facility, The Pennsylvania State University.

Linkage Analysis

We calculated linkage and order of molecular markers using a DOS computer environment for each test cross using MAPMAKER/EXP 3.0 (Lander et al., 1987; Lincoln et al., 1992) with error detection invoked (Lincoln and Lander, 1992). This program assumes a 1:1 segregation ratio; however, as the ratio changes to 3:11 as in the autotetraploid model, the accuracy of the recombination fraction decreases. Nevertheless, the orders of the markers in a linkage map will not be affected. Data were analyzed by the "[F.sub.2] backcross" option and presence of marker and aposporous phenotype were assigned to "A" = heterozygous dominant, and absence of marker and sexual phenotype were assigned to "H" = homozygous recessive. In a few instances, marker data were missing and were analyzed as such by MAPMAKER/EXP 3.0. Map distances are given in Kosambi cM units.


The cross B-2s x Higgins yielded 15 sexual and 38 aposporous progeny (aaaa x AAaa, 3: 11 expected, [chi square] = 1.49, P = 0.22). The cross B-2s x B-12-9 yielded 20 sexual and 42 aposporous progeny (aaaa x Aaaa, 15: 13 expected, [chi square] = 11.3, P [is less than] 0.001; or aaaa x AAaa, 3:11 expected, [chi square] = 4.31, P = 0.38). Therefore, both aposporous parents appeared to be genotype AAaa. We do not know why the greenhouse cross of B-2s x B-12-9 failed to provide a segregation similar to that reported in an earlier crossing in the field (Sherwood et al., 1994). The two crosses produced a total of 115 progeny. Of these, 75 (65.2%) were classified in each of two successive years; 69 of these had the same reproductive type both years; six of these appeared to be facultative in the second year. Thirty three of the 115 (28.7%) were classified in one year only (1993, 1994, or 1995). The remaining 12 progeny were classified in each of three successive years, with no change in classification.

Screening for Markers

Subsamples of 40 (10 sexual, 30 aposporous) and 30 (10 sexual, 20 aposporous) progeny from the B-2s x B-12-9 and B-2s x Higgins crosses, respectively, were screened for informative (polymorphic) molecular markers by bulk segregant analysis. DNAs of one sexual and two aposporous bulked samples from the Higgins cross, one sexual and three aposporous bulked samples from the B-129 cross, and each parent were amplified by PCR with each of 500 decanucleotide primers and the UGT197 primer pair. The amplification products of the seven bulked samples and the three parents produced by 500 primers were compared by agarose gel electrophoresis. Out of 5000 reactions, five primers produced potentially informative molecular markers not previously reported: A20, B14, J16, M02, and N15. Previously reported markers C04-600 and UGT197-144 are compared in Table 1 with the new markers. The band patterns produced by primers A20, B14, J16, M02, and N15, as well as by C04 and UGT197, were reproducible when analyzed by three different persons in two different laboratories using DNA from at least two separate plant extractions. Markers A20-730 (A20 for primer OPA20, 730 for the marker size in base pairs), B14-550, and M02-680 were the only ones associated with apomixis and not with sexual reproduction in the bulked samples and parents (Table 1), and thus were the most apparently informative markers. However, DNA from only one of the 10 genomes represented in the bulked genomic DNA is sufficient to produce a marker during amplification. Conversely, although one or more members in a bulk may test positively for a marker when tested individually, the marker is not necessarily detected when the member is included in a bulk. Therefore, for linkage analysis, DNA from individuals in the seven bulks was amplified separately with the primers selected through bulk segregant analysis.

Table 1. Detection (+) or non-detection (-) of molecular markers in bulked apomictic or sexual buffelgrass progeny and their parents.
                                Markers (Primerbp)

DNA([dagger])      Phenotype     UGT197-144     J16-800

Bulked progeny

B-2s x Higgins     Aposporous       +               -
B-2s x Higgins     Aposporous       +               +
B-2s x Higgins     Sexual           -               -
B-2s x B-12-9      Aposporous       +               +
B-2s x B-12-9      Aposporous       +               +
B-2s x B-12-9      Aposporous       +               +
B-2s x B-12-9      Sexual           +               -


B-12-9            Aposporous       +               +
B-2s              Sexual           -               -
Higgins           Aposporous       +               +

                         Markers (Primerbp)

DNA([dagger])       M02-680    A20-730    B14-550

Bulked progeny

B-2s x Higgins          +          +         +
B-2s x Higgins          +          +         +
B-2s x Higgins          -          -         -
B-2s x B-12-9           +          +         +
B-2s x B-12-9           +          +         +
B-2s x B-12-9           +          +         +
B-2s x B-12-9           -          -         -


B-12-9                  +          +         +
B-2s                    -          -         -
Higgins                 +          +         +

                          Markers (Primerbp)

DNA([dagger])          N15-370    C04-600

Bulked progeny

B-2s x Higgins              +         +
B-2s x Higgins              +         +
B-2s x Higgins              -         -
B-2s x B-12-9               +         +
B-2s x B-12-9               +         +
B-2s x B-12-9               +         +
B-2s x B-12-9               +         +


B-12-9                      +        +
B-2s                        -        +
Higgins                     +        +

([dagger]) No progeny plant assigned to a bulk was present in any other bulk.

Ozias-Akins et al. (1993) reported the primer C04 generated an approximately 600-base pair marker associated with apomixis. In our material, a C04-600 marker was expressed by the sexual parent and both aposporous parents. Because it was expressed by all parents, it was not appropriate to test segregation and distance of C04-600 in our two progeny lines. The N15-370 marker was of interest in that the parents and the bulks (with one exception) fit the pattern of expression expected for a linked marker, and furthermore because its pattern of expression duplicated that of UGT197-144, which was linked to apospory (Ozias-Akins et al., 1993).

Linkage Analysis

Genomic DNAs from the 70 individuals that made up the bulked samples were screened for the RAPD marker corresponding to each primer listed in Table 1. Genomic DNAs of some individuals not included in the bulks were also examined: three sexual and five aposporous genotypes from the Higgins cross, and two sexual and two aposporous genotypes from the B-12-9 cross.

All but marker N15-370 could be shown to be coupled to the apospory gene, and thus on the same chromosome, by estimating linkages for coupling and repulsion phases (Wu et al. 1992). The coupling phase recombination fractions (r) of the seven markers in progeny of the two crosses (Higgins and B-12-9, respectively) are given in Table 2. All markers had r values less than 0.5 in progeny; r was less than 0.1 for markers J 16-800 and UGT197-144 in both crosses. The calculated recombination frequency for each marker in repulsion phase ranged from 0.9 to 1.9 (data not shown), which excludes the repulsion phase from our analyses. In coupling phase, the upper confidence limit was 0.5 for N15-370, indicating that this marker is not closely linked. In addition, chi-square analysis was used to test whether markers were linked to the apospory locus (Table 2). At P = 0.05, marker N15-370 was unlinked in both crosses and marker B14-550 was unlinked in the Higgins cross (Table 2).

Table 2. Recombination fractions (r), confidence intervals (C.I.) of r, and chi-square test of linkage for markers of two buffelgrass crosses.
Marker                 r       C.I.([dagger])

B-2s x B-12-9
UGT197-144          0.023          0-0.080
J 16-800            0.071      0.015-0.195
M02-680             0.159      0.066-0.301
A20-730             0.163      0.068-0.307
B14-550             0.256      0.135-0.412
N15-370             0.477      0.274-0.500

B-2s x Higgins
UGT197-144         0.027           0-0.095
J 16-800           0.055       0.007-0.187
M02-680            0.162       0.045-0.288
A20-730            0.257       0.125-0.433
B14-550            0.343       0.169-0.493
N15-370            0.378       0.140-0.499

Marker            [chi square]([double dagger])    Linked?

B-2s x B-12-9
UGT197-144              40.091(***)                  Yes
J 16-800                30.857(***)                  Yes
M02-680                 20.455(***)                  Yes
A20-730                 19.558(***)                  Yes
B14-550                 10.256(**)                   Yes
N15-370                  0.091(ns)                   No

B-2s x Higgins
UGT197-144              33.108(***)                  Yes
J 16-800                28.444(***)                  Yes
M02-680                 16.892(***)                  Yes
A20-730                  8.257(**)                   Yes
B14-550                  3.457(ns)                   No
N15-370                  2.189(ns)                   No

(*), (**), and (***) indicate significance at the 0.05, 0.01, and 0.001 probability levels, respectively; (ns) indicates not significant.

([dagger]) Lower and upper C.I. of r based on equations 1 and 2 from the appendix of Wu et al. (1992).

([double dagger]) Chi-square calculations based on equation of Mather (1951) as used by Wu et al. (1992).

A preliminary linkage map was constructed using multipoint linkage analysis of the seven markers with MAPMAKER/EXP 3.0. Markers J16-800, UGT1971-44, and M02-680 bracketed the apospory locus and were linked within 5.7 cM (Kosambi) and 10.1 cM (Kosambi) of each other in the Higgins and B-12-9 crosses, respectively (Fig. 1). The analysis placed UGT 197-144 at the apospory locus in the B- 12-9 progeny and within 2.9 cM of the apospory locus in the Higgins progeny. The order of these three markers and the apospory locus was the same for both crosses. MAPMAKER/EXP 3.0 analysis indicated that A20-730 was about 24 and 36 cM from the apospory locus in the B-12-9 and Higgins crosses, respectively, on the same side as M02680, and the analysis indicated two misclassified individuals. Larger populations of progeny are necessary to establish its position. B14-550 was loosely linked to the apospory locus in the two crosses. Thus, A20-730 and B14550 were not placed on the map. As indicated by the data in Table 2, MAPMAKER analysis also showed that marker N 15-370 was unlinked. In analyzing linkages for these markers, we considered alternate orders, but this produced software-generated candidate errors. Since this result suggested mistyping of the reproductive phenotype of eight individuals, the alternate orders were rejected and excluded from the final analysis. The orders and linkages shown in Fig. 1 did not produce candidate errors. Log-likelihood values were several orders of magnitude better than the rejected alternate orders. Thus, the reported linkage maps represent the smallest log-likelihood values unaccompanied by candidate errors.



We have established linkage of two RAPD markers and one STS marker to the apospory locus in autotetraploid buffelgrass (Fig. 1). Lubbers et al. (1994) reported marker UGT197-144 as present in aposporous buffelgrass, but did not give any data for sexual P. ciliare. They reported weak association of C04-600 and strong association of UGT197-144 with apospory in a survey of 19 Pennisetum species. Our results are consistent with their findings, even though we could not include C04-600 in linkage analysis because it was expressed in the maternal parent, B-2s. We report here the first linkage map for the apospory-bearing chromosome in buffelgrass. The markers UGT197-144, J16-800, and M02-680 will be useful in developing a more detailed map and in locating sites close enough to the apospory locus to allow "walking the chromosome" to the gene itself. This seems to be the most direct route to cloning the gene.

SDRF markers represent a single dominant phenotypic allele in one parent only (e.g., Aaaa) and are either present or absent in each progeny (Sorrells, 1992). On the other hand, DDRF markers, which represent two alleles in one parent only (e.g., AAaa), will produce a marker in recombinant progeny with either one or two dominant alleles. Because we could not distinguish between SDRF and DDRF markers, we therefore considered the RAPD and STS markers equivalent to SDRF markers (single dominant allele) for purposes of linkage analysis. Hence, we based selection of markers on presence or absence (not detected) only, and since heterozygous loci were not specified in the analysis, marker linkages could be estimated with MAPMAKER/EXP 3.0. Nevertheless, the polyploid genetics of buffelgrass make linkage analysis complex. Cytologically, buffelgrass appears to be a segmental allotetraploid (Hignight et al., 1991; Snyder et al., 1955). Because of previous genetic studies, we considered buffelgrass autotetraploid at the apospory locus. Wu et al. (1992) addressed the problem of estimating molecular marker linkages in polyploids. In the case of autopolyploids, increasing ploidy level requires increasingly larger population size to calculate repulsion phase linkages at the 1% confidence level, even with SDRFs. On theoretical grounds, Wu et al. (1992) established that calculating coupling phase linkages for autopolyploids is independent of ploidy level. Using the Wu et al. (1992) equation, we calculated the maximum detectable recombination fractions for autotetraploid buffelgrass progeny populations of 34 and 40 to be 0.30 and 0.32 (37 and 40 cM), respectively. The recombination fractions shown in Fig. 1 (as cM) are significantly less than 0.32, as the greatest distance is 5.1 cM, and all of the linkage distances are less than the calculated maximum detectable recombination fraction values. Although MAPMAKER/EXP 3.0 overestimates recombination fractions of markers in crosses of duplex tetraploids, the order of the markers is correct. Thus, it is appropriate to use MAPMAKER/EXP 3.0 for multiple pairwise analysis of linkages and map construction with our crossing data.

A disadvantage of using RAPD markers is that amplified fragments within and between progeny cannot be assumed to represent homologous chromosome loci. For a given decanucleotide, the same complimentary primer sites may be present on nonhomologous chromosome segments. This situation could result in amplification of nonhomologous DNA of about the same base pair size. Thus, Southern blot hybridization data are necessary to establish marker relationship. In the case where there is relationship, detection by hybridization would result in assignment of most of our "linked" RAPD markers in apomictic and sexual individuals as homologous, but some could be nonhomologous. Where no relationship exists, detection by hybridization would result in assignment of the "linked" RAPD marker in apomictic and sexual individuals as nonhomologous. For the first case, one would expect to retain linkage, but with larger Kosambi distances between neighboring markers; for the second case, linkage would not exist. Primarily for this reason, we present the linkage maps here as preliminary evidence for presence of three markers on homologous chromosome segments. However, in spite of this disadvantage in working with RAPD data, our evidence clearly supports previous conclusions that the apomixis gene is located at a single locus of one linkage group. Analysis of markers in larger population sizes of 75 to 100 progeny will provide better estimates of linkage order and distances, and would indicate whether A20-730 and B14-550 should be included as markers linked to the apospory locus.

Establishing these markers for the apospory gene will be a valuable aid to positioning other markers near the apomixis gene locus and could be useful in tracking the homologous chromosomes in meiotic cells during aposporous embryo sac development. Markers linked to the chromosomes of the Aaaa and aaaa genotypes but not part of the apospory gene itself could be developed for tracking this aaaa chromosome in sexual embryo sac development, and thus provide a direct comparison of chromosome behavior for the two modes of reproduction. Finally, markers physically near the apospory gene are critical to walking to the gene.


We thank Dr. Clyde C. Berg for conducting the crosses of B-12-9 and Higgins with B-2s. We also thank Paul Forlano for running PCR reactions, agarose gel electrophoresis, and evaluation of the data.

Abbreviations: DDRF, double-dose restriction fragment; PCR, polymerase chain reaction; RAPD, random amplified polymorphic DNA; RFLP, restriction fragment length polymorphism; SDRF, single-dose restriction fragment; STS, sequence-tagged site.

(1) Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.


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D. L. Gustine,(*) R. T. Sherwood, and D. R. Huff

D.L. Gustine and R.T. Sherwood, USDA-ARS, Pasture Systems and Watershed Management Res. Lab., US Regional Pasture Res. Lab., Curtin Road, University Park, PA 16802; D.R. Huff, Dep. of Agronomy, The Pennsylvania State Univ., University Park, PA 16802. Received 29 April 1996. (*) Corresponding author (E-mail:
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Author:Gustine, D.L.; Sherwood, R.T.; Huff, D.R.
Publication:Crop Science
Date:May 1, 1997
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