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Rapid prediction of mating system of Vicia species.

SEVERAL Vicia species are of considerable economic importance as grain, green manure, cover, and forage crops. Management and use of germplasm collections of this genus has become difficult because of increases in the collection size, and many species are wild with little information available on their reproductive biology. Determination of the mating systems of the species is essential to choose the regeneration method that minimizes genetic drift or erosion of their potentially unique characteristics.

Recently, Zhang and Mosjidis (1995) determined that V. articulata, V. benghalensis, V. errilia, V. lutea, and V. sativa were self-fertilizing. However, Hanelt and Mettin (1989) indicated that about 10% cross-fertilization may occur in V. sativa. Mlyniec (1962) determined that V. villosa did not set fruits unless visited by bees; however, McGregor (1976) indicated that some self-fertilization might occur. Zhang and Mosjidis (1995) determined that V. villosa ssp. villosa and V. villosa ssp. varia were cross-fertilizing. Hence, they concluded that regeneration procedures for these two species should provide isolation from foreign pollen. Zhang and Mosjidis (1995) also reported that flowers of V. pannonica needed tripping to set fruit at a high percentage; thus, they deemed it to be a species with mixed mating.

The distribution of genetic variation is primarily determined by interactions among selection, mating systems, and effective population sizes (Hamrick and Godt, 1989). In self-fertilizing species, isozyme variation within populations is lower than that among populations (Hamrick, 1989). In contrast, cross-fertilizing species have higher variability within populations than among populations (Hamrick, 1989).

The use of controlled-pollination experiments in the field to determine mating system is costly and time consuming (Zhang and Mosjidis, 1995). Furthermore, Brown et al. (1997) pointed out the importance of determining the in situ and the ex situ mating systems of a population to design adequate regeneration strategies. It would be highly desirable to have a simple method whereby the mating system of a population could be determined once a sample had arrived at the gene bank. The objective of this research was to predict the mating system of 12 Vicia species through estimation of variability within and among accessions by assessing polymorphism for seven enzymes. Results were corroborated with the data from controlled pollination experiments reported by Zhang and Mosjidis (1995).

MATERIAL AND METHODS

Sample Preparation

Thirty-one accessions of V. articulata, V. benghalensis, V. cracca, V. errilia, V. lutea, V. narbonensis, V. pannonica, V. peregrina, V. pisiformis L., V. satira, V. villosa ssp. villosa, and V. villosa ssp. varia, were obtained from the U.S. Plant Genetic Resources Conservation Unit, Griffin, GA. These accessions were randomly chosen from the few that had enough viable seed for distribution.

Crude extracts were obtained from 10 randomly chosen seedlings from each accession of the species that were deemed self-fertilizing and 40 seedlings from each accession of the cross-fertilizing species (Zhang and Mosjidis, 1995; Rosa and Jouve, 1992). Seedlings from each accession were grown in a growth chamber at 18 [degrees] C and 10-h daylength. About 100 mg of cotyledonary tissue with shoots were excised from each 7- to 10-d-old seedling. The tissue was ground with a powerdriven pestle in a 1.5-mL centrifuge tube containing 80 [Micro]L of extraction buffer consisting of 16.7% (w/v) sucrose and 8.3% (w/v) sodium ascorbate in 50 mMpH 7.4 Tris-HCl (Stuber et al., 1988). The extracts were centrifuged at 2940 x g for 5 rain and either used immediately or stored at -24 [degrees] C for several hours.

Electrophoresis and Staining

A slight modification of the microslab isoelectric focusing gel described by Mulcahy et al. (1981) was used to prepare the slabs. A mixture of 4 mL [H.sub.2]O, 1 mL of acrylamide and bisacrylamide stock (4.75 g acrylamide and 0.25 g bisacrylamide in 10 mL [H.sub.2]O), 1 mL of ammonium persulfate stock (0.025 g in 10 mL [H.sub.2]O), 0.5 mL of ampholyte (pH 4-9), and 0.2 mL of TEMED stock (0.20 mL TEMED in 10 mL [H.sub.2]O) was used to make four slabs. Filter paper wicks (1 by 1 mm, Whatman No. 3) were soaked in the homogenate and loaded onto the gels. A sample from V. articulataPI 220879 was simultaneously loaded on both ends of each gel as a standard. Running buffers were the same as those described by Mulcahy et al. (1981). The loaded gels were run in five voltage steps (50, 100, 200, 300, and 400 V) for 15 min each. The gels were stained for acid phosphatase (ACP, E.C. 3.1.3.2), esterase (EST, E.C. 3.1.1.1), isocitrate dehydrogenase (IDH, E.C. 1.1.1.42), realate dehydrogenase (MDH, E.C. 1.1.1.37), malic enzyme (ME, E.C. 1.1.1.40), peroxidase (PRX, E.C. 1.11.1.7), and shikimate dehydrogenase (SKD, E.C. 1.1.1.25) by the modified procedures of Wendel and Weeden (1989). The modifications were (i) Fast Blue BB salt was used instead of Fast Blue RR and Fast Garnet GBC for EST; (ii) NADP instead of NAD was used for IDH; (iii) 50 mMTris-HCl at pH 9.1, instead of 8.5, was used for MDH; and (iv) NBT instead of MTT was used for IDH, MDH, ME, and SKD.

Data Measurement and Analysis

Isozyme bands were scored as present (1) or absent (0) to establish binomial data. Bands of a given enzyme for an individual plant were numbered from the most anodal to the most cathodal. Polymorphism index, which estimates the degree of variability of the accessions and species was defined by Skroch et al. (1992) as

[1] [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

where i, j = 1 ..... n is the number of individuals (seeds or plants) within each accession k = 1...m is the number of bands detected, and [p.sub.i] and [p.sub.j] take the values 1 or 0 when the band is present or absent in Individuals i and j, respectively.

A polymorphism index (PX) was calculated for each enzyme as

[2] [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

where k = 1 ..... m is the number of bands detected for a given enzyme in individuals i or j. The [PX.sub.e.w] is the mean of the absolute values of all possible pairwise differences among n individuals of an accession and range from 0 to 1. According to this equation, two plants with the same bands have [PX.sub.e.w] = 0, i.e., there are no differences between them. Within-accession polymorphism ([PX.sub.w]) was calculated as the mean [PX.sub.e.w] (across enzymes), i.e.,

[3] [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

where c = 1 ..... l is the number of enzymes studied. Larger values of [PX.sub.w] represent greater average variation within the accession.

Among-accession polymorphism for each enzyme ([PX.sub.e.a]) was calculated as the mean of the absolute values of all the pairwise differences among accessions within a given species. Equation [2] was used to make the calculations after replacing [p.sub.ki], and [p.sub.kj] by [f.sub.ki'] and [f.sub.kj'], which are frequencies of the kth band (number of individuals that carry it divided by the total number of individuals measured in the accession) for a given enzyme in accessions i' and j', respectively, where i', j' = 1 ..... n' is the number of accessions. Among-accession polymorphism ([PX.sub.a]) was calculated as the mean [PX.sub.e.a] across enzymes within a species with Eq. [3] after replacing [PX.sub.e.w] by [PX.sub.e.a]. Larger values of [PX.sub.a] represent greater average variation among accessions within a species. Total polymorphism of a species is the sum of mean [PX.sub.w] (across accessions within the species) and [PX.sub.a].

RESULTS AND DISCUSSION

The seven enzyme systems produced a total of 144 different bands across all accessions: five for ME, 10 for IDH and SKD, 25 for MDH, 26 for ACP, 33 for EST, and 35 for PRX. Banding patterns varied from only one band in most accessions for ME to more than 10 bands for ACP in 14 of 31 accessions. The overall mean number of bands per accession for the seven enzymes was 32.9, with a range of 21.4 in V. pisiformis PI 314124 to 43.1 in V. errilia PI 252053.

More than 90% of Vicia accessions (28 of 31) were polymorphic for at least one of the seven enzymes studied. Accessions of V. villosa and V. villosa ssp. varia had largest [PX.sub.e.w] for ACP, EST, IDH, MDH, and PRX (Table 1). Vicia peregrina PI 230346 and V. lutea PI 250797 had the largest [PX.sub.e.w] for ME and V. ervilia PI 203145 had the largest [PX.sub.e.w], 0.20, for SKD (Table 1).

Table 1. Polymorphism index for each of seven enzymes ([PX.sub.e.w]) for 31 Vicia accessions and within-accession polymorphism ([PX.sub.w]).
                                        Enzyme([dagger])

Accession and origin            ACP      EST      IDHI     MDH
V. articulata Hornem

PI 206390--Cyprus              0.00      0.00     0.00    0.00
PI 220879--Belgium             0.00      0.00     0.00    0.00
PI 449362--Ecuador             0.00      0.00     0.00    0.00

V. benghalensis L.

PI 298001--Australia           0.07      0.01     0.02    0.08
PI 298003--Australia           0.19      0.00     0.05    0.04
PI 449330--Chile               0.00      0.00     0.00    0.00

V. cracca L.

PI 371785--Alaska              0.14      0.01     0.00    0.08
PI 372763--Alaska              0.01      0.05     0.14    0.07

V. ervilia (L.) Willd.

PI 203145--Jordan              0.12      0.10     0.05    0.04
PI 252053--Turkey              0.04      0.00     0.02    0.00
PI 426201--Afghanistan         0.02      0.00     0.00    0.00

V. lutea L.

PI 201994--Turkey              0.06      0.01     0.00    0.00
PI 249880--Crete               0.00      0.01     0.00    0.00
PI 250797--Afghanistan         0.14      0.07     0.00    0.00

V. narbonensis L.

PI 206927--Turkey              0.03      0.00     0.00    0.00
PI 294298--Israel              0.00      0.00     0.00    0.00
PI 294301--Israel              0.12      0.03     0.07    0.01

V. pannonica Crantz

PI 220887--Belgium             0.07      0.06     0.00    0.03
PI 220888--Belgium             0.07      0.07     0.11    0.07

V. peregrina L.

PI 230346--Iran                0.00      0.05     0.00    0.00
PI 234766--Spain               0.00      0.01     0.00    0.03

V. pisiformis L.

PI 314124--Alaska              0.02      0.00     0.00    0.02

V. saliva L.

`Cahaba White'--cv. Alabama    0.00      0.00     0.00    0.01
`Warrior'--cv. Alabama         0.21      0.00     0.00    0.00
PI 284563--Germany             0.00      0.00     0.00    0.03

V. villosa Roth

PI 201883--Iran                0.32      0.18     0.50    0.20
PI 206493--Turkey              0.32      0.20     0.46    0.24
PI 222217--Afghanistan         0.29      0.19     0.35    0.19

V. villosa Roth ssp.
varia (Host) Corbiere

PI 210431--Greece              0.34      0.18     0.38    0.28
PI 212044--France              0.32      0.13     0.47    0.27
PI 234051--Spain               0.33      0.18     0.43    0.23

                                        Enzyme([dagger])

Accession and origin
V. articulata Hornem           ME       PRX     SKD    ([PX.sub.w])

PI 206390--Cyprus              0.00     0.00    0.00      0.000
PI 220879--Belgium             0.04     0.07    0.04      0.021
PI 449362--Ecuador             0.00     0.03    0.00      0.004

V. benghalensis L.

PI 298001--Australia           0.00     0.07    0.19      0.063
PI 298003--Australia           0.00     0.07    0.00      0.050
PI 449330--Chile               0.00     0.00    0.00      0.000

V. cracca L.

PI 371785--Alaska              0.22     0.11    0.00      0.080
PI 372763--Alaska              0.00     0.06    0.06      0.056

V. ervilia (L.) Willd.
PI 203145--Jordan              0.00     0.05    0.20      0.094
PI 252053--Turkey              0.00     0.00    0.00      0.009
PI 426201--Afghanistan         0.00     0.00    0.00      0.003

V. lutea L.

PI 201994--Turkey              0.00     0.05    0.09      0.030
PI 249880--Crete               0.00     0.01    0.00      0.003
PI 250797--Afghanistan         0.36     0.08    0.08      0.104

V. narbonensis L.

PI 206927--Turkey              0.08     0.03    0.00      0.002
PI 294298--Israel              0.00     0.00    0.00      0.000
PI 294301--Israel              0.00     0.00    0.00      0.003

V. pannonica Crantz

PI 220887--Belgium             0.00     0.09    0.08      0.047
PI 220888--Belgium             0.16     0.05    0.00      0.076

V. peregrina L.

PI 230346--Iran                0.41     0.02    0.00      0.069
PI 234766--Spain               0.16     0.04    0.00      0.034

V. pisiformis L.

PI 314124--Alaska              0.09     0.16    0.00      0.041

V. saliva L.

`Cahaba White'--cv. Alabama    0.00     0.02    0.00      0.004
`Warrior'--cv. Alabama         0.00     0.01    0.00      0.031
PI 284563--Germany             0.00     0.01    0.00      0.006

V. villosa Roth

PI 201883--Iran                0.12     0.16    0.16      0.230
PI 206493--Turkey              0.29     0.16    0.15      0.260
PI 222217--Afghanistan         0.16     0.16    0.06      0.214

V. villosa Roth ssp.
varia (Host) Corbiere

PI 210431--Greece              0.03     0.17    0.12      0.214
PI 212044--France              0.26     0.15    0.12      0.246
PI 234051--Spain               0.19     0.47    0.19      0.289


([dagger]) ACP, acid phosphatase; EST, esterase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; PRX, peroxidase; SKD, shikimate dehydrogenase.

The [PX.sub.w] of each accession ranged from zero to 0.289. V. villosa ssp. varia PI 234051 had the largest [PX.sub.w] (0.289), whereas V. articulata PI 206390, V. benghalensis PI 449330, and V. narbonensis PI 294298 had no variation ([PX.sub.w] = 0) (Table 1). Variability within each species, as measured by mean [PX.sub.w] of each species (across accessions), ranged from 0.008 for V. articulata to 0.250 for V. villosa ssp. varia. (Table 1).

Among-accession polymorphism for each enzyme ranged from [PX.sub.e.a] = 0.00 for SKD in V. peregrina and for ME in V. satira to [PX.sub.e.a] = 0.52 for IDH in V, narbonensis (Table 2). Mean [PX.sub.e.a] across enzymes within a species ranged from [PX.sub.a] = 0.127 (V. villosa ssp. varia) to [PX.sub.a] = 0.301 (V. ervilia) (Table 2). A greater proportion of the total polymorphism (total PX) of V. villosa and V. villosa ssp. varia resulted from within- rather than among-accession polymorphism. The other species had larger [PX.sub.a] than mean [PX.sub.w] (Table 3). These results indicate that V. villosa and V. villosa ssp. varia are predominantly cross-fertilizing, whereas V. articulata, V. benghalensis, V. ervilia, V. lutea, V. pannonica, and V. sativa are predominantly self-fertilizing. This is in agreement with the results of controlled-pollination experiments in the field obtained by Zhang and Mosjidis (1995). The species V. cracca, V. narbonensis, and V. peregrina, which were lost in the experiment of Zhang and Mosjidis (1995) because of lack of adaptation to the southeastern USA environments, were also determined to be predominantly self-fertilizing. Furthermore, [PX.sub.w] of each accession of the species known to be predominantly selffertilizing ranged from 0.000 to 0.104, whereas [PX.sub.w] of each accession of the species known to be predominantly cross-fertilizing ranged between 0.214 and 0.289. These results indicate that the mating system of the only accession of V. pisiformis tested in this study, but not tested by Zhang and Mosjidis (1995), is predominantly self-fertilizing ([PX.sub.w] = 0.041). Similar results were found for each of the accessions of V. cracca, V. narbonensis, and V. peregrina; however, the manner in which the accession was collected may influence its degree of variability.

Table 2. Among-accession polymorphism index for each of seven enzymes ([PX.sub.e.a]) within 12 Vicia species and their mean ([PX.sub.a]).
                                  Enzyme([dagger])

Species                  ACP      EST       IDH      MDH

V. articulata           0.31     0.18      0.20      0.21
V. benghalensis         0.35     0.27      0.31      0.22
V. cracca               0.26     0.15      0.29      0.12
V. ervilia              0.34     0.39      0.19      0.25
V. lutea                0.32     0.28      0.13      0.24
V. narbonensis          0.38     0.24      0.52      0.24
V. pannonica            0.27     0.32      0.26      0.13
V. peregrina            0.27     0.34      0.10      0.09
V. sativa               0.18     0.18      0.33      0.06
V. villosa              0.20     0.09      0.13      0.16
V. villosa
 ssp. varia             0.12     0.07      0.19      0.16

                                     Enzyme([dagger])

Species                  ME       PRX      SKD     ([PX.sub.a])

V. articulata           0.14     0.22      0.17      0.204
V. benghalensis         0.27     0.26      0.17      0.264
V. cracca               0.20     0.19      0.03      0.173
V. ervilia              0.44     0.32      0.22      0.301
V. lutea                0.25     0.17      0.19      0.226
V. narbonensis          0.22     0.30      0.11      0.287
V. pannonica            0.18     0.17      0.05      0.197
V. peregrina            0.31     0.22      0.00      0.190
V. sativa               0.00     0.18      0.06      0.141
V. villosa              0.09     0.09      0.15      0.130
V. villosa
 ssp. varia             0.13     0.07      0.15      0.127


([dagger] ACP, acid phosphatase; EST, esterase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; PRX, peroxidase; SKI), shikimate dehydrogenase.

Table 3. Total polymorphism index and the percent contribution of mean within-accession polymorphism (P[X.sub.w]) and among-accession polymorphism (P[X.sub.a]) in each of 11 Vicia species.
                                           Mean
Species                      Total PX    P[X.sub.w]    P[X.sub.a]

V. articulata                   0.21          4            96
V. benghalensis                 0.30         14            86
V. cracca                       0.24         29            71
V. ervilia                      0.34         10            90
V. lutea                        0.28         17            83
V. narbonensis                  0.30          4            96
V. pannonica                    0.26         23            77
V. peregrina                    0.24         21            79
V. sativa                       0.15          9            91
V. villosa                      0.37         64            36
V. villosa ssp. varia           0.38         66            34


Since among-accession polymorphism was greater than within-accession polymorphism in V. articulata, V. benghalensis, V. cracca, V. ervilia, V. lutea, V. narbonensis, V. pannonica, V. peregrina, and V. sativa, genetic diversity of these species will be better represented in germplasm banks by increasing the number of accessions than by increasing the sample size of each accession. Seed may be increased from relatively small number of individuals per accession. However, in the predominantly cross-fertilizing species V. villosa and V. villosa ssp. varia, in which about two thirds of the variability resided within the accessions (Table 3), a relatively large sample size of each accession would be required to maintain genetic variability. Total PX in V. villosa and V. villosa ssp. varia was larger than that in the predominantly self-fertilizing species by more than 10%. This value is similar to results of a survey of 473 species conducted by Hamrick and Godt (1989), where they compared variability in self- and cross-fertilizing species.

In summary, most of the accessions studied were variable for at least one of the seven enzymes. Within-accession polymorphism was larger than that among accessions in V. villosa and V. villosa ssp. varia, whereas within-accession polymorphism was smaller than that among accessions of V. articulata, V. benghalensis, V. cracca, V. ervilia, V. lutea, V. narbonensis, V. pannonica, V. peregrina, V. pisiformis, and V. sativa. Total polymorphism in V. villosa and V. villosa ssp. varia was larger than that in the other species. Although the number of accessions per species used to measure polymorphism was too small to determine unequivocally the mating system per species, the results indicate that accessions of V. villosa and V. villosa ssp. varia are predominantly cross-fertilizing species, whereas the accessions of other species are mainly self-fertilizing; therefore, seed regeneration should differ between these two groups. The use of isozyme bands to make a preliminary determination of the mating system proved accurate. Advantages of this method are the small seed sample, low expense, and simplicity. A limitation of this procedure is that it does not provide information on the amount of mixed mating (mixture of selfing and outcrossing), yet it helps to broadly categorize the accessions as predominantly self- or cross-fertilizing.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the support of Mr. Gilbert Lovell, Curator, Plant Genetic Resources Conservation Unit, Griffin, GA 30223-1797. This research was partially funded by the USDA-ARS, Specific Cooperative Agreement 58-43YK-9-0017.

Abbreviations: ACP, acid phosphatase; EST, esterase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; PRX, peroxidase: [PX.sub.a], mean among-accession polymorphism across enzymes; [PX.sub.e.a], among-accession polymorphism within a species for a given enzyme; [PX.sub.e.w], within-accession polymorphism for a given enzyme; [PX.sub.w], within-accession polymorphism; SKD, shikimate dehydrogenase.

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Wendel, J.F., and N.F. Weeden. 1989. Visualization and interpretation of plant isozymes, p. 5-45 In D.E. Soltis and P.S. Soltis (ed.) Isozymes in plant biology. Dioscorides Press, Portland, OR.

Zhang, X., and J.A. Mosjidis. 1995. Breeding systems of several Vicia species. Crop Sci. 35:1200-1202.

Dep. of Agronomy and Soils and Alabama Agric. Exp. Stn., Auburn Univ., AL 36849-5412, USA. J. Series No. 3-975784. Received 30 March 1997. J.A. Mosjidis, Corresponding author (jmosjidi@acesag.auburn.edu).
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Publication:Crop Science
Date:May 1, 1998
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