Genetic control of leucine aminopeptidase and esterase isozymes in the interspecific cross Cucurbita ecuadorensis x C. maxima

Biochemical Genetics 5:223-229 (1971)

Genetic Control of Leueine Aminopeptidase and Esterase Isozymes in the Interspecific Cross Cucurbita ecuadorensis x C. m a x i m a J. R. Wall s and Thomas W. Whitaker 2

Received 9 Oct. 1970 Final24 Nov. 1970

Starch gel electrophoretic analyses of crude seed extracts of Cucurbita ecuadorensis, C. maxima, their F1 and F2, and three of the four possible interspecific backcrosses reveal that the genus is polymorphie for alpha-naphthyl acetate esterases (Est) and leucine aminopeptidase (LAP). The two electrophoretie forms of both Est and LAP are controlled by codominant alleles. The two loci do not exhibit linkage. Neither the LAP nor the Est phenotypes exhibit a significant deviation from the expected 1:1 ratio in interspecific backcrosses when the donor parent alleles are transmitted through female gametes, but there is a significant deviation for Est when transmission is through male gametes. Differential gametic selection involving the Est-1 locus suggests structural differences between the genomes of the parental species for the chromosomal region in which this locus occurs. No structural differences are indicated between the parental genomes for the chromosome region bearing the Lap-1 locus. INTRODUCTION The study of electrophoretic variants of enzymes and proteins has furnished a valuable means of investigating the genetic structure of populations (Prakash et al., 1969; Brown and Allard, 1969), biosystematic relationships (Hunziker, 1969), and the evolution of the gene itself (Shows et al., 1969). In this paper we report yet another application of isozyme gel zone electrophoresis to evolutionary genetics, namely, the application of the method to a higher plant speciation problem involving two related South American species of the New World genus Cucurbita. The center of diversity of the genus Cucurbita is in the region south of Mexico City extending as far south as the Mexico-Guatemala border (Whitaker and Bemis, 1965). It is a relatively small genus comprised of about 25 species, only three of which 1 Department of Biology, George Mason College of the University of Virginia, Fairfax, Virginia. 2 U.S. Department of Agriculture, ARS, Crops Research, La Jolla, California. 223

224

Wall and Whitaker

are exclusively South American. These three have been termed the "Maxima group" by Hurd and Linsley (1970). A fourth species, C. moschata, of the Moschata group, has inhabited both North and South America disjunctly since before the presence of man in the New World (Hurd and Linsley, 1970). All other species are indigenous to North America. The Maxima group is comprised of two closely related species, C. andreana and C. maxima, and a more distantly related species, C. ecuadorensis. This investigation concerns the latter two species, their F1, Fz, and backcross progenies. C. maxima is known today only as a cultivated species, but there is ample archaeological evidence of its former natural distribution in the temperate regions of Peru, Chile, and Argentina (Whitaker and Cutler, 1965). C. ecuadorensis is a recently discovered and described species of limited distribution in the lowlands of Ecuador (Cutler and Whitaker, 1969). It is unclear when members of the genus first became established in South America. However, based upon evidence from the coevolution of the squash bees of the genus Peponapis with the Maxima group, this group apparently was first isolated from the North American gene pool well before the Moschata group invaded South America in the early Tertiary (Hurd and Linsley, 1967, 1970). Starch gel electrophoretic surveys of Cucurbita have been reported for esterases (Schwartz et al., 1964; Wall, 1969) and leucine aminopeptidase (Wall, 1969). This paper describes the genetic control of alpha-naphthyl acetate esterases (E.C. 3.1.1-) and leucine aminopeptidase (E.C. 3.4.1.1) in the interspecific cross C. ecuadorensis x C. maxima and presents evidence for genome divergency between the two species. A preliminary report of this work has been published (Wall and Whitaker, 1968). MATERIALS

AND METHODS

The plant materials used were C. ecuadorensis (2n = 40), collected in Ecuador by Dr. and Mrs. A. E. Michelbacher, University of California at Berkeley, and C. maxima cv. "Pink Banana" (2n = 40). Cross-pollinations were performed in the field at Brawley, California. Pollen stainability was used as the measure of fertility. Enzyme preparations for electrophoresis were obtained from individual mature seeds first soaked in distilled water for 2-4 hr at 22 C. The seed coats were removed and the embryos were ground in porcelain mortars in 0.15 Mphosphate buffer, pH 7.0, at about 4 C. Sufficient buffer was added to make a slurry which was freeze-thawed at least once to enhance enzyme release. Vertical starch gel electrophoresis was conducted at 4 C. All gels contained 13 % hydrolyzed starch. Two buffer systems were used for the two enzymes. Leucine aminopeptidase (LAP) separation was achieved using the discontinuous system, pH 8.6, developed by Poulik (1957) and modified by K. A. Ferguson (Ashton and Braden, 1961) with a voltage gradient of 7 v/cm for about 5 hr. Esterases were separated using the continuous triple buffer system, pH 8.6, of Boyer et al. (1963) with a voltage gradient of 6 v/cm for 14 hr. Although LAP isozymes can be separated by the EBT buffer system, the resolution is inferior to the Poulik-Ferguson system. Enzyme visualization was achieved for LAP by placing the cut gels in a reaction mixture consisting of 100 ml of 0.05 M tris-maleate buffer at pH 6.0, 25 mg of L-leucyl ]~-naphthylamide HC1, and 50 mg of Black K salt. The reaction mixture for the

225

Genetic Control of Isozymes in Cucurbita

+

Est-l-a [st-l-b

Phen.- b

ab

a

b

ab

ab

a

b

ab

Fig. 1. Seed Est phenotypes observed in Cucurbita. Left to right: C. maxima cv. "Pink Banana," F1 hybrid, C. ecuadorensis, and six individual Fzs.

esterases consisted of 100 ml of 0.05 M tris-maleate buffer at p H 7.0, 25 mg of alphanaphthyl acetate, and 50 mg of Fast Garnet GBC salt. R E S U L T S AND D I S C U S S I O N Genetic Control of Est and L A P Variants

Esterase and LAP phenotypes of seed extracts are illustrated in Figs. 1 and 2. Both loci exhibit codominance, and three phenotypes are present for each enzyme system. The gene symbols assigned to the Est variants are Est-1 a for the enzyme migrating more anodally, and Est-1 b for the more slowly migrating enzyme. The LAP variants are assigned the symbols L a p - 1 a for the faster and L a p - 1 b for the more slowly migrating enzymes.

+

LAP-I-a LAP-I-b

Phen.- b

ab

a

ab

a

b

ab

ab

b

Fig. 2. Seed LAP phenotypes observed in Cucurbita. Left to right: C. ecuadorensis, F1 hybrid, C. maxima cv. "Pink Banana," and six individual F2s.

Wall and Whitaker

226

Table I. Summary of Crosses Demonstrating the Inheritance of Est-1 Phenotypes and Differential Elimination o f Donor's Allele in Reciprocal Backcrosses Est-1 phenotypes ofprogeny

Generation Pi P2 F1 F2 F1BC (a)

(b)

Female parent

Male parent Est1-a

Est1-ab

Est1-b

Total

df

Za

C. ecuadorens~ C. maximacv."P.B." C. ecuadorensis F1

C. ecuadorens~ C. maximacv."P.B.'" C. maxima cv."P.B." F1

25 0 0 27

0 0 25 53

0 22 0 24

25 22 25 104

2

0.21(1:2:1)

F1 C. ecuadorensis F1

C. ecuadorensis F1 C. maximacv."P.B."

32 33 0

28 17 101

0 0 94

60 50 195

1 1 1

0.26(1:1) 5.12(1:1) 0.25(1:1)

P

P=0.90

0.70>P>0.50 0.025>P>0.020 0.70>P>0.50

The seven matings demonstrating the mode of inheritance of the Est-1 variants are summarized in Table I. The phenotypic ratios of P~, P2, their F~, F 2, and backcross generations indicate that C. eucadorensis and C. maxima are homozygous for the faster and more slowly migrating forms, respectively, at the Est-1 locus. All segregating populations give a satisfactory fit to their expected phenotypic ratios with the exception of the backcross C. ecuadorensis ~ x F1 c~, the deviation of which, as will be shown below, is evidence of genome divergency between C. ecuadorensis and C. maxima at the Est-1 locus. The inheritance of the LAP variants is summarized in Table II. Again, C. ecuadorensis and C. maxima are homozygous at the Lap-1 locus, but for the slower and faster forms, respectively. All segregating populations give a satisfactory fit for their expected phenotypic ratios. The LAP phenotypes, like the Est phenotypes, are controlled by two codominant alleles. There is no evidence of linkage between the Est-1 and Lap-1 loci. Table III, which summarizes the combined F 2 data for these loci, gives clear evidence of their independent assortment (0.99 > P > 0.95).

Table II. Summary o f Crosses Demonstrating the Inheritance o f LAP-1 Phenotypes and Lack of Differentia1 Elimination o f Donor's Allele in Reciprocal Backcrosses LAP-1 phenotypes o f progeny

Generation PI P2 F1 Fz F1BC (a)

(b)

Female parent

Male parent

Total

df

Z2

P

LAP- LAP- LAPl-a 1-ab 1-b C. ecuadorensis C. maxima cv. "P.B." C. ecuadorensis F~

C. ecuadorensis C. maxima cv. "P.B." C. maxima cv. "P.B." F1

0 22 0 29

0 0 25 50

25 0 0 25

25 22 25 104

2 0.47(1:2:1)

0.90> P > 0.70

F1 C. ecuadorensis F1

C. ecuadorensis F1 C. maxima ev. "P.B."

0 0 106

31 23 89

29 27 0

60 50 195

1 0.07(1:1) 1 0.32(l:1) 1 1.48(1:1)

0 . 9 0 > P > 0.70 0 . 7 0 > P > 0.50 0 . 3 0 > P > 0.20

Genetic Control of Isozymesin Cucurbita

227

TableIII. F2 SegretationRatiofor Est-1 and Lap-I Lociand Chi-SquareAnalysisfor IndependentAssortment(seefootnote a for notation) Genotypes E.Ia/E-la; E-la/E-la; E-la/E-lb; E-ia/E-Ib; E.la/E-la; E-la/E-lb ; E-lO/E-lb; E.lb/E-lb; E-lb/E.lb; L-Ib/L-1 b L-lb/L-1 a L.lt'/L.1 ~ L.lb/L-1 a L.Ia/L.I a L.la/L.1 a L-Ib/L-I ~ L-lb/L.1 a L.la/L-1 a

Genotypicfrequency Observedfrequency Expectedfrequency Deviation Dev.Z/exp.

1/16

a E = E~t: L = Lap

2/16

2/16

4/16

1/16

2/16

1/16

2/16

1116

6

11

14

25

9

14

5

14

6

6.5 0.5 0.04

13 2.0 0.31

13 1.0 0.08

26 1.0 0.04

6.5 2.5 0.96

13 1.0 0.08

6.5 1.5 0.35

13 1.0 0.08

6.5 0.5 0.04

Z~~

1.98(8dr); 0.99>P>0.95.

Genome Divergency and Species Relationships Table IV presents d a t a on the fertility o f the p a r e n t a l species, the F 1, F 2, a n d b a c k c r o s s generations. A l t h o u g h the p o p u l a t i o n s supplying the d a t a are small, the i n f o r m a t i o n available is useful for illustrating the relative extent o f genetic isolation between C. ecuadorensis and C. maxima. These species hybridize with relative ease a n d p r o d u c e F l s o f n o r m a l vigor a n d o f r e a s o n a b l e fertility. N o instance o f c o m p l e t e pollen sterility has been f o u n d in the F~. However, the F 2 gives evidence o f h y b r i d b r e a k down, having some individuals with c o m p l e t e pollen sterility. Also, 25 ~ (six o f 24 individuals) o f one F2 p o p u l a t i o n exhibited a b n o r m a l development, having chlorotic leaves, stems, a n d petioles. The m o s t plausible e x p l a n a t i o n for h y b r i d b r e a k d o w n is still Stephens' (1949, 1950) hypothesis that infertility in an F 2 is the result o f small c h r o m o s o m a l structural differences between p a r e n t a l species. Such differences for the c h r o m o s o m e region c a r r y i n g the E s t - 1 locus o f the C u c u r b i t a materials u n d e r discussion here are suggested by the differences in transmission o f the d o n o r E s t allele in reciprocal backcrosses to

Table IV. Fertility of Parents, F1, Fz, and Backcross Generations Percent stainable pollen Generation P1 (C. ecuadorensis) P2 (C. m a x i m a cv. "P.B.") F I (C. eeuadorensis~ x C. m a x i m a

cv. "P.B,"o~) F2 (FI~ x FI~) F1 BC (a) (F1 ~ x C. ecuadorensisc?) (C. ecuadorensis$ x F~9) (b) (FI~ x C. m a x i m a cv. "P.B."ff)

N Range

Mean

4 4 7 16

84-94 88-97 51-68 0-75

90.3 95.0 57.7 29.9

I0

0-96 43-67 58-92

31.8 56.0 78.6

4 8

228

Wall and Whitaker

C. ecuadorensis (Table I). When the Est-1 b allele from C. maxima is transmitted through female gametes (via the F1 in the backcross F1 ~- x C. ecuadorensis ¢)), the backcross ratio is 1 : 1. But when this allele is transmitted through F1 male gametes (C. ecuadorensis ~ x F 1 c?), there is a significant deviation from the 1:1 ratio, suggesting differential elimination of the Est-1 b allele. Based upon the view that closely related species may differ by small structural differences in one or more of their chromosomes, it is clear how reciprocal differences in Est-1 ratios might occur (see Stephens, 1949). In the F1, crossing over in a region of the genome lacking complete homology will lead to the formation of crossover and noncrossover chromatids. The latter would be fully viable in gametes, while the former would not be because they carry small deficiencies and duplications. Selection would tend to eliminate some of the gametes carrying these recombination chromatids and would be much more intense for male gametes than for female gametes because of pollen competition. The noncrossover chromatids would be of two types, namely, C. maxima and C. eeuadorensis. In backcrosses to C. ecuadorensis, the donor parent (C. maxima) chromatids would be at a selective disadvantage to the ecuadorensis chromatids. It is concluded that the Est-1 locus is located in a region of a chromosome which lacks complete heterospecific homology. The possibility that the differences in reciprocal backcross ratios are determined by the linkage of a pollen lethal to the Est-I locus in C. maxima, with the lethal active only in C. ecuadorensis stylar tissue, is contraindicated by the ease of obtaining the F1 hybrid with C. maxima as male parent. Since there is hybrid breakdown in the recombination generations and only partial exclusion of the donor Est-I allele in the backcross C. ecuadorensis ~ x F~ ~, the possibility of linkage between the Est-1 locus and a gametophytic factor (Ga vs. ga), such as reported by Bemis (1959) in lima beans, where Ga gametophytes effect fertilization to the almost complete exclusion of ga gametophytes, is also rejected. Since differential elimination of the donor species LAP allele did not occur in reciprocal backcrosses to C. eeuadorensis (Table II), it is concluded that the Lap-1 locus occurs in a chromosomal region having high heterospecific homology. Although we have limited this study to two loci controlling isozymes of the mature seed, it should be possible to achieve a more complete analysis of genome homologies for parental species by using additional enzyme variant markers. Small portions of seed may be used for seed isozyme assay and the remainder germinated for the analysis of electrophoretic markers of the developmental stages of individual genotypes, so long as the formal genetics of each marker is understood. Since dominant and recessive marker systems require progeny testing of heterozygotes, a difficult and time-consuming operation in higher organisms, the simplest markers to use are those determined by codominant alleles. Genome analysis by the use of isozyme markers is a promising means for studying species relationships, especially for closely related species which can be readily hybridized. Unlike conventional cytological studies of F 1 meiotic figures, it is a method which can be easily applied to genera with numerous small chromosomes, such as Cueurbita, and which can give evidence of cryptic chromosomal structural differences. In addition, this method can reveal a much more detailed picture of chromosome block and loci differences and similarities than can genome analysis

Genetic Control of Isozymes in Cucurbita

229

using " t o t a l " seed storage proteins, such as r e p o r t e d by J o h n s o n a n d H a l l (1965) for Triticum species.

ACKNOWLEDGMENTS The a u t h o r s w o u l d like to express their a p p r e c i a t i o n to Drs. P. D. H u r d , Jr., W. P. Bemis, a n d Y. H o t t a for r e a d i n g the m a n u s c r i p t a n d m a k i n g helpful comments.

REFERENCES

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