The Two B Genes of Cucurbita are Unlinked

Cucurbit Genetics Cooperative Report 14:116-122 (article 41) 1991

Oved Shifriss
21 Walter Avenue, Highland Park, NJ 08904

The available data (in reference 4 and in the present report) support the conclusion that the two B genes of Cucurbita are unlinked. One of the B genes originated in C. pepo and the other in C. maxima.

In order to acertain the nature of the relationship between the two B genes by breeding tests, these genes were transferred to C. moschata. As a result, two different inbreds were established in C. moschata: NJ-B carries the B of C pepo and IL-B carries the B of C. maxima. (1). The evidence for their unlinked relationship is based on inheritance studies of the cross NJ-B X IL-B as well as of testcrosses.

Each of the B genes conditions precocious depletion of chlorophyll. This effect is presumably due to blockage of normal transformation of proplastids into chloroplasts, indirectly inhibiting chlorophyll synthesis.

The two genes were designated years ago by symbol B because they are responsible for the bicolor variations that potentially effect fruit pigmentation. Since they are now believed to be unlinked, the B of C. pepo is designated as B₁ and the B of C. maxima, as B₂.

B₁ and B₂ are similar in two respects. First, their primary target is the fruit, Specifically, the fruit “skin” (the outer layer of cells) is the site at which they are active in all known genetic backgrounds. Second, when these genes are inserted in certain backgrounds they effect some or all other aerial organs.

B₁ and B2 also differ from one another. First, the expression of B₁ is strictly limited to pre-anthesis stages, whereas the expression of B₂ can be manifested over a wide range of time during fruit development,. from very early pre-anthesis stages to post-anthesis stages. Second, apart from fruit, B₁ can effect a relatively few other organs (e.g., cotyledons and true leaf blades), whereas B₂ can effect all other aerial organs, depending on the genetic background. Particularly relevant to the discussion here is the fact that, unlike B₁, B₂ can bring about precocious depletion of chlorophyll in stems.

The objective of the present report is to summarize and interpret the cumulative data bearing upon the relationship between the B genes. It should be pointed out, however, that the long-term aim is to identify the major genetic elements that play a role in sustaining the capacity of plants to synthesize chlorophyll throughout development. This broader aim includes testing the HPT (the nuclear homostat of plastid transformation) hypothesis (2).

In a preliminary study of inheritance (1), the F₁ plants of NJ-B X IL-B exhibited precocious depletion of chlorophyll, essentially as in IL-B, and the F₂ varioed widely, ranging from lethal seedlings (albino or yellow seedlings died at the cotyledon stage) to completely green plants. The intermediate plants manifested chlorophyll deficiency selectively in different organs, at different times and in varying extent during development. especially striking were the variegated patterns of green and yellow that effected fruits and leaves.

This preliminary study ws conducted under greenhouse conditions that were not very favorable to plant growth; classification of phenotypes was often difficult or uncertain; and no firm conclusions were drawn from it. Nevertheless, the 4.1% of completely green plants in the F2 (9 out of 217) did not disagree with the hypothesis of duplicate genes (for 15:1 ratio, chi-square = 1.6372 and P = 0.20 – 0.30), alluding that these plants might be conditioned by a homozygote for two standard (“wild-type”) unlinked genes.

Subsequent studies of inheritance were conducted under field conditions in four growing seasons. During this period no lethal seedlings were evident in the F₂. But semi-lethal (particularly yellow) plants consistently occurred in the F₂, albeit in varying proportions and in varying degrees of yellowing. The majority of these plants survived in each season, but they were difficult to reproduce. Although clear-cut lethals appeared in one environment, and “semi-lethals” appeared in other environments, the overall picture of the F₂ variation was the same in its complexity. For sake of simplicity the present study of inheritance was limited to the effect of chlorophyll deficiency on fruits and stems exclusively.

Results and interpretation. The system of classification of individual plant phenotypes is described in Table 1. The data in Table 2 are based on the use of this system. The hypothetical relationship between genotypes and phenotypes is shown in Figure 1.

The data in Table 2 are of special significance. These data were gathered under field conditions during the fourth growing season, after some experience in classification was gained in the previous three seasons. In obtaining these data each plant was observed at least three times before its phenotype was determined. Furthermore, segregates of questionable phenotypes received special examination. For example, no plant was classified as completely green (GO, GS) unless it produced in sequence over 15 green fruits, and exhibited green stems throughout the season. This examination was essential because some potentially bicolor-fruited individuals produce green fruits (not more than 10) early in development, andbecause other individuals that potentially can exhibit chlorophyll deficiency in their stems have completely green stems early in life.

The hypothesis on the relationship between genotypes and phenotypes in Figure 1 tells us that the data in Table 2 should give a good fit to 12:3:1 ratio as well as to 15:1 ratio. This is true if the phenotypic classes are grouped according to theory (Figure 1). A good fit to 12:3:1 ratio is obtained if classes are arranged into three groups of 1 to 4, 5 to 6, and 7, i.e., 275:61:23 (X² = 0.73, df = 2, P = 0.50-0.70). An excellent fit to 15:1 ratio is obtained if classes are arranged into two groups of 1 to 6 and 7, i.e., 336:23 (X² = 0.0150, df = 1, P = 0.90-0.95).

The grouping of the phenotypic classes (1 to 7) according to theory is supported by several observations. First, there is little doubt that the overwhelming majority if not all the persistently green plants of class 7 (GO, GS) breed true.

Second, field notes showed that thephenotypes of classes 5 and 6 reflect respectively the expression of homozygotes and heterozygotes of gene B of C pepo (as represented here by NJ-B). These observations are supported by the excellent fit to the 12:1:2:1 ratio (275:21:40:23, X² = 0.7586, dr = 3, P = 0.80-0.90).

Third, the lumping of classes 1 to 4 is justified by two considerations. (i) The F₁ phenotype shows that the expression of the B of C. maxima (as represented here by IL-B) is epistatic to the B of C. pepo with respect to chlorophyll deficiency in stems., (ii) The evidence in the following report shows that the phenotypes of clsses 2, 3 and 4 are conditioned by the heterozygotes of class 2 varies greatly and that some of their phenotypes may be mistaken occasionally with the phenotype of class 1.

Finally, the data summation in Tables 3 and 4 further strengthens the conclusion that the two B of C. pepo and C. maxima are unlinked.

Acknowledgment. I thank Baldwin Miranda, R.B. Volin and T.V. Williams of Northrup King Co. for providing land and technical assistance to conduct my research in Naples, Florida from 1988 to 1990.

Table 1. The system employed in phenotypic classification of ovaries, fruits and stems.

Class	Phenotypic symbolsz	Description
1	PDC-UO, PDC-S	Ovaries are subject to precocious depletion of chlorophyll over their entire surface. All ovaries and fruits are uniformly pigmented. Stems are also subject to precocious depletion of chlorophyll and appear yellow or golden.
2	GOT, PDC-SL	Ovaries are green. But sometime following anthesis the immature fruits are subject to chlorophyll depletion. Usually only their upper region is affected, resulting in bicolor (green and yellow) pigmentation. Stems are subject to precocious depletion of chlorophyll, but expressivity is low.
3	GOT, GS	Ovaries and fruits are as in class 2. Stems are consistently green.
4	GO, PDC-SL	Ovaries are green. Stems are as in class 2.
5	PDC-UO, GS	Ovaries are subject to precocious depletion of chlorophyll over their entire surface. All ovaries and fruits are uniformly pigmented. Stems are consistently green.
6	PDC, BiO, GS	Ovaries are subject to precocious depletion of chlorophyll ove their upper region, resulting in bicolor (green and yellow) pigmentation prior to anthesis. But some ovaries on the same plant are unaffected, being completely green. Stems are consistently green.
7	GO, GS	Ovaries and stems are consistently green.

Key to symbols:

Bi = bicolor; G = green; L = low expressivity; O = ovaries prior to anthesis; PDC – precocious depletion of chlorophyll; S = stems; T = turning, i.e., the fruit turns from green to yellow sometime following anthesis; U = uniformly pigmented.

Table 2. Inheritance of precocious depletion of chlorophyll, based on a cross between two “precocious” inbreds of Cucurbita moschata. Seed was sown in a greenhouse on February 15 and seedlings were transplanted into a field on March 2, 1990. Naples, Florida.

Phenotypic Classes^z

Breeding Material	1 PDC-UO PDC-S	2 GOT PDC-SL	3 GOT GS	4 GO PDC-SL	5 PDC-UO GS	6 PDC-BiO GS	7 GO GS	Number of classified plants
P1 , NJ-BY	0	0	0	0	5	0	0	5
P2 , IL-BY	5	0	0	0	0	0	0	5
F1 , P1, X P2	5	0	0	0	0	0	0	5
F2	249	22	1	3	21	40	23	359

^z See Table 1 in the present report for the system of classification.

^ySee Table 1 in reference 3 for the origin and description of this inbred.

Figure 1. Hypothetical relationship between genotypes and phenotypes in the F₂ of NJ-B X IL-B. See classes of phenotypes in Table 1 and Table 2 (above).

Table 3. Tests of the hypothesis that precocious depletion of chlorophylls in two inbreds of Cucurbita moschata is conditioned by two unlinked genes, B1 and B₂ .. Summary of field data obtained in Naples, Florida from fall of 1988 to spring of 1990.

	Classificationz
Breeding materials and tests	B1-B2 plus B1+ B1+ – B2	B1– B2+ B2+	B1+ B1+ B2+ B2+	Number of classified plants
P1 , NJ-By	0	27	0	27w
P2, IL-Bx	27	0	0	27w
F1, P1 x P2	53	0	0	53w
F2 pooled	832	184	82	1098w
	X²	df	P
Deviation (12:3:1)	5.02	2	0.05 – 0.10
Heterogeneity	1.82	6	0.90 – 0.95
F1 x standards x	210	85	112	407u
pooled
	X²	df	P
Deviation (2: 1: 1)	4.00	2	0.10 – 0.20
Heterogeneity	4.79	4	0.30 – 0.50
F1 x P1	47	43	0	90t

^z See Figure 1 for the relationship between genotypes and phenotypes.
^y The genotype of NJ-B is B1 B1 B₂+ B₂+
^x The genotype of IL-B is B1+ B1+ B₂ B₂ .
^w Total plants of 4 tests made in 4 seasons from fall of ’88 to spring ’90. The data from fall ’88 to fall ’89 are in Table 3 of reference 4. The data from spring ’90 are in Table 2 of the present report.
^vThe genotype of “standards” is B1+ B1+ B2+ B2+ .
^u Based on the sum of 3 tests made in 1989. See Table 3 in reference 4.
^t Based on a single test made in the fall of 1988. See Table 3 in reference 4.

Table 4. F₂ data obtained from the cross of NJ-B and IL-B. These two inbreds exhibit precocious depletion of chlorophyll in similar as well as different ways.

Growing Season	Number of plants that exhibited precocious depletion of chlorophyll	Number of completely green plants	Total	X² (15:1)
Winter of 1985z	208	9	217	1.6372
Fall of 1988y	196	18	214	1.7059
Spring of 1989y	204	15	219	0.1343
Fall of 1989y	280	26	306	2.6362
Spring of 1990y	336	23	359	0.0150
				6.1286
F2 pooled	1224	91	1315	1.0079
				5.1207
	X²	df	P
Deviation	1.01	1	0.30 – 0.50
Heterogeneity	5.12	4	0.20 – 0.30

^z Grown in New Brunswick, NJ, under greenhouse conditions (see reference 1)
^y Grown in Naples, Florida, under field conditions.

Literature Cited

1. Shifriss, O. 1986. Relationship between the B genes of two Cucurbita species. Cucurbit Genetics Coop. 9: 97-99.
2. Shifriss, ). 1989. Control of chlorophyll during plant development. Cucurbit Genetics Coop. 12: 82 – 83.
3. Shifriss, O. 1990. Relationship between the B genes of two Cucurbita species, III. Cucurbit Genetics Coop. 13: 50 – 54.
4. Shifriss, O., R.B. Volin and T.V. Williams. 1990. Relationship between the B genes of two Cucurbita species, IV. Cucurbit Genetics Coop. 13: 55-57.