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• Christina Dian Kurniawati

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Applied genetics I GENETIC ANALYSIS IN PEA

Introduction Pea (Pisum sativum) is an excellent crop for genetic analyses, because it is a strictly self fertilizing crop though artificial crossing is easy. Several easily recognisable characters are known to have simple inheritance. In fact Mendel also chose pea for his classic experiments which led to the discovery of the famous laws of inheritance. In the present experiment attention is paid to three different characters each of which shows a simple monogenic dominant/recessive inheritance and which are present in several common Dutch varieties (N.b. The choice of cultivars is not very relevant as long as the same characters are studied). The following information about the various cultivars is important.

Cultivar

Anthocyanin

Colour of cotyledons

Nature of seed coat

Genotype

Finale

-

green

transparent

aa gg dd

Paloma

-

yellow

transparent

aa GG dd

Gastro

+

yellow

opaque

AA GG DD

Crosses have been made between these cultivars and further generations have been obtained by selfing. In this practical, we will predict and check the segregation ratios for the F2 and subsequent generations of two traits: colour of cotyledons and nature of seed coat.. The character "anthocyanin" will not be included in this year’s practical. It shows in early plant development stages in the leaf-axils of the plants. In mature plants, the presence or absence of anthocyanin can also be observed in the flowers (coloured vs white flowers). This enables us to classify plants in the vegetative stage and to test the correctness of this classification with the flowers. Both other characters, i.e. colour of cotyledons and colour of seed-coat, must be observed on the seed. There is, however, one complication. The seed-coat belongs to the maternal tissue and thus represents the genotype of the mother plant on which the seed is formed. The cotyledons, however, already belong to the next generation. They are genotypically determined by the genotype of the zygote, and hence, also by the pollinator. This aspect of the genotype can therefore be evaluated already in the pod that grows on the mother plant (see figure). Such deviant seeds (because the seed shows traits contributed by the pollinator) as well as the phenomenon as such are indicated with the word "xenia". (Greek plural; literally meaning: guest friendship). Xenia enable a quick check for a successful cross, especially when the seed-coat is transparent. In case of an opaque seed-coat, the colour of the cotyledons can be made visible by scraping away part of the coat with a knife or by putting the seeds on a glassplate with illumination from below. 1

For a better understanding of the above phenomenon an example is given. Take the cross gg dd x GG DD which results in an F1 with the genotype Gg Dd. Seeds with the F1constitution have yellow (G.) cotyledons but are harvested on a mother plant with the genotype gg dd, i.e. with a translucent seed-coat. The Dd-genotype will only become apparent in the seeds that grow on the F1-plants. Those seeds will all show an opaque seed-coat. Question: What will be the colour of the cotyledons of the seeds harvested if those F1 plants are self-pollinated?

Pollen grains G

G

g Mother genotype (gg)

Gg

Seed coat

Gg

gg

Cotyledons of zygote

Maternal tissue

Fig 1. Schematic presentation of fertilisation in pea

2

Assignment 1. Calculate the expected phenotypic segregation ratios for the two characters per crossing combination and per generation (Table 1). 2. Investigate the F2 and F3 seeds collected from different crosses of selfed F1 and F2 plants. All participants should check the samples of this material for the type of seed-coat and the colour of the cotyledons, keeping in mind the information presented in the introduction. Fill out the observed numbers of phenotypes in Table 2. 3. Determine the expected numbers of each phenotypes on the basis of the total number of seeds that were scored, and the segregation ratio in Table 1 for that cross and generation. (Fill out the expected numbers in Table 2). 4. The pooled results collected by all participants should be entered in Table 3. 5. Calculate on the basis of your expected segregation and the observed segregation (Table 2) whether the results are in agreement with your expectation. The chi-square test, needed for this exercise is based on true numbers of seeds in each phenotypic class, and not on percentages or proportions. 6. Calculate also for the pooled data (Table 3) whether the actual segregation deviates significantly from the expectation. 7. Give at least 5 different hypotheses why results may significantly deviate from the expected segregation ratio. 8. How would you organize an experiment to investigate the percentage of out-crossing in a field crop of pea?

References BLIXT, S., 1974. The Pea. In: R.C. King (ed.). Handbook of genetics, Vol. 2, Plenum Press, New York: 181-221. GOTTSCHALK, W. and G. WOLFF, 1983. Induced mutation in plant breeding. Monographs on theoretical and applied genetics, Vol. 7, Springer Verlag, 283 pp.

Chi-square test Formula:

  2

O  E 2 E

; in which O = observed number, E = expected number

Suppose: you expect a 3: 1 segregation (for example red versus white), your population is 20 plants. In that case, you expect: 15 red and 5 white. Suppose you observe: 17 red and 3 white What is your X2? 3



2

2 2  17  15 3  5  

15

5

 1.067

In the table below you can find out whether the X2 is high enough to support the judgement that your expectation was incorrect. From the formula, you can conclude that the closer your observation matches your expectation, the smaller (O – E), and the lower the calculated X2 value. So, if the calculated X2 value is higher than the value in the table, the observation indicates that your expectation was incorrect.

Degrees of freedom 1 2 3 4

P (2 > X2) 0.50

0.25

0.10

0.05

0.025

0.010

0.005

0.45 1.39 2.37 3.36

1.32 2.77 4.11 5.39

2.71 4.61 6.25 7.78

3.84 5.99 7.81 9.49

5.02 7.38 9.35 11.14

6.63 9.21 11.34 13.28

7.88 10.60 12.84 14.86

Degrees of freedom = (number of categories scored) - 1

4

Results Table 1

Expected ratios of seeds classified according to the characters colour of cotyledons and nature of seed-coats in samples of seed harvested from selfed F1- and selfed F2 plants of pea from different crosses (F = cv. Finale, P = cv. Paloma, G = cv. Gastro).

Phenotypic segregation of seeds Crossing combination

Seed opaque, generation yellow

FxP

F2

:

:

:

F3

:

:

:

F2

:

:

:

F3

:

:

:

F2

:

:

:

F3

:

:

:

PxG

FxG

opaque, green

transparent, yellow

transparent, green

5

Table 2

Expected and observed segregation (numbers) for the characters colour of cotyledons and nature of seed-coats in samples of F2and F3 seeds of pea (F = cv. Finale, P = cv. Paloma, G = cv. Gastro). (Individual results). Phenotypic segregation of seeds (Observed numbers)

Phenotypic segregation of seeds (Expected numbers)

Crossing parents

Seed Total opaque, opaque, transparent, transparent, opaque, opaque, transp. Generation number of yellow green yellow green yellow green yellow seeds

FxP bag:

F2

transp. green

X2

Sign.*

F3 bag: PxG bag:

F2

F3 bag: FxG bag:

F2

F3 bag: *

S (significant) or NS (no significant) deviation from expected segregation ratio. 6

Table 3

Expected and observed segregation (numbers) for the characters colour of cotyledons and nature of seed-coats in samples of F2and F3 seeds of pea (F = cv. Finale, P = cv. Paloma, G = cv. Gastro). (Pooled results).

Phenotypic segregation of seeds (Observed numbers)

Phenotypic segregation of seeds (Expected numbers)

Crossing parents

Seed Total opaque, opaque, transparent, transparent, opaque, opaque, transparent transparent X2 Sign.* Generation number of yellow green yellow green yellow green yellow green seeds

FxP

F2

F3

PxG

F2

F3

FxG

F2

F3

7

8