The DNA polymorphism analysed according to the RAPD-PCR method

and useful characteristics of hens

Magdalena Maria Gryzińska

Department of Biological Basis of Animal Production,

University of Life Sciences in Lublin, 13 Akademicka Street, 20-950 Lublin

An academic study financed from the research fund for 2005-2008, as research project no. 2 PO6Z 28

Classic gene mapping developed very slowly. This status quo lasted until advanced techniques of molecular biology and cytogenetics were devised. They enabled the identification of new groups of genetic markers that were exceptionally useful in mammal genome mapping [18]. DNA techniques are still being perfected, which creates new possibilities for a quick elaboration of complete genome maps of the eukaryotic genome. The chief aim in mapping the animal genome is to identify the location and distances between genes in chromosomes, as well as to search for genetic markers which constitute distinguishing features of genes and determine their useful characteristics. Another stage in animal genome mapping is the identification of the main genes which condition economically vital quantitative characteristics [21].

Hens are considered to be good objects in the elaboration of genetic maps owing to a short interval between their generations and the possibility to generate a significant number of related offspring at the same time and in the same environmental conditions. Of importance is also the accessibility of a considerable amount of DNA which can be obtained from lymphocytes and nuclear erythrocytes [18]. The hen genome consists of 39 chromosome pairs that are made up of 8 pairs of macrochromosomes, one pair of sex chromosomes (Z and W), as well as 30 pairs of microchromosomes [5, 6], which amounts to between 30 and 50 thousand genes [8]. At present, the hen genome map includes 135 loci, out of which 35 are located in the 8 large autosomal chromosomes and sex chromosomes. An international cooperation has been initiated to elaborate molecular maps of the hen genome by using two verified populations for this purpose. The presentation of an international stripe pattern of the hen karyotype and a facilitation of its genetic mapping is impossible due to the lack of techniques to identify chromosomes and microchromosomes (n = 29 + 8 + 2) [18, 20]. Particular difficulty in assigning respective genes to microchromosomes is presented by the lack of a model of the hen karyotype [18]. The first hen chromosome map which showed the position of 7 genes in a sex chromosome and 11 ones in four autosomal conjugated groups was published by Hutt in 1936. At the onset of the nineties the first international programs to generate marker genome maps of other farm animals were created.

The use of DNA markers is one of the most effective applications of the molecular biology techniques [20]. RAPD has found multiple practical uses in poultry breeding. All over the world research is conducted on the uses of RAPD markers and the determination of diversity in genetic similarities, genetic variability and polymorphism [1, 14]. Sharma et al. conducted research which required the use of RAPD markers in order to detect polymorphism between two hen populations: Aseel and Kadaknath and to assess genetic diversity between them [15]. Levin et al. used RAPD in order to create the genetic map of the hen Z chromosome. RAPD markers are widespread in the whole Z chromosome and are likely to affect the majority or all the characteristics contained in this chromosome [10]. An appraisal of the genetic similarity of eight parental flocks based on the RAPD-PCR methodology was also performed by Bednarczyk et al. [3], as well as by A. Okumus i M. Kaya [13]. The former isolated DNA from blood drawn from the alar veins of 25 randomly selected adult hens from each group. The reaction was performed using three different 10-nucleotide starters. 126 stripes were obtained. 8 monomorphic products with the molecular mass of 316-883 base pairs were identified. A considerable polymorphism of the remaining amplification products was also determined.

The RAPD analysis was performed on Polbar hens. The variety was created by crossbreeding Greenleg hens with Plymouth Rocks. The Polbar hen is an autosexing breed, which constitutes its most important characteristic.

The aim of the research was to determine the interrelation between phenotypic DNA forms identified by the RAPD method and selected useful characteristics of Polbar hens.

THE MATERIAL AND METHODS

The material for analysis was provided in the form of blood drawn form the alar veins of 50 Polbar hens bred in the Laura Kaufman Didactic and Small Animal Research Station which belongs to the Department of Biological Basis of Animal Production of the University of Life Sciences in Lublin, as well as in the form of records of useful characteristics in hens between the 28 and 33 week of life. The blood was collected in sterile Vacuette test tubes with 4 mL of Medlab Products blood containing the EDTA-K₂ anticoagulant in the proportion of 1,8 g of EDTA in 1 mL of the blood.

DNA isolation from complete blood was performed with the DNA Isolation Kit from 0.1 – 1 mL of fresh blood, 0.1 mL of frozen blood and with the Kucharczyk Blood DNA Prep Plus from blood traces, allowing for modifications such as reducing the blood volume to 50mL and refilling to the volume of 100mL with the TRIS buffer solution. 1.5 mL test tubes were filled successively with: 50mL of the TRIS buffer solution, 200mL of the LT solution, 50mL of the blood and 20mL of proteinase K.

The isolated DNA was amplified according to the RAPD-PCR method. Proglio starters were used in the reaction. The volume of the assayed material was 25mL, i.e. 20mL of the mixture and 5mL of the DNA matrix.

The reaction mixture prepared for 10 assays consisted of: H₂O - 87,5mL; QIAGEN PCR Buffer - 25mL; Q-Solution - 37,5mL; MgCl₂ - 37,5mL; dNTP Mix - 10mL; primer 1 ABI-01 – 5’gTTTCgCTCC3’ - 2mL; primer 2 ABI-05 – 5’ TgCgCCCTTg3’ - 2mL; Taq polymerase - 2,5mL.

The RAPD-PCR reaction was performed in the MJ Research PTC thermocycler – 225 Peltier Thermal Cycler. In the following customized program a thermal profile was used that lasted 4h 46 min and began with a preliminary denaturation of bifilar DNA carried out at the temperature of 94°C and lasting 5 minutes.

Next, a repeating program was employed that consisted of 46 cycles, each of which was composed of three stages: a 1 minute denaturation at 94°C; the starter addition at 36°C for 2 minutes; a 1 minute elongation at 72°C. The program was completed by: a 10 minute cycle at 72°C and the final one running until the end at 4°C.

After amplification the experimental product was split up using electrophoresis on 1,8% Fermentas agarose gel – TopVision™ LE GQ Agarose, with an addition of ethidium bromide: 10mL of ethidium bromide in 100 mL of the agarose gel. Marking with ethidium bromide as the intercalating substance consists in its photoactivation with light whose wavelength is 302 nm. The molecular mass benchmark was the Fermentas GeneRuler™ 50bp DNA Ladder marker which contained thirteen visible fragments with the following lengths: 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 and 1031 base pairs. On the gel were placed the 25mL DNA samples mixed with 2mL of a loading buffer which consisted of a 30% glycerol solution and bromophenol blue. Apart from the samples the gel was also covered with the marker.

The electrophoresis was performed in a TBE buffer onboard BIORAD Sub-cell ^® GT at the voltage of 80V for 210 minutes. Data backup was made using the CCD system. Scion Image was used as the processing program.

The research documentation was made up of the photos of the gel on which the electrophoretic splitting took place. The analysis of the electrophoresis results was made using the PopGen 32 Ver. 1.0 (Beta) software. A single allele chart was prepared for all the analysed birds and then each individual bird was in turn compared with it in order to determine the stripe pattern (phenotype). The names of the phenotypes were determined depending on the number of stripes and their spacing.

The farm documentation provided data on the useful characteristics of the hens. The hens came from one brood. The following characteristics were determined:

- the number of eggs laid in the period under analysis

- the weight of the eggs with the accuracy of 0.01g

The hens were divided into groups depending on the phenotypic stripe pattern obtained in the RAPD-PCR reaction. In such a group juxtaposition the mean values for a given characteristic were compared using the Duncan test on the basis of one-way analysis of variance.

THE RESULTS AND DISCUSSION

The isolated DNA was submitted to the RAPD-PCR reaction. On the basis of the RAPD-PCR method it was concluded that a polymorphism is present in Polbar hens. Stripes were identified in thirty three birds, which amounts to 66% of those under analysis. The obtained DNA profiles of the analysed birds are presented in the following electrophoregrams:

Fig 1. The electrophoregram of the DNA stripe patterns of the Polbar hens according to the RAPD-PCR method. M: marker, 1-18: individual birds 1 to 18

Fig 2. The electrophoregram of the DNA stripe patterns of the Polbar hens according to the RAPD-PCR method. M: marker, 19-36: individual birds 19 to 36

Fig 3. The electrophoregram of the DNA stripe patterns of the Polbar hens according to the RAPD-PCR method. M: marker, 37-50: individual birds 37 to 50

The electrophoregram analysis (Fig. 1) provided grounds for the identification of eighteen phenotypes: A – one stripe is visible; B1, B2, B3 – two stripes; C1, C2, C3 – three stripes; D1, D2 – four stripes; E1, E2 – five stripes; F – six stripes; G – nine stripes; H- ten stripes; I1, I2 – eleven stripes, J – twelve stripes. In seventeen cases the PCR reaction produced no effect.

The presence of between one and twelve stripes at the height of between approximately 50 and 1000 base pairs was observed (Tab. 1). In the case of all the birds in which amplification took place a characteristic stripe was observed at the height of 300 base pairs. Similar conclusions were arrived at by Wiśniewska et al. [12] who conducted research on a duck population, as well as by Bednarczyk and Siwek [3] in the case of hens and Wei et al. [19]. A close corelation was observed between the identified phenotype and useful characteristics, the egg weight in this case. The lack of amplification does not testify to poor useful characteristics of the birds in which it was observed. On the contrary, in many cases the hens laid a considerable number of eggs with the mean weight of 42,54 g. Although, as a rule, hens laying a large number of eggs are characterized by a lower weight of their eggs and the other way round, the table shows that the highest number of eggs which were at the same time the heaviest were laid by birds belonging to the B1 phenotype, i.e. those with two stripes.

Table 1. The number of stripes and the identified Polbar hen phenotypes determined on the basis of the analysis of the electrophoregrams obtained from the RAPD-PCR reaction

Bird number	Stripe number	Identified phenotype	Bird number	Stripe number	Identified phenotype
1	no	-	26	3	C2
2	4	D1	27	no	-
3	5	E1	28	4	D2
4	2	B1	29	1	A
5	1	A	30	no	-
6	1	A	31	10	H
7	1	A	32	no	-
8	no	-	33	11	I1
9	no	-	34	2	B3
10	no	-	35	1	A
11	no	-	36	11	I2
12	2	B2	37	1	A
13	no	-	38	9	G
14	1	A	39	3	C3
15	no	-	40	1	A
16	1	A	41	3	C3
17	no	-	42	1	A
18	1	A	43	no	-
19	1	A	44	no	-
20	12	J	45	no	-
21	12	J	46	no	-
22	12	J	47	no	-
23	3	C1	48	1	A
24	no	-	49	6	F
25	1	A	50	5	E2

Table 2. The correlation between the phenotypes and useful characteristics of the Polbar hens

Identified phenotype	Egg number	Mean weight of an egg (g)
A	15,08	41,30^b
B1	24,00	44,99^a
C1	16,00	35,76^b
C2	7,00	39,15^c
C3	17,50	43,58^a
D1	16,00	39,79^a
D2	16,00	39,85^c
E1	12,00	45,33^a
E2	9,00	43,62^b
F	1,00	43,70^b
G	13,00	41,67
H	6,00	39,50^c
I1	21,00	39,34^b
I2	17,00	44,39^a
J	15,00	40,47^a
No amplification	15,29	42,54^b

a, b, c – the means marked with different letters exhibit a significant statistical variance with P ≤ 0,05

Table 2 presents the means of the analysed useful characteristics in the hens divided into groups depending on the identified phenotypes which were determined according to the RAPD-PCR method. The Duncan test was performed on the basis of one-way analysis of variance for the eighteen identified phenotypes. Three groups containing the following phenotypes were singled out:

a) B1, C3, D1, E1,I2, J,

b) A, C1, E2, F, I1, X (no amplification),

c) C2, D2, H.

Statistically significant differences were observed between the above identified three groups. The hens from the first group with B1, C3, D1, E1, I2 and J phenotypes laid bigger eggs than the hens from the other groups did, with the mean egg weight of 43,09g, whilst those from the second group with A, C1, E2, F and I1 phenotypes, as well as those not susceptible to amplification laid eggs that weighed 41,04 g on average. The lightest eggs were laid by the hens from the third group that included C2, D2 and H phenotypes. The mean weight of eggs in this group amounted to
39,50 g.

Szwaczkowski et al.[17] assessed the association of selected egg protein forms and the serum of the N88 hen breed with its useful characteristics. In the research conducted between 1995 and 1996 on 300 birds which came from the Laying Brood Hen Farm in Rujsca the following protein genotypes were analysed: for prealbumins, albumins (Alb), postalbumins (Paa), transferrine oraz pretransferrine (Ptf), postransferrine (Potf), ovalbumin (Ov-a), ovoglobulins (G2, G3, G4) and conalbumin (Co). In order to identify the protein genotypes an electrophoresis was carried out on polyacrylamide gel. Useful characteristics taken into consideration involved: the body weight, the egg weight, the sexual maturity age, as well as the initial laying capability. Monomorphism was observed in the following proteins: Alb, Paa, Ptf, Potf, Ov-a, G3 and Co. Similar research was performed by Brodacki et al. [4] in order to identify polymorphism and to determine the interrelation between the polymorphic protein forms of the yolk and white of eggs and the body and egg weight in Greenleg Partridge hens.

The obtained results confirmed that RAPD can be employed in the role of a genetic marker in order to determine useful characteristics of hens. The author`s research concerned the laying capability. However, research was also done to analyse meat capabilities. Siwek and Bednarczyk [3] did research using the RAPD-PCR method to evaluate the genetic similarity of eight parental flocks of meat hens. According to the producer they came from one and the same breeding stock. The DNA was isolated from blood drawn from the alar veins of twenty five randomly selected mature hens from each flock. In the PCR reaction 126 stripes were identified. Their number ranged from three to ten depending on the starter used and the flock of birds. The resultant eight monomorphic products with the molecular masses between 316 and 883 base pairs and a considerable polymorphism of the remaining amplification products indicates a heterogenous origin of the bird flocks under comparison.

The RAPD-PCR method was proved effective in assessing the genetic diversity between five hen varieties [8]. The varieties were: the Greenleg Partridge hen (Z11), the Yellowleg Partridge hen (Ż33), the Leghorn (G99 and H22), the Rohde Island Red (R11) and the Sussex (S66). Out of ten starters two were chosen. They rendered different, repeatable amplifications and created a polymorphic pattern in one or more varieties. The obtained results suggest the presence of DNA polymorphism in the analysed hen varieties. Similar results in poultry were obtained by Bednarczyk & Siwek [3], Smith et al. [16] and Wężyk et al. [20]. The orientation, duration and intensity of selection resulted in differences in the genotypic, quantitative and qualitative characteristics of the hen varieties under analysis. The genetic diversity was affected by breeding practices and the value of the similarity index can stem from the common phylogenetic origin of the analysed hen varieties. The research showed that both the Leghorn varieties (G99 i H22) do not have very close genetic similarity. On the other hand, there is little genetic disparity between the Z11 and Ż33 hen varieties. They stem from a common stock. The closest genetic similarity is present in the R11 and S66 varieties which over several generations were selected in the same direction and using the same method. The employment of the RAPD-PCR method to determine the genetic diversity and similarity proved exceptionally effective. The obtained results justify the continuation of protection of the analysed genetic hen resources against extinction.

In order to determine the DNA (RAPD) polymorphism of a flock of the Japanese quail Gajewska [7] used 8 starters consisting of 10 nucleotides. Six of them produced PCR polymorphism. Depending on the applied starter between 2 and 14 amplified matrix DNA fragments were obtained for a single individual bird under analysis. The conclusion that the RAPD-PCR method is effective in determining polymorphism in poultry was also arrived at by Horn et al [9] who analysed DNA polymorphism in a geese population consisting of twenty individual birds. In similar research performed on the White Leghorn Singh and Sharma [24] also used twelve starters and observed a 22 % polymorphism. The varieties used in the research were laying hen varieties. Mollah et al. also proved that RAPD markers can be useful in determining polymorphism [12]. Out of 39 fragments amplified with four starters 25 showed polymorphism. The number of observed stripes ranged from 9 to 11. Wiśniewska et al. [21] performed a RAPD-PCR molecular analysis in selected duck populations. The aim of the research was to provide genetic profiles of the selected duck groups on the basis of an analysis of the DNA polymorphism observed using the RAPD-PCR method. The molecular analysis of the selected groups was based on the following parameters: the number of the amplified DNA fragments counted, the number of stripes common for the profiles under comparison in pairs and the PG factor. The mean genetic similarity was within the range of between 0,68 and 0,78, which suggests a high level of similarity between the analysed groups. Consequently, their common phylogenetic origin can be assumed. Similar results were obtained in parallel studies of domestic geese varieties. In these groups genetic similarity had the following values: 0,71-0,80 [2] and 0,59-0,68 [11].

Amplification did not occur in 32% of the birds under analysis. One stripe was observed in 28%, while two, three, four, five and six stripes were identified in 22%, nine, ten, and eleven in 8% and twelve stripes in 6% of the hens.

Table 3. The phenotypic diversity of Polbar hens on the basis of electrophoregrams obtained with the RAPD-PCR method

Stripe number	Bird number	% of birds	Phenotype number
No amplification	16	32	1
1	14	28	1
2	3	6	3
3	4	8	3
4	2	4	2
5	2	4	2
6	1	2	1
9	1	2	1
10	1	2	1
11	2	4	2
12	3	6	1
Total	50	100	15

Birds with the A genotype or those with one stripe are phenotypically identical since the stripe was observed at the same height, i.e. 300 base pairs. Disparity in this respect was also not observed in birds with the following numbers of stripes: six, nine, ten and twelve. Two different phenotypes were observed in hens with four, five and six stripes. On the other hand, the presence of three different phenotypes was observed in birds with two and three stripes.

An analysis of genetic similarity was also performed by Okumus A. and Kaya M. [13] who used twelve primers to identify it. The research was carried out in the following hen populations: Rir I, Rir II, Barred I, Barred II, Colombian Rock, Line-54, Blue Line, Maroon Line, Black Line and Brown Line. Out of twelve primers nine amplified the DNA genome in ten meat hen samples. 35 stripes were obtained. A polymorphism was identified in 42%. The greatest genetic similarity was detected between: the Barred I and the Maroon (0, 3733) and the smallest between the Colombian Rock and the Barred II (0, 0899).

CONCLUSION

The RAPD-PCR reaction product was observed in thirty three birds, i.e. 66%. No amplification was observed in seventeen hens. On the basis of the numbers (ranging from none to twelve) of stripes eighteen phenotypes were identified.

A correlation was observed between the presence of polymorphism identified by RAPD-PCR and the laying performance (the number of eggs laid between the 28 and 33 week of life and the weight of those eggs) of Polbar hens. The Duncan test was performed on the basis of one-way analysis of variance for the eighteen identified phenotypes. Three groups containing the following phenotypes were singled out:

a) B1, C3, D1, E1, I2, J,

b) A, C1, E2, F, I1, X (no amplification),

c) C2, D2, H.

Statistically significant variance was identified for the weight of eggs laid between the 28 and 33 week of life. The hens from the first group with B1, C3, D1, E1, I2 and J phenotypes laid the biggest eggs with the mean egg weight of 43,09g, whilst those from the second group with A, C1, E2, F and I1 phenotypes, as well as those not susceptible to amplification laid eggs that weighed 41,04 g on average. The lightest eggs were laid by the hens from the third group that included C2, D2 and H phenotypes. The mean weight of eggs in this group amounted to 39,50 g.

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