PCR Based RFLP Genotyping of Bovine Lymphocyte Antigen DRB3.2 in Iranian Holstein Population
Seyed Ali Ghorashi
Major Histocompatibility Complex (MHC) class II locus DRB3 was investigated
by PCR based restriction fragment length polymorphism (PCR-RFLP) assay.
A total of 262 Holstein cows participating in the national recording system
were sampled from 10 herds. A two-step polymerase chain reaction was carried
out in order to amplify a 284 base-pair fragment of exon 2 of the target
gene. Second PCR products were treated with three restriction endonucleas
enzymes RsaI, BstYI and HaeIII. Digested fragments
were analyzed by polyacrylamid gel electrophoresis. Twenty-eight BoLA-DRB3
alleles were identified. Identified alleles are: BoLA-DRB3.2 *3, *6, *7,
*8, *9, *10, *11, *12, *13, *14, *15, *16, 20, *21, *22, *23, *24, *25,
*26, *27, *28, *32, *36, *37, *40, *51, *iaa and *ibb. The BoLA-DRB3.2*40
allele that was observed in this study has not been reported previously.
The calculated frequencies were as follows: 2.29, 1.34, 0.19, 14.5, 0.38,
3.05, 12.21, 1.34, 2.29, 1.34, 2.48, 9.16, 0.95, 0.77, 6.68, 9.16, 17.94,
1.15, 0.57, 1.15, 0.95, 0.57, 0.38, 1.91, 0.38, 5.73, 0.19 and 0.95% respectively.
The six most frequently observed alleles (BoLA-DRB3.2 *8, *11, *16, *22,
*23 and *24) accounted for 69.65% of the alleles in these 10 herds. The
results of this study confirm the allelic distribution of six most frequent
alleles in Holstein population`s worldwide.
Breeding goals for dairy cattle have focused mainly on increasing
the productivity and have ignored health traits such as disease resistance.
Development of molecular techniques has resulted in identification of
new genetic markers which enables researchers to characterize genes responsible
for host immunity and productivity traits (Van Dorp et al., 1999).
Major histocompatibility complex (MHC) genes of cattle, known as the bovine
leukocyte antigen (BoLA), have received attention because of their association
with immunity, reproductive and productive traits (Schukken et al.,
1994; Lewin, 1996; Dietz et al., 1997a,b; Sharif et al.,
1998a,b; Park et al., 2004). The MHC is a large cluster of tightly
linked genes that constitute the most important genetic component of the
mammalian immune system (Klein, 1986). The MHC encodes cell surface glycoproteins
that bind antigens derived from pathogens or parasites and present them
to T-lymphocytes which trigger the appropriate immune response (Sommer,
2005). Three major classes of MHC genes can be distinguished. MHC class
I genes play an essential role in the immune defense against intracellular
pathogens. In contrast, MHC class II genes are predominantly involved
in monitoring the extracellular environment by presenting peptides mainly
derived from parasites to the T-cells (Klein et al., 1997). The
class III genes encode secreted proteins, some of which are associated
with the immune regulation. This complex has been mapped to chromosome
23 and spans approximately 2.5 Mb of the cattle genome (Lewin, 1996).
Bovine Lymphocyte Antigen class II genes are distributed in two distinct
regions, IIa and IIb. The DRA, DRB, DQA and DQB genes are located in the
IIa region, while the DOB, DIB, DYA and DYB genes are located in the IIb
region. The class IIb of DRB gene includes DRB1, DRB2 and DRB3. Of the
class II genes, cattle express one DR gene pair (DRA and DRB3) and one
or two DQ gene pairs per haplotype (Amills et al., 1998). In particular,
the DRB3 locus and its gene product are among the best defined in cattle,
functionally important and highly polymorphic. The product of the BoLA-DRB3
gene is a beta chain of an MHC class II molecule, expressed on antigen
presenting cells (APCs). Correspondingly, most research focuses on the
second exon of the DRB3 gene. As a result of the role in antigen presentation,
DRB3 alleles have been examined for associations with various autoimmune
and infectious diseases. In cattle, the number and the significance of
the MHC-disease associations and production traits increased dramatically
following the development of class II typing techniques. Van Eijk et
al. (1992) developed a PCR-RFLP class II typing technique and detected
thirty different BoLA-DRB3.2 alleles based on evaluation of 168 animals
representing 10 cattle breeds. Fourteen additional novel BoLA-DRB3.2 alleles
were identified by Gelhaus et al. (1995). In the other studies,
Lewin (1996) and Dietz et al. (1997a) identified 35 and 22 BoLA-DRB3.2
alleles in Holstein cows respectively. In a larger study involving BoLA-DRB3.2
genotyping of 1100 Holstein cows from 93 commercial dairy farms in the
United States, 24 previously described alleles and five new alleles were
found (Dietz et al., 1997b). After that, twenty-one BoLA-DRB3
alleles with two novels were identified in 176 individuals using sequence
based polymorphism technique (Takeshima et al., 2002). To date
103 BoLA-DRB3 alleles have been identified exploiting several DNA
typing techniques from various breeds of cattle, including Holstein, Red
Pied, Martinique, Brahman, Japanese Black, Jersey, Hereford, Ethiopian
Arsi, N`Dama, Boran, Swedish Red-and-White, Simmental and Brazilian Gir
(Van Eijk et al., 1992; Gilliespie et al., 1999; Takeshima
et al., 2003; Da Mota et al., 2004). In the earlier report
we also identified six novel alleles in BoLA-DRB3.2 locus of Sarabi cattle
not reported previously (Pashmi et al., 2006). Looking through
the previous studies, alleles *8, *11, *16, *22, *23, *24 and *28 have
been reported as the most effective and frequently detected alleles in
cattle breeds. Up to the present time there is not any comprehensive study
on the allelic distribution of DRB3.2 gene within Iranian dairy farms.
Corresponding to well documented association of BoLA allelism with dairy
production, in this project we aimed to study of the variability of the
motif and determine the frequency of DRB.3.2 alleles in the Iranian Holstein
MATERIALS AND METHODS
DNA isolation: Blood samples of 262 Holstein cows were born during 1995-2003 from ten
herds of Tehran and suburb dairy farms, participating in the national
recording system were collected and transferred to the Animal Genetics
Division of NIGEB. Blood samples (approximately 5-10 mL) were taken from
each cattle via the jugular vein and stored at -20°C. The genomic
DNA extracted from samples by phenol-chloroform procedure with minor modifications.
DRB3.2 Gene amplification: Two-step polymerase chain reaction (PCR) was carried out in order to
amplify a 284 base-pair fragment of BoLA-DRB3.2 gene according to Van
Eijk et al. (1992) procedure with some modification. Total volume
of reaction 1 was 25 μL containing: 1X PCR buffer, 200 μM dNTP
mix, 2.5 mM MgCl2, 0.5 μM of each primer (HL030 and HL031),
1 unit of Taq DNA polymerase and 25 ng of genomic DNA. Then 2 μL
of the first PCR product was used for the second PCR reaction. Total volume
and concentration of reaction 2 was the same mentioned above with the
exception of the primers (HL030 and HL032).
Restriction endonuclease digestion: The PCR-amplified DNA fragments from the second PCR reaction were digested
with three restriction endonuclease enzymes RsaI, HaeIII
and BstYI (MBI Fermentas GMBH). For each restriction endonuclease
digestion, 5 μL of the second PCR reaction product was used. PCR
products were digested with RsaI and HaeIII separately for
3 h at 37°C and with BstYI for 3 h at 50°C. The total volume
of each digestion was 25 μL.
Gel electrophoresis: Five micro liter of the restriction enzyme digested samples were electrophoresed
in 8% polyacrylamide with TBE buffer (0.9 M Tris base, 0.09 M boric acid,
2.5 mM EDTA; pH = 8.1). Gels were run at 80 V for 4 h and stained with
silver nitrate. The BoLA-DRB3.2 allelic nomenclature as described by Van
Eijk et al. (1992) was considered to scoring digestion patterns.
RESULTS AND DISCUSSION
The 284 base-pair PCR amplified fragment of the BoLA-DRB3.2 gene
was digested with RsaI, HaeIII and BstYI restriction
enzymes. Frequency analysis of BoLA-DRB3.2 alleles of 262 Iranian Holstein
cattle summarized in Table 1. A total of twenty-eight
BoLA-DRB3.2 alleles were observed in the experimental population. From
these alleles, one allele (BoLA-DRB3.2 *40) was the novel allele type
not reported previously (Fig. 1). Although this allele
was rare with a frequency of 0.38, we were interested in the observation
of a new allele in the universal Holstein cattle. The six most frequent
observed alleles (BoLA-DRB3.2 *8, *11, *16, *22, *23 and *24) accounted
for 69.65% of the alleles in the experimental population.
Allele frequencies for BoLA-DRB3.2 of Holstein cows
as identified by PCR-RFLP analysis
The patterns of restriction enzyme digestion of three
alleles were electrophoresed in 8% polyacrylamide that one of them
(uu aa bb lanes) is novel alleles type (BoLA-DRB3.2 *40) not reported
previously. The second PCR products were digested with RsaI,
HaeIII and BstYI. M is 50 bp ladder and R, H and B are
first letters of endonuclease enzymes
Similarly, Dietz et al. (1997b) reported that the six most frequently detected alleles
(BoLA-DRB3.2 *8, *11, *16, *22, *23 and *24) accounted for 70.3% of the alleles in a population
of Holstein cows. In another study, Sharif et al. (1998a) showed
BoLA-DRB3.2 *8, *11, *16, *22, *23 and *24 alleles are the most frequent
in a population of Holstein cows in Canada, as such their frequency were
83.5%. Our results were completely in accordance with aforementioned investigation
outcomes. The six most frequently detected alleles in the study of Kelm
et al. (1997) and Ledwidge et al. (2001) were similar to
Dietz et al. (1997b) and Sharif et al. (1998a). In a comprehension
study Dietz et al. (1997b) reported 29 BoLA-DRB3.2 allele based
on genotyping 1100 Holstein cows in the United States. Also they pointed
out that BoLA-DRB3.2 *8 and *11 alleles have frequencies of 21 and 18%
respectively. Similarly Kelm et al. (1997) reported the frequencies
of aforementioned alleles as 21 and 17%, respectively. In the present
study the frequencies of BoLA-DRB3.2 *8 and *11 alleles were 14.5 and
Previous studies indicate that there is significant association between
infectious disease of cattle and BoLA genes. Sharif et al. (1998b) reported that there is no significant association between BoLA-DRB3.2
*16 and *23 alleles and production traits in Holstein cattle. In contrary
BoLA-DRB3.2 *8 allele was significantly associated with increased 305-days
milk, fat and protein yields and BoLA-DRB3.2 *22 was associated with decreased
milk and protein yield. The frequency of these alleles (BoLA-DRB3.2 *8
and *22) in our study were 14.5 and 6.68% respectively. Sharif et al.
(2000) reported significant relationship between the presence of glutamic
acid at position β74 and occurrence of mastitis caused by Staphylococcus
sp. This motif is present in BoLA-DRB3.2 *22, *23 and *24 alleles.
Presence of arginine or lysine at position 13 also showed a tendency (p
= 0.1) towards an association with a higher risk of clinical mastitis
caused by the same bacteria. This motif is present in BoLA- DRB3.2 *8
and *23 alleles (Sharif et al., 2000). The frequency of BoLA-DRB3.2
*8, *22, *23 and *24 alleles in present study were 14.5, 6.68, 9.16 and
17.94% respectively. Dietz et al. (1997b) have identified BoLA-DRB3.2
*16 as a potential risk factor for acute intramammary infection. Also
they have mentioned there is genetic relationship between BoLA-DRB3.2
alleles and several indicator traits of innate and adaptive immunity in
127 periparturient Holstein cows. In a comprehensive study Starkenburg
et al. (1997) surveyed associations of some DRB3.2 alleles with
production traits and reported substantial differences in allelic frequencies
of milk selected and control lines of Holstein cows, as BoLA-DRB3.2 *24
was associated with increased fat yield during first lactation and *8
was related to decreased milk and protein yields. Also BoLA-DRB3.2 *7
was associated with a significant increase in protein yield during first
and second lactation as well as a significant association in chronically
elevated SCS and acutely elevated SCS during second lactation. They also
reported that the frequencies of *24, *8 and *7 in milk selected lines
were 28, 9 and 5%, respectively. In contrary, the frequencies of aforementioned
alleles in control lines were 7, 23 and 10%, respectively. At a glance,
a positive relationship between milk selected and control lines in production
related traits was observed. Interestingly the frequencies of *24, *8
and *7 alleles in the present study were 17.94, 14.5 and 0.19%, respectively.
Ascending or descending trends of these allele frequencies is in coincidence
with milk selected lines in Starkenburg`s study (1997). An explanation
for this accordance could be successive years of artificial insemination
with imported elite sire`s semen in dairy farms of the country.
Results of the present study substantiate that minor differences exist
between Iranian, Canadian and USA Holstein population with regard to BoLADRB3.2
allelic frequency. Our results also confirm the allelic distribution of
6 most frequent alleles in Holstein population`s worldwide. Studies are
in progress to evaluate relationship between BoLA-DRB3.2 allele types
and SCC and some production traits in Iranian Holstein cattle. The significance
of this report is that it offers interesting perspectives for the incorporation
of molecular genetic techniques to animal breeding in Iran.
This study was conducted at the Animal Genetics Division of National
Institute of Genetic Engineering and Biotechnology and was supported financially
by the University of Tehran. The authors wish to express their gratitude
to the personnel of National Animal Breeding Center especially Mr. Olyaei,
Mr. Kazemi and Mr. Mansouri. The authors would also like to appreciate
the kindly technical assistance of Mrs. Reyhaneh Salehi-Tabar.
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