Microsatellite DNA contains that microsatellite noumenon and both
sides of its flanking sequence regions and it is 1~6 bp. Flanking sequences
enable microsatellite to be located on a particular position of the genome
specificity. The character of microsatellites include highly polymorphic,
plenty and well-distributed in the genome. For that, this technology became
the second generation molecular markers after the first generation molecular
markers (restriction fragment length polymorphism, RFLP) (Nakamura et
al., 1987) and had been widely applied in many studies territory of
natural populations. Recently microsatellite markers has been used to
establish genetic map of chromosome of the human, mouse, rat, silkworm
and chicken and many other species and has been widely used in gene location,
clone, disease diagnosis, phylogenetic analysis, identification of species,
animal breeding, evolutionary trees and other areas. With the development
of research, the abundant number of microsatellites polymorphisms has
been used to analyze the significant difference among different species
in order to search for genetic markers that link main production traits
which serve as the scientific bases for molecular breeding and Marker-Assisted
Wan line Angora rabbits is a developed Angora breed which was bred from
Chinese Angora and German Angora. In this study, Wan line Angora rabbits
were selected randomly which were healthy and one year old. Wool yield
at age of one year were measured. Using microsatellite markers, the relationships
between 4 polymorphism of microsatellite markers and wool yield at age
of one year will be studied to obtain the theory evidence of marker assisted
selection for Angora rabbit`s wool yield.
MATERIALS AND METHODS
When this study was conducted, 51 Wan line Angora rabbits (11♂-40♀)
were from the rabbit farming of the Institute of Animal Science and Veterinary,
Anhui Academy of Agricultural Science (Anhui, China). Approximately 3
mL blood was collected from each individual (from the central artery vein
of the ear) in tubes containing ACD (Citric acid, Sodium citrate, Dextrose)
as anticoagulant and immediately transferred to the ice-box.
Genomic DNA were extracted according to the literature (Xin-Sheng et
al., 2005). The 4 pairs of microsatellite primers were synthesized
by the Shenggong biological engineering technology company and primer
sequences has been shown in Table 1.
The total 25 μL volume of PCR reaction mixture comprised as follows:
100 ng of template DNA, 10 x PCR buffer 2.5 μL, 25 mM MgCl2,
2.0 μL of dNTP (200 μM), 1.0 μL of each primer (10 pmo1
L-1), 1 U of TakaRa Taq enzyme.
||Primer information of four microsatellites
PCR conditions was as follows: initial denaturation at 94 °C for 5
min; followed by denaturation at 94 °C for 1 min, annealing at 55 to
60 °C for 1 min, extension at 72 °C for 1 min for 34 cycles and
final extension at 72 °C for 10 min.
PCR products were electrophoresed on denaturing polyacrylamide (arcylamide:
bisacrylamide = 29: 1) gel to separate PCR products with different sizes
at 200V for 6~8 h. The gels were silver stained to visualize gene fragments.
According to the different rate of migration of different gene fragments,
the length of fragments were analyzed by Kodak imaging software KDSZD2.0.
Statistical methods: Allele frequencies were calculated using
the following formula.:
Pi = [2(ii)+(ij1)+(ij2)+
In the formula, Pi represent frequency of the ith allele;
i represent the ith allele at a certain seat of microsatellite; J1.
J2 ...... Jn represent alleles from the first to
the nth which show the codominance with i; n represent the
seat number of microsatellite.
Microsatellite is codominant inheritance, therefore, allele frequency
can be attained by simple statistic measurement of the detected genotype.
Heterozygosity (H) was calculated using the following formula:
In the formula, n represent the seat number of microsatellite; Pi
represent frequency of the ith allele.
Polymorphism Information Content (PIC) is a indicatrix which reflected
variation of microsatellite DNA mutation, reflected polymorphic of microsatellite
DNA level, the formula as follows:
In the formula, n represent the seat number of microsatellite; Pi
represent frequency of the ith allele and Pj represent
frequency of the jth allele.
Data analysis: Using SPSS11.5 Generalized Linear Model (GLM) to
analyze the relationships between polymorphism of microsatellite markers
and wool yield at age of one year in Wan line Angora rabbits and analyzed
with least square equation.
The PCR product and polymorphism of microsatellite: The amplified
results of 4 pairs of microsatellite primers were as shown in Fig.
In Wan line Angora rabbits, the total number of alleles was 18 at 4 microsatellite
loci. The number of alleles per locus ranged from 3 to 6 (Table
2). The average number of alleles of 4 microsatellite loci was 4.5 ± 1.29.
Heterozygosity and polymorphism information content of Wan line Angora
rabbits: According to the allele frequencies at 4 microsatellite loci,
heterozygosity and genetic polymorphism information content of Wan line
Angora rabbits were calculated in Table 3. Table 3 indicated
that the average heterozygosity of Wan line Angora rabbits was 0.680.
The highest heterozygosity level was 0.721 at Sol33 locus; the lowest
heterozygosity level was 0.630 at Sat4 locus. The average polymorphism
information content of Wan line Angora rabbits was 0.642, the highest
polymorphic information content level was 0.705 at Sol33 locus; the lowest
polymorphic information content level was 0.559 at Sat4 locus.
The relationships between microsatellite locus and wool yield: The
relationships between polymorphism of microsatellite markers and wool
yield at age of one year in Wan line Angora rabbits were analyzed by least
square equation (Table 4). The relationship between Sol33
locus and wool yield was significant (p<0.05). The relationships were
no significant between Sat4, Sat13, So144 loci and wool yield (p>0.05).
Five genotypes were discovered at Sol33 locus and wool yield of genotype
AD and BD were
||PAGE of PCR product of Sat4 locus
||PAGE of PCR product of Sat13 locus
||PAGE of PCR product of Sol33 locus
||PAGE of PCR product of Sol44 locus
significantly higher than that of other genotypes (p<0.05); the differences
of wool yield between genotype AD and CE was 78 g at age of one year.
||Allele frequencies at 4 microsatellite
polymorphism information content of Wan line Angora rabbits
Least squares means
and standard errors for wool yield of different genotypes of 4 microsatellite
loci in Wan line Angora rabbit
The different superscripts
within the same column differ significantly (p<0.05)
Gene heterozygosity of colony can be measured by use of polymorphism
of molecular genetic markers. Heterozygosity is also known as gene diversity.
The average gene gheterozygosity of a colony is the optimum parameters
to measure the variation of a colony (Bin et al., 1999). The average
gene heterozygosity can reflect the variation level of the genetic structure
approximately. The average gene heterozygosity of Wan line Angora rabbits
at 4 microsatellite loci was 0.680 and indicated the colony with high
genetic diversity. In this study, the average gene heterozygosity of Wan
line Angora rabbits was higher than that of JIRONG rabbit (Chun-Mei et
al., 2005) and lower than that of Angora rabbit (Jin et al.,
2006). The reason may be caused by using of the different rabbit species,
microsatellite markers and the number of microsatellite loci.
Polymorphic Information Content (PIC) is a better indicator which can
measure the polymorphism of gene fragment. When the PIC>0.5, indicated
the locus of high polymorphism; When 0.25<PIC<0.5, indicated the
locus of medium polymorphism and when PIC<0.25, indicated the locus
of low polymorphism. Meanwhile polymorphic information content relate
to the availability and efficiency of the locus, the polymorphic information
content is higher, the proportion of heterozygous is greater and could
provide more genetic information. In this study, the average polymorphism
information content of Wan line Angora rabbits was 0.642 and the polymorphic
information content at each microsatellite locus was higher than 0.5,
indicated they were high polymorphic loci and that are same to the results
of Angora rabbit (Jin et al., 2006).
The tests analyzed by least square equation, showed that the relationship
was significant between Sol33 locus and wool yield at age of one year.
Perhaps, Sol33 locus linked with the major gene which control rabbits
wool yield. At present, there are not literature about the research of
rabbits, but there are many reports about the research in other species.
Van Kaam et al. (1999) indicated the relationship was significant
between LEI166 microsatellite locus and the 48 day old weight. Van Kaam
et al. (1998), Tatsuda et al. (2000) and Talsuda and Fujinaka
(2001) indicated that maybe there were some QTLs in chicken`s chromosome
1 which could control the weight of chickens. Qun-Lan et al. (2005)
reported that the relationship was significant between ADL278, LEI166,
MCW 222 microsatellite loci which were located in the 3rd and 8th chromosome
and the weight of Luyuan chickens at age of 12 weeks. Gen-Bao et al.
(2005) indicated the relationship was significant between some microsatellite
locus in the 13th chromosome and pork quality of Taihu pigs. Ming-Xing
et al. (2005) reported that there were different genotypes of six
microsatellite loci could help Beijing Holstein cow to resistance mastitis.
These results indicated that microsatellite marker assisted breeding livestock
as a choice of molecular marker and could accelerate animal breeding progress.
In this study, five genotypes were detected at Sol33 microsatellite locus
of Wan line Angora rabbits, which genotype of BD and AD individual`s wool
yield were significantly higher than the other three genotypes which did
not have gene D. This may indicated that the gene D could synergize wool
yield of Wan line Angora rabbits, but the study of 51 Anhui rabbits could
not detect DD genotype of the individual. Therefore, whether the gene
D could synergize wool yield of Wan line Angora rabbits still need further
This research was supported by High Technology Research Program
of Jiangsu Province, China (No. BG2007325).