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*Part of Ph.D. thesis of first author
Present address: 1Subject Matter Specialist (Animal Science)
(dasugenvet@gmail.com), Howrah Krishi Vigyan Kendra,
Jagatballavpur, Howrah, West Bengal. 2Principal Scientist
(skgicar@gmail.com). 3Research Associate (choudhary633
@gmail.com), Artificial Breeding Research Centre, National
Dairy Research Institute, Karnal, Haryana. 4Assistant Director
(kokatels@gmail.com), MAFSU Sub-Centre, Udgir, Latur,
Maharashtra.
Microsatellite markers are extensively used in assessing
genetic structure, genetic diversity and relationship analyses
(Zhou et al. 2008). They are ideal for deciphering genetic
variability (Zhou et al. 2008) and provide a powerful tool
for marker-assisted selection (MAS) and QTL research
(Sewalem et al. 2002). The Rhode Island Red (RIR) chicken
population brought at the Central Avian Research Institute
almost 3 decades ago was well adopted, acclimatized and
genetically improved over last 33 years covering 29
generations of selection and being maintained as selected
line. The population has shown positive response for egg
production on long term selection based on part-period egg
production (Anonymous 2011), which however has been
slowing down in the last few generations, probably due to
reduction in genetic variability (Das et al. 2015a). Faster
genetic progress is possible using genomics data, which
may impact on layer breeding in the future (Albers and Van
Indian Journal of Animal Sciences 86 (9): 1021–1024, September 2016/Article
Association study between microsatellite genotypes and layer
performances in Rhode Island Red chicken*
ANANTA KUMAR DAS1, SANJEEV KUMAR2, ABDUL RAHIM3 and LAXMIKANT SAMBHAJI KOKATE4
ICAR-Central Avian Research Institute, Izatnagar, Uttar Pradesh 243 122 India
Received: 19 January 2016; Accepted: 17 February 2016
ABSTRACT
This investigation aimed to study association between microsatellites and layer performances in Rhode Island
Red selected line chicken. Genomic DNA samples isolated from the 12 randomly selected birds were investigated
at 24 microsatellite loci. The microsatellite alleles were separated on 6% urea-PAGE and their molecular sizes
were estimated. Locus specific alleles were identified according to their sizes, and their association with layer
performance traits was assessed by least squares analysis of variance. Analysis revealed that age at sexual maturity
of the birds had significant influence of 180bp/190bp and 184bp/196bp microsatellite genotypes in MCW0075
locus. Egg weight at 28th week of age was significantly associated with 210bp/244bp, 216bp/216bp, 216bp/238bp,
222bp/244bp genotypes in MCW0005; and 173bp/173bp, 175bp/175bp, 177bp/177bp in MCW0014. Egg production
upto 40 weeks of age was also significantly associated with some genotypes in MCW0044 (133bp/151bp, 136bp/
160bp), ADL0102 (136bp/166bp, 146bp/174bp, 166bp/166bp) and ADL0158 (178bp/214bp, 184bp/184bp, 184bp/
214bp, 184bp/222bp). MCW0051 (90bp/118bp, 105bp/118bp, 118bp/118bp), MCW0014 (173bp/173bp, 175bp/
175bp, 177bp/177bp) and ADL0176 (200bp/200bp, 200bp/236bp, 202bp/202bp) demonstrated significant influences
on body weight at 40th week of age. Findings suggested faster genetic progress in RIR flocks by adapting
microsatellite genotype based selection.
Key words: Association, Body weights, Egg production, Egg weights, Microsatellite genotypes, RIR chicken
Sambeek 2002). Hence, the present investigation was
carried out with the avowed objective of association study
between microsatellites and layer performances in RIR
selected line chicken.
MATERIALS AND METHODS
Birds (12) were randomly chosen from RIR selected line
chicken maintained at the Central Avian Research Institute,
Izatnagar. Their genomic DNA samples were extracted as
detailed in earlier literatures (Das et al. 2015a, 2015b) and
PCR ready DNA samples were prepared at a concentration
of 50 ng/µl. FAO (2011) recommended 24 microsatellite
loci as detailed in earlier literatures (Das et al. 2015a, 2015b)
were used for present study. The chicken specific
microsatellite synthesized primers (Custom Oligos, 0.01
µM) were obtained commercially and their annealing
temperatures were optimized as per Wimmers et al. (2000).
The PCR reactions and amplifications were carried out using
these DNA samples for each microsatellite marker as
detailed in earlier literatures (Das et al. 2015a, 2015b). The
molecular sizes of amplified products were adjudged for
their probable sizes through 1.4% horizontal agarose gel
electrophoresis (Das et al. 2015a, 2015b). The microsatellite
alleles were then identified by running the amplified
products on vertical denaturing polyacrylamide gel
electrophoresis (6% urea-PAGE) (Das et al. 2015a, 2015b)
1022 DAS ET AL.[Indian Journal of Animal Sciences 86 (9)
50
followed by silver staining (Beidler et al. 1982). Molecular
sizes of various alleles at different microsatellite loci were
determined using the Quantity One software on GelDoc
2000. The observed alleles in each sample at each
microsatellite loci and its probable genotypes were recorded.
Locus specific alleles were identified according to their
molecular sizes and assigned names from alphabet A to H
in ascending order of their molecular sizes.
The layer performances i.e. body weight at 20th weeks
of age (BW20), age at sexual maturity (ASM), egg weights
at 28 and 40th week of age (EW28, EW40), body weight at
40th week of age (BW40) and part period egg production
upto 40 weeks of age (EP40) were recorded.
The performance data recorded on the experimental birds
was analyzed for assessing their association with
microsatellite alleles by least squares analysis of variance
(Harvey 1990), incorporating microsatellite locus as fixed
effect in the statistical model:
Yjk = µ + Mj + ejk
where, Yjk, observation of kth individual of jth
microsatellite locus; µ, population mean; Mj, fixed effect
of jth microsatellite locus; ejk, random error associated with
mean zero and variance ó2. Critical Difference (CD) test at
the 5% level of probability of significance was performed
for assessing critical differences among the least squares
means under microsatellite genotypes.
RESULTS AND DISCUSSION
The Least squares analysis of variance elucidates that
some specific microsatellite genotypes in loci out of 24 loci
investigated in this study had significant (P<0.05) influence
on performance traits and are presented in Table 1.
Part period egg production upto 40 weeks of age (EP40)
was found to be significantly associated with some specific
microsatellite genotypes of loci MCW0044, ADL0102 and
ADL0158 (Table 1) in agreement to the earlier report (Das
et al. 2013). Similarly, body weight at 40th week of age
(BW40) was significantly influenced by loci MCW0051,
MCW0014 and ADL0176; age at sexual maturity (ASM)
by MCW0075; and egg weight at 28th week of age (EW28)
by MCW0005 and MCW0014 (Table 1) in agreement to
the earlier report (Das et al. 2013). In accordance to these
present findings, previously few workers also reported
significant association of some microsatellite alleles/
genotypes with age at sexual maturity (Van Kaam et al.
1999, 1998), body weights (Boschiero et al. 2009, Jennen
et al. 2006, Pandya et al. 2005, Van Kaam et al. 1999, 1998),
egg weights (Chatterjee et al. 2008, Van Kaam et al. 1999,
1998), and egg production (Chatterjee et al. 2010,
2008,Wardecka et al. 2002, Van Kaam et al. 1999, 1998) in
different chicken genotypes.
Critical Difference test (Table 2) demonstrated that the
microsatellite genotype DG heterozygote of MCW0044
locus had significantly higher EP40 than CF heterozygote
of the locus. BD and DD heterozygotes of MCW0051 locus
were statistically indifferent for BW40, though they had
significantly higher BW40 than CD heterozygote of the
locus. CF heterozygote of MCW0075 locus had
significantly lower ASM than BE heterozygote of the locus.
AE and BD heterozygotes of MCW0005 locus were
statistically indifferent for EW28, though they had
significantly higher EW28 than either CE heterozygote or
BB homozygote of the locus, CE and BB being statistically
indifferent. AA homozygote of MCW0014 locus had
significantly higher EW28 than either BB or CC
homozygotes of the locus, BB and CC being statistically
indifferent. Again, AA homozygote of MCW0014 locus had
significantly the highest BW40 followed by CC and BB
Table 1. Least squares analysis of variance of various layer performance traits under different microsatellite
loci in RIR selected line chicken
Source of variation df Mean sum of squares
BW20 ASM EW28 BW40 EW40 EP40
MCW 0044 1 19837.5 600.0 0.04 30104.2 0.04 1410.7**
Remainder 6 28875.0 170.3 7.1 17013.9 14.1 82.6
MCW 0051 2 8583.8 128.4 7.2 50093.8* 8.6 398.4
Remainder 5 35184.0 273.0 5.7 6400.0 13.5 221.8
MCW 0075 1 104.2 864.0* 12.0 8437.5 15.0 600.0
Remainder 6 32163.9 126.3 5.1 20625.0 11.6 217.7
MCW 0005 3 17470.8 170.0 11.9* 25104.2 15.6 292.0
Remainder 4 35168.8 278.0 1.8 14218.8 9.5 257.5
MCW 0014 2 12223.2 71.3 9.1* 36160.7* 10.7 185.1
Remainder 4 38781.3 338.4 1.3 2812.5 13.5 360.8
ADL 0102 2 4580.4 470.5 10.5 13348.2 15.6 595.7*
Remainder 4 41781.3 125.6 2.7 14218.8 9.1 75.5
ADL 0158 3 15640.3 368.1 0.13 21423.6 2.1 563.8*
Remainder 4 36541.7 129.4 10.6 16979.2 19.7 53.6
ADL 0176 2 18223.2 90.8 8.1 33348.2* 10.7 215.7
Remainder 4 35781.3 328.6 1.8 4218.8 13.5 345.5
* P<0.05; ** P<0.01; *** P<0.001. BW20/BW40, body weight in g at 20/40th week of age; ASM, age at sexual maturity in days;
EW28/EW40, egg weight in g at 28/40th week of age; EP40, part period egg production in numbers upto 40 weeks of age.
September 2016] MICROSATELLITES-PERFORMANCE ASSOCIATION IN RIR CHICKEN 1023
51
homozygotes of the locus. DD homozygote of ADL0102
locus had significantly higher EP40 than either BE or AD
heterozygotes of the locus, BE and AD being statistically
indifferent. DD homozygote of ADL0158 locus had
significantly higher EP40 than either CG or DH or DG
heterozygotes of the locus, CG and DH or DH and DG being
statistically indifferent. CE heterozygote of ADL0176 locus
had significantly higher BW40 than either DD or CC
homozygotes of the locus, DD and CC being statistically
indifferent. The present findings were in the line of earlier
reports of few researchers namely Chatterjee et al. (2010,
2008) and Pandya et al. (2005). One-to-one correspondence,
in the form of significance, between microsatellites and
phenotypes like age at sexual maturity, body weights, egg
weights and egg production traits may be the informative
indicator for elucidating QTL and microsatellite
relationships (Chatterjee et al. 2010). The genetic principle
of significant association of microsatellites and phenotypes
is possibly due to the phenomenon of linkage and if the
microsatellite be very closely linked (about 20 cM) with a
certain phenotype, it will specifically be observed in terms
of a significant association (Chatterjee et al. 2010) which
was observed in the present study, though the linkage
analysis (using the CRI-MAP program package) was not
carried out in this study because it would thrive to carry
out a research with a large number of samples to associate
microsatellite alleles with performance traits in more
accuracy.
It may be concluded that microsatellite alleles are
associated with performance traits suggesting faster genetic
progress in layer flocks by adapting microsatellite genotype
based selection. The results paved way for utilization of
microsatellite markers in molecular breeding for rapid
genetic improvement in layer chicken performance.
However, further study may be taken on larger sample size
to reach definite conclusion.
ACKNOWLEDGEMENT
The Indian Veterinary Research Institute-Fellowship for
pursuing first author’s Ph.D. programme is sincerely
acknowledged. The authors are thankful to the Head of the
Division of Avian Genetics and Breeding, CARI, Izatnagar
for providing the necessary facilities for this work.
REFERENCES
Albers G A A and Van Sambeek F M J P. 2002. Breeding strategies
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Table 2. The estimated least squares means of various layer performance traits under different
microsatellite (MS) genotypes in RIR selected line chicken
Microsatellite MS genotypes Least squares means ± standard errors
loci
Code allele:allele BW20 ASM EW28 BW40 EW40 (g) EP40
bp : bp (g) (days) (g) (g) (g) (nos.)
MCW 0044 CF 133:151 1600.00±69.37 163.50±5.33 44.83±1.09 1808.33±53.25 53.17±1.54 85.33±3.71b
DG 136:160 1715.00±120.16 143.50±9.23 45.00±1.89 1950.00±92.23 53.00±2.66 116.00±6.43a
MCW 0051 BD 90:118 1750.00±187.57 146.00±16.52 48.00±2.39 2050.00±80.00a57.00±3.68 112.00±14.89
CD 105:118 1616.00±83.89 162.40±7.39 44.00±1.07 1760.00±35.78b52.60±1.65 85.60±6.66
DD 118:118 1600.00±132.64 155.00±11.68 45.50±1.69 1950.00±56.57a52.50±2.60 102.0±10.53
MCW 0075 BE 180:190 1635.00±126.82 176.50±7.95b47.00±1.60 1900.00±101.55 55.50±2.41 78.00±10.43
CF 184:196 1626.67±73.22 152.50±4.59a44.17±0.93 1825.00±58.63 52.33±1.39 98.00±6.02
MCW 0005 AE 210:244 1520.00±187.53 169.00±16.67 49.00±1.35a2050.00±119.24 56.00±3.08 84.00±16.05
BB 216:216 1680.00±187.53 141.00±16.67 42.0±1.35b1850.00±119.24 49.00±3.08 120.0±16.05
BD 216:238 1750.00±132.61 165.00±11.79 46.50±0.95a1900.00±84.32 56.00±2.18 92.00±11.35
CE 222:244 1582.50±93.77 157.00±8.34 43.75±0.67b1762.50±59.62 52.00±1.54 89.00±8.02
MCW 0014 AA 173:173 1750.00±196.93 146.00±18.40 48.00±1.15a2050.00±53.03a57.00±3.67 112.0±19.00
BB 175:175 1565.00±139.25 159.50±13.01 44.50±0.81b1725.00±37.50c52.00±2.60 92.50±13.43
CC 177:177 1657.50±98.47 158.50±9.20 43.25±0.57b1800.00±26.52b52.00±1.84 90.75±9.50
ADL 0102 AD 136:166 1635.00±144.54 176.50±7.93 47.00±1.16 1900.00±84.32 55.50±2.14 78.00±6.14b
BE 146:174 1582.50±102.20 157.00±5.60 43.75±0.82 1762.50±59.62 52.00±1.51 89.00±4.35b
DD 166:166 1680.00±204.41 141.00±11.21 42.00±1.64 1850.00±119.24 49.00±3.02 120.00±8.69a
ADL 0158 CG 178:214 1565.00±135.17 159.50±8.04 44.50±2.31 1725.00±92.14 52.00±3.14 92.50±5.18b
DD 184:184 1715.00±135.17 143.50±8.04 45.00±2.31 1950.00±92.14 53.00±3.14 116.00±5.18a
DG 184:214 1750.00±191.16 184.00±11.38 45.00±3.26 1750.00±130.30 55.00±4.44 72.00±7.32c
DH 184:222 1573.33±110.37 159.33±6.57 45.00±1.88 1883.33±75.23 53.33±2.56 85.00±4.23bc
ADL 0176 CC 200:200 1582.50±94.58 157.00±9.06 43.75±0.67 1762.50±32.48b52.00±1.84 89.00±9.29
CE 200:236 1750.00±189.16 146.00±18.13 48.00±1.35 2050.00±64.95a57.00±3.67 112.0±18.59
DD 202:202 1715.00±133.76 162.50±12.82 43.50±0.95 1800.00±45.93b52.00±2.60 96.00±13.14
Means within a microsatellite locus having different superscripts differ significantly (P<0.05); BW20/BW40, body weight in g at
20/40th week of age; ASM, age at sexual maturity in days; EW28/EW40, egg weight in g at 28/40th week of age; EP40, part period egg
production in numbers upto 40 weeks of age.
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