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HayKOBHH BicHHK .HbBiBCbKoro Ha^oHaibHoro ymBepcurery BeTepHHapHoi' mcahuuhh Ta 6ioTexHonoriH iMeHi C.3. I^H^Koro Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies named after S.Z. Gzhytskyj
doi: 10.15421/nvlvet7414
ISSN 2519-2698 print ISSN 2518-1327 online
http://nvlvet.com.ua/
UDC 636.4.082.25 : 575.22
Genetic polymorphism of the Landrace pig based on microsatellite markers
S.I. Lugovoy, S.S. Kramarenko, V.Ya. Lykhach Lugsergey23@gmail.com
Mykolayiv National Agrarian University,
Georgy Gongadze Str., 9, Mykolayiv, Ukraine, 54020
The aim of this study was to analyze the genetic variability and population structure of the Landrace population by using 12 microsatellite markers. A total of 90 pigs representing one commercial breed (Landrace) were sampled. Twelve microsatellite loci (SW24, S0155, SW72, SW951, S0386, S0355, SW240, SW857, S0101, SW936 SW911 and S0228) were selected and belong to the list of microsatellite markers recommended by FAO/ISAG.
GenAIEx software was used to calculate the allele frequencies, effective number of alleles (Ae), observed (Ho) and expected (He) heterozygosity, within-population inbreeding estimate (Fis), Shannon's information index (ISh). Overall allele frequency values ranged from 0.006 to 0.9333 (at allele SW951120). The number of observed alleles (Na) detected ranged from 5 (S0155 and SW911) to 13 (SW72), with an overall mean of 9.00 ± 0.80 and a total of 108 alleles were observed at these loci. However, the effective number of alleles (Ae) ranged from 1.57 (SW951) to 5.49 (SW240) with a mean of 3.29 ± 0.33. Shannon's information index (ISf) which measures the level of diversity, was sufficiently high -from 0.79 (for SW951) to 2.01 (for SW240) - with a mean of 1.43 ± 0.09. The overall means for observed (Ho) and expected (He) heterozygosities were 0.578 ± 0.009 and 0.662 ± 0.004, respectively, which ranged from 0.307 (SW951) to 0.814 (SW857) and 0.361 (SW951) to 0.818 (SW240), respectively. Of the 12 microsatellites analyzed using Fisher's exact test, 50% were in Hardy-Weinberg equilibrium, and 6 were out of equilibrium (P < 0.05).
Three mutation models namely, infinite allele model (I.A.M.), two phase model (T.P.M.), stepwise mutation model (S.M.M.) were estimated using the BOTTLENECK software. The results are indicated that the Landrace pig population is non-bottlenecked and remained at mutation-drift equilibrium.
The study stands first in genetic characterization of the Ukrainian Landrace pig population through microsatellite markers. The various parameters and values used to quantify genetic variability, such as the high mean (and effective) number of alleles and the expected and observed heterozygosities, indicated high genetic variability in the Ukrainian Landrace pigs. The population has not undergone any recent and/or sudden reduction in the effective population size and remained at mutation-drift equilibrium.
Key words: genetic polymorphism, microsatellite loci, pigs, Landrace breed
Генетический полиморфизм свиней породы ландрас на основе микросателлитных маркеров
С.И. Луговой, С.С. Крамаренко, В.Я. Лихач Lugsergey23@gmail.com
Николаевский национальный аграрный университет, ул. Георгия Гонгадзе, 9, г. Николаев, 54020, Украина
Цель этого исследования состояла в том, чтобы проанализировать генетическую изменчивость и структуру популяции свиней породы ландрас с использованием 12 микросателлитных локусов. Всего было исследовано 90 свиней, представляющих одну коммерческую породу (ландрас). Были выбраны 12 микросателлитных локусов (SW24, S0155, SW72, SW951, S0386, S0355, SW240, SW857, S0101, SW936 SW911 и S0228), которые входят в список микросателлитных маркеров, рекомендованных ФАО/ISAG.
Citation:
Lugovoy, S.I., Kramarenko, S.S., Lykhach, V.Ya. (2017). Genetic polymorphism of the Landrace pig based on microsatellite markers. Scientific Messenger LNUVMBT named after S.Z. Gzhytskyj, 19(74), 63-66.
Для расчета частот аллелей, эффективного числа аллелей (Ae), наблюдаемой (Ho) и ожидаемой (He) гетерозиготно-сти, оценки инбридинга (Fis), информационного индекса Шеннона (ISh) использовалось программное обеспечение GenAIEx. В целом частота аллелей варьировала от 0,006 до 0,9333 (для аллеля SW951120). Число аллелей (Na) варьировало от 5 (S0155 и SW911) до 13 (SW72) при среднем значении 9,00 ± 0,80. В общей сложности, в этих локусах было обнаружено108 аллелей. Однако эффективное число аллелей (Ae) варьировало от 1,57 (SW951) до 5,49 (SW240) при среднем значении 3,29 ± 0,33. Информационный индекс Шеннона (ISh), который свидетельствует об уровне разнообразия, был достаточно высоким - от 0,79 (для SW951) до 2,01 (для SW240) при среднем значении 1,43 ± 0,09. Средние значения оценок наблюдаемой (Ho) и ожидаемой (He) гетерозиготности составляли 0,578 ± 0,009 и 0,662 ± 0,004 соответственно и варьировали от 0,307 (SW951) до 0,814 (SW857) и от 0,361 (SW951) до 0,818 (SW240) соответственно. Из 12 локусов микросателлитов, проанализированных с использованием точного теста Фишера, 50% находились в состоянии равновесия Харди-Вайнберга, а еще 6 - отклонялись от него (Р < 0,05).
С использованием программного обеспечения BOTTLENECK оценивались три мутационные модели, а именно: модель бесконечного числа аллелей (I.A.M.), двухфазная модель (T.P.M.), модель пошаговой мутации (S.M.M.). Результаты показывают, что для популяции свиней породы ландрас не отмечено bottleneck-эффекта и она находится в состоянии равновесия между мутационным процессом и дрейфом генов.
В исследовании впервые приведена генетическая характеристика украинской популяции свиней породы ландрас с использованием полиморфизма микросателлитных локусов. Различные параметры, используемые для количественной оценки генетической изменчивости, дают высокие оценки для среднего (и эффективного) числа аллелей, ожидаемой и наблюдаемой гетерозиготности, что указывает на высокую генетическую изменчивость украинской популяции свиней породы лан-драс. Популяция не претерпела каких-либо недавних и/или внезапных сокращений эффективной численности и оставалась в состоянии равновесия между мутационным процессом и дрейфом генов.
Ключевые слова: генетический полиморфизм, микросателлитные локусы, свиньи, порода ландрас
Генетичний полiморфiзм свиней породи ландрас на 0CH0Bi мжросател^них маркер1в
С.1. Луговий, С.С. Крамаренко, В.Я. Лихач Lugsergey23@gmail.com
Миколтвський нацюнальний аграрний утверситет, вул. Георггя Гонгадзе, 9, м. Миколтв, 54020, Украша
Мета цього дослгдження полягала в тому, щоб проаналгзувати генетичну мгнливгсть i структуру популяцп свиней породи ландрас з використанням 12 мжросателтних локуЫв. Всього було до^джено 90 свиней, яю представляють одну комерцшну породу (ландрас). Було обрано 12 мжросателтних локуЫв (SW24, S0155, SW72, SW951, S0386, S0355, SW240, SW857, S0101, SW936 SW911 i S0228), як входять до списку мжросателтних маркерiв, рекомендованих ФАО /ISAG.
Для розрахунку частот алелей, ефективного числа алелей (Ae), фактичноi (Ho) i оч^вано! (He) гетерозиготностi, ощ-нки тбридингу (Fis), тформацшного тдексу Шеннона (ISh). використовувалося програмне забезпечення GenAIEx. В цыому, частота алелей варжвала вiд 0,006 до 0,9333 (для алелi SW951120). Число алелей (Na) варжвало вiд 5 (S0155 i SW911) до 13 (SW72) iз середтм значенням 9,00 ± 0,80. Загалом для цих локуЫв було виявлено108 алелей. Однак ефективне число алелей (Ae) варiювало вiд 1,57 (SW951) до 5,49 (SW240) iз середтм значенням 3,29 ± 0,33. 1нформацшний тдекс Шеннона (ISh), який свiдчить про рiвень рiзноманiтностi, був досить високим - вiд 0,79 (для SW951) до 2,01 (для SW240) - iз середтм значенням 1,43 ± 0,09. Середт значення фактичноi (Ho) та оч^ваноь (He) гетерозиготностi становили 0,578 ± 0,009 i 0,662 ± 0,004, вiдповiдно i варювали вiд 0,307 (SW951) до 0,814 (SW857) i вiд 0,361 (SW951) до 0,818 (SW240) вiдповiдно. 1з 12 локуЫв мж-росател^в, проаналiзованих з використанням точного тесту Фiшера, 50% перебували в сташ рiвноваги Хардi-Вайнберга, арешта - вiдхилялися вiд нього (Р < 0,05).
З використанням програмного забезпечення BOTTLENECK ощнювалися три мутацтт моделi, а саме: модель нескт-ченного числа алелей (I.A.M.), двохфазна модель (T.P.M.), модель покроковоi мутацп (S.M.M.). Результати свiдчать, що для популяцп свиней породи ландрас не вiдзначено bottleneck-ефекту i вона перебувала в стан рiвноваги мiж мутацшним про-цесом i дрейфом гетв.
У до^дженю вперше наведена генетична характеристика укра!нськоi популяцп свиней породи ландрас з використанням полiморфiзму мжросателтних локуЫв. Рiзнi параметри, як використовуються для ктьюсноi ощнки генетичноi мни-востi, дають висот ощнки середнього (i ефективного) числа алелей, очiкуваноi i фактичног гетерозиготностi, що вказуе на високу генетичну мнивкть украiнськоi популяцп свиней породи ландрас. Популящя не зазнала будь-яких недавтх i/або раптових скорочень ефективноi чисельностi i залишалася в стан рiвноваги мiж мутацшним процесом i дрейфом гетв.
Ключовi слова: генетичний полiморфiзм, мкросателтн локуси, свит, порода ландрас
Introduction
In recent years, knowledge of the commercial and local pig resources of the Ukraine has increased, following a similar trend in other countries. The productive lifespan, good maternal ability and high meat production of the Landrace pigs is remarkable. In addition, the superiority of crossbreeding these pigs with Large White breeds in
terms of the growth stage and weight gain of offspring is also noteworthy. However, the genetic characterization of the Landrace pigs from Ukrainian populations is still insufficient. In Ukraine, the first studies using molecular markers in pigs were done by V. Topiha et al. (Topiha et al., 2010) who analyzed a panel of five microsatellites in 241 Large White pigs.
The aim of this study was to analyze the genetic vari-
ability and population structure of the Landrace population by using 12 microsatellite markers.
Material and Methods
A total of 90 pigs representing one commercial breed (Landrace) were sampled. The animals belonged to the breeding group of the public joint-stock company (PJSC) «Plemzavod «Stepnoy» located in Zapovitne village, Kamensko-Dniprovsky district of Zaporozhia region, Ukraine.
PCR analysis was carried out on DNA extracted from 90 ethanol-fixed small pieces of ear tissue samples. A DNA extraction using the Nexttec Clean Column kit (Nexttec, Germany) according to the manufacturer's instructions was performed.
Twelve microsatellite loci which presented reliable amplification standards (SW24, S0155, SW72, SW951, S0386, S0355, SW240, SW857, S0101, SW936 SW911 and S0228) were selected and belong to the list of microsatellite markers recommended by FAO/ISAG. The adopted strategy for the selection of the loci was to represent most of the autosomic pig chromosome.
Electrophoresis was carried out using an ABI 3130*l Genetic Analyzer (Applied Biosystems, USA). Allele sizes of each microsatellite were determined using GeneMapper ver. 4.0 (Applied Biosystems).
All the samples were stored in the DNA Bank of the Laboratory of All-Russian Science Institute of Animal Husbandry named after L.K. Ernst (VIJ), Dubrovitzy (Russian Federation), where this experiment was developed.
GenAIEx version 6.5 (Peakall and Smouse, 2006) was used to calculate the allele frequencies, effective number of alleles (Ae), observed (Ho) and expected (He) heterozygosity, within-population inbreeding estimate (Fis), Shannon's information index (ISh).
GENEPOP version 4.2 (Rousset, 2008) was used to perform deviations form Hardy-Weinberg equilibrium (HWE) per locus using Markov chain algorithm implemented according to authors recommendation with 10,000 dememorizations, 200 batches and 5,000 interactions per batch.
The BOTTLENECK (version 1.2.03) (Cornuet and Luikart, 1996) analysis was performed to know whether this pig population exhibits a significant number of loci with excess of heterozygosity.
Results and Discussion
The genetic diversity parameters in the Landrace pig, such as allele number, effective number of allele, observed and expected heterozygosity, within-population inbreeding estimate (Fis) and Shannon's information index are presented in Table 1.
All the loci studied were polymorphic. Overall allele frequency values ranged from 0.006 to 0.9333 (at allele SW951120). The number of observed alleles (Na) detected ranged from 5 (S0155 and SW911) to 13 (SW72), with an overall mean of 9.00 ± 0.80 and a total of 108 alleles were observed at these loci. However, the effective number of alleles (Ae) ranged from 1.57 (SW951) to 5.49 (SW240) with a mean of 3.29 ± 0.33.
Shannon's information index (ISh) which measures the level of diversity, was sufficiently high - from 0.79 (for SW951) to 2.01 (for SW240) - with a mean of 1.43 ± 0.09. The overall means for observed (Ho) and expected (He) heterozygosities were 0.578 ± 0.009 and 0.662 ± 0.004, respectively, which ranged from 0.307 (SW951) to 0.814 (SW857) and 0.361 (SW951) to 0.818 (SW240), respectively. Of the 12 microsatellites analyzed using Fisher's exact test, 50% were in Hardy-Weinberg equilibrium, and 6 were out of equilibrium (P < 0.05).
Table 1
Microsatellite analysis in the Landrace pig population
Locus n Na Ae Ish Ho He Fis HWE
SW24 69 10 4.22 1.64 0.797 0.763 -0.045 ns
S0155 90 5 2.52 1.14 0.589 0.603 0.024 ns
SW72 85 13 2.97 1.49 0.671 0.664 -0.010 ns
SW951 88 7 1.57 0.79 0.307 0.361 0.151 ***
S0386 90 10 2.75 1.46 0.578 0.637 0.093 ns
S0355 85 10 3.04 1.42 0.412 0.671 0.386 ***
SW240 77 12 5.49 2.01 0.675 0.818 0.174 ***
SW857 86 10 5.22 1.85 0.814 0.809 -0.007 ns
S0101 87 6 3.29 1.30 0.747 0.696 -0.074 ns
SW936 85 12 2.67 1.43 0.435 0.625 0.304 ***
SW911 45 5 2.53 1.19 0.422 0.605 0.303 *
S0228 86 8 3.25 1.44 0.488 0.693 0.295 ***
* - P < 0.05; *** - P < 0.001; ns - P > 0.05.
The within-population inbreeding estimates (Fis) observed at 8 loci were positive and 4 loci revealed negative with a mean of 0.133 ± 0.046 is indicating significant heterozygosity shortage in the Landrace pig population.
Three mutation models namely, infinite allele model (I.A.M.), two phase model (T.P.M.), stepwise mutation model (S.M.M.) were estimated using the BOTTLENECK software (Table 2). The results are indicated that
the Landrace pig population is non-bottlenecked and remained at mutation-drift equilibrium.
The effective number of alleles found in this study (3.29 ± 0.33) was higher than that in the German Land-race (2.27), the Vietnam Landrace pigs (2.83) (Thuy et al., 2006). This value for the Ukrainian Landrace pigs was comparable to than that found by Vicente et al. (Vicente et al., 2008) in the Landrace pigs from Portugal (3.47).
The average expected (0.662) and observed (0.578) heterozygosities of the Ukrainian Landrace pigs indicated a high degree of variability. The heterozygosity observed here resembled the values found for other breeds, such as the Vietnam Landrace pigs (He = 0.600) (Thuy et al., 2006), the Spanish Landrace pigs (He = 0.640) (Boitard et
The Fis values in the sample of the Ukrainian Land-race pigs ranged from -0.074 to 0.386. The mean within population inbreeding estimate (Fis) was 0.133. The deficiency of heterozygotes (13.3 percent) in the Ukrainian Landrace pig population is higher to heterozygote shortfall observed in the Portuguese Landrace pig (3.8 percent) (Vicente et al., 2008) and the Spanish Landrace pigs (6.0 percent) (Boitard et al., 2010).
Conclusions
The study stands first in genetic characterization of the Ukrainian Landrace pig population through microsatellite markers. The various parameters and values used to quantify genetic variability, such as the high mean (and effective) number of alleles and the expected and observed heterozygosities, indicated high genetic variability in the Ukrainian Landrace pigs. The population has not undergone any recent and/or sudden reduction in the effective population size and remained at mutation-drift equilibrium.
Researcher's perspectives
The evaluation of pig genetic resources and the conservation of key pig populations will be important to enable the Ukrainian agriculture and food industries to respond to future changes in consumer needs. Two breeds are mainly used as maternal lines in the Ukrainian swine breeding programs: Landrace and Large White, which represent 37.3 percent and 51.8 percent of the overall germplasm used for pork production, respectively (Gladiy et al., 2015). Evaluating the genetic diversity within a breed is also a requisite for genetic conservation.
Acknowledgement
This study was performed with financial support of the Ministry of Education and Science of Ukraine (No. 0116U004760).
al., 2010) and the Portuguese Landrace pigs (He = 0.670) (Vicente et al., 2008). The observed heterozygosity found in present study is also in close agreement with the reported values in Landrace (0.522) by Swart et al. (Swart et al., 2010) for the Southern African domestic pigs.
Table 2
References
Topiha, V.S., Lugovoy, S.I., Kramarenko, S.S. (2010). Analiz geneticheskogo raznoobraziia svinei krupnoi beloi porody na osnove multilokusnykh genotipov mikrosatellitov. Visnik agrarnoy nauki Prichornomor'ia. 1(52), T. 2, 3-11 (In Russian).
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Rousset, F. (2008). GENEP0P'007: a complete reimplementation of the GENEPOP software for Windows and Linux. Molecular Ecology Resources. 8, 103-106.
Cornuet, J.M., Luikart, G. (1996). Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics. 144, 2001-2014.
Thuy, N.T.D., Melchinger-Wild, E., Kuss, A. W., Cuong, N.V., Bartenschlager, H., Geldermann, H. (2006). Comparison of Vietnamese and European pig breeds using microsatellites. Journal of animal science. 84 (10), 2601-2608.
Vicente, A.A., Carolino, M.I., Sousa, M.C.O., Gmja, C., Silva, F.S., Martinez, A.M., Vega-Pla, J.L., Carolino, N., Gama, L.T., (2008). Genetic diversity in native and commercial breeds of pigs in Portugal assessed by microsatellites. Journal of animal science. 86(10), 2496-2507.
Boitard, S., Chevalet, C., Mercat, M.J., Meriaux, J.C., Sanchez, A., Tibau, J., Sancristobal, M. (2010). Genetic variability, structure and assignment of Spanish and French pig populations based on a large sampling. Animal genetics. 41(6), 608-618.
Swart, H., Kotze, A., Olivier, P. A. S., Grobler, J. P. (2010). Microsatellite-based characterization of Southern African domestic pigs (Sus scrofa domestica). South African Journal of Animal Science. 40(2), 121-132.
Gladiy, M.V., Ruban, S.Y., Getya, A.A., Pryima, S.V. (2015). Porody sil'skogospodars'kykh tvaryn Ukrainy. Istoriya, stan, perspektyvy rozvytku. Rozvedennia i genetyka tvaryn. 49, 44-57 (In Ukrainian).
Cmammn nadiuMxa do peda^ii 7.02.2017
Bottleneck analysis in the Landrace pig population
Model Sign rank test - Number of loci with heterozygosity excess
Expected Observed Probability
I.A.M. 7.16 4 0.060
T.P.M. 7.14 4 0.061
S.M.M. 7.10 1 < 0.001
