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11РАЗВЕДЕНИЕ, СЕЛЕКЦИЯ И ГЕНЕТИКА
THE USE OF ISSR MARKERS FOR CHARACTERIZATION OF GENETIC DIFFERENTIATION OF CATTLE BREEDS
Kosovsky G.Yu., Glazko T.T., Arkhipov A.V., Khovankina A.V., Babii A.V., Kornienko E.V., Kovalchuk S.N., Glazko V.I.
Scientific Institution Center of Experimental Embryology and Reproductive Biotechnologies, Moscow, Russian Federation
ABSTRACT. To date, different DNA markers and methods have been developed and widely employed for the determination of genetic polymorphisms and evaluation of the genetic structure of farm animal populations. But despite the diversity of the developed methods, it is difficult to choose the most reliable and the least labor-consuming approach with easily interpretable results. One of the promising and reliable methods that meet the requirements of multilocus genotyping is the method of inter-simple sequence repeat (ISSR) markers. As ISSR markers in the current work, 6 microsatellites have been used as primers in polymerase chain reaction (PCR) to evaluate genetic differentiation of dairy breeds and non-specialized cattle (holsteinized Black-and-White, Estonian Red, Ayrshire, Kazakh Whiteheaded, Tagil native cattle, Yakutian cattle, zeboid cattle, 10 animals of each breed). According to obtained results, the most polymorphic spectra were obtained by (GAG)6C and (AGC)6G primers. The dendrogram built by ISSR-PCR evaluation of different cow breeds demonstrated the close link between discovered polymorphism of DNA fragments under study and the animals' differentiation by trend of productivity (dairy or non-specialized). The presented data demonstrate that every cattle group has specific features in amplicon spectrum polymorphism revealed by the use of microsatellite sequences as PCR primers (ISSR-PCR markers).The obtained data suggest the possibility to use the approach of ISSR-PCR merkers for development of breed-specific test systems. These would improve the selection and breeding of animals with desirable phenotypes and help consolidate the groups of cattle with respect to their breed affiliation.
Keywords: cattle breeds, genetic differentiation, DNA technologies, genetic markers, microsatellites Problemy biologii productivnykh zhivotnykh - Problems of Productive Animal Biology, 2016, 3: 91-97
To date, the different DNA markers and methods have been developed and widely employed for the determination of genetic polymorphisms and evaluation of the genetic structure of farm animal populations. But despite the diversity of the developed methods, it is difficult to choose the most reliable and the least labor-consuming approach with easily interpretable results (Lenstra et al., 2012). In the last years, the creation of test systems for multilocus genotyping (genome scanning) of farm animals involves the DNA microarrays for single nucleotide polymorphism (SNP) detection. However, these SNPs remain difficult to detect in genome, demand expensive equipment and expendable materials. Furthermore, in spite of high amount of data obtained in the present time and the creation of SNP databases for human, cattle, pigs and some other species, a simple test systems for individual animals genotyping have not been created. Also, there is no evidence about possibility of their use for evaluation of breed affiliation and the link between SNP markers and the genes involved in development of economically valuable traits (Zhan et al., 2011; Utsunomiya et al., 2014). The less complicated approach for genome scanning is the development of the simultaneous genotyping by a set of loci of anonymous DNA fragments flanked by microsatellite inverted repeats. Such multilocus spectra allow to obtain DNA profiles for individual genomes when, based on composition of DNA fragments (anonymous DNA) with different length, the reliable evaluation of breed affiliation and desirable traits is possible. The development of such a test system requires the preliminary analysis of
DNA fragments polymorphism flanked by microsatellite sequences. These markers are among the best candidates that meet the requirements of multilocus genotyping method due to their multi-copy and relatively high level of polymorphism in the genomes. This method was developed for more than two decades ago (Zietkiewicz et al., 1994) and has been called the method of genomic ISSR-PCR (ISSR - inter-simple sequence repeat; PCR - polymerase chain reaction) markers. Soon after it had been proposed, the method of ISSR-PCR scanning became widely explored for genotyping of many plant and animal organisms (Stolpovskii et al., 2013; Askari et al., 2014; Costa et al., 2016).
The aim of the current work was to perform the genome scanning on a group of dairy and non-specialized cattle breeds using as PCR primers the following microsatellite fragments (AG)9C, (GA)9C, (GAG)6C, (CTC)6C, (AGC)6G and (ACC)6G.
Materials and methods
The work was performed on DNA samples isolated from whole blood of the following breeds animals: 1) holsteinized Black-and-White cattle from "Zarya" farm; 2) Estonian Red cattle (Dubrovy, Pskov region); 3) Ayrshire cattle (Moscow region); 4) Kazakh whiteheaded cattle (Volgograd); 5) Tagil native cattle; 6) Yakutian cattle (Sakha Republic); 7) zeboid cattle (Snegiri, Moscow region); 8) holsteinized Black-and-White cattle from Moscow Timiryazev Agricultural Academy vivarium (MTAAV).
Amplification of individual DNA fragments was performed by means of ISSR PCR in which DNA fragments flanked by inverted primer repeats are amplified. PCR was executed with the following thermal cycle: 1. 94°C 1 min.; 2. 94°C 30 s, 55°C 30 s., 72°C 2 min., 35 cycles; 3. final elongation 72°C 10 min. The reaction was carried out in NYX Technik amplifier. Incubation mix contained 1 ^l of 10X PCR buffer, 1 ^l of dNTP mix (10 mM), 1 ^M of primer, 2 ^l (0.5-1 ^g) of DNA matrix, 1 ^l (5 u.) of Taq DNA polimerase. Electrophoresis of the amplification products was performed on Voltronyx power supply. The samples, mixed with loading buffer, were applied into wells of 1.5% agarose gel with ethidium bromide at concentration of 0.5 ^g/ml. Electrophoresis was carried out in TBE buffer (89 mM Tris, 89mM boric acid, 2mM EDTA, pH 8.3); 100 - 3000 bp and 0.25-10 kb DNA Ladders (SibEnzyme) were used as the DNA fragments length markers. Scanning was executed under UV light (^=312 nm) on ECXF20.M transilluminator. Only the clearly recognizable and reproducible DNA fragments were included into analysis. The mathematical processing, including dendrogram building, was carried out with the use of TPFGA program (Miller, 1997). PIC (Polymorphic Information Content) index was calculated using the following formula: PIC=2f(1-f), where f is the frequency for one of the alleles. Because ISSR-PCR markers are dominant by amplification product presence, f was estimated as vr, where R is the fraction of animals lacking the DNA fragment of a certain length in the amplification spectrum. R value was considered the fraction of homo-zygous recessives.
Results and Discussion
The analysis of interbreed genetic differentiation of the holsteinized Black-and-White, Estonian Red, Ayrshire, Kazakh Whiteheaded, Tagil native cattle, Yakutian cattle and zeboid cattle was performed on the basis of the results of ISSR-PCR with (AG)9C, (GA)9C, (GAG)6C, (AGC)6G and (ACC)6G primers. The amount of reproducibly detected amplicons in the animals of dairy and non-specialized breeds varied between different microsatellites and between animals of different breeds. For example, this amount ranged from 4 to 11 for (AG)9C-spectra, from 5 to 10 for (GA)9C -spectra, from 4 to 9 for (GAG)6C-spectra, from 5 to 11 for (CTC)6C--spectra, from 3 to 13 for (AGC)6G-spectra, from 4 to 11 for (ACC)6G-spectra.
By the fraction of polymorphic loci in all the amplicons, the representatives of the different breeds were similar except for holsteinized cattle from Moscow region. At the same time, the interbreed difference was observed in contribution to such generalized spectrum for amplicons derived
from different primers. In general, amplicon spectra polymorphism for primers based on dinucleotide microsatellites was higher than for primers based on trinucleotide microsatellites except for amplicon spectra of: i) (GAG)6C primer in beef Kazakh and zeboid cattle, dairy Ayrshire cattle and holsteinized cattle from Moscow region; ii) amplicon spectra of (AGC)6G primer in Ayrshire cattle, Estonian Red cattle, zeboid cattle. The summary of polymorphism evaluation by the microsatellite-derived amplicon spectra PICs for cattle groups under study is shown in Table 1. The values of average PICs in all the amplicon spectra grouped the Yakutian, the Kazakh and the Tagil breeds; the other breeds have much higher PIC values (see Table). The unique is the PIC value for (CTC)6C-derived amplicon spectra in Ayrshire cows which is 0.0922 as opposite to 0.0000 in other breeds.
Polymorphic information content (PIC) of amplification product spectra of di- and trinucletide microsatellite inverted repeat-flanked DNA fragments (ISSR markers) in the cattle breeds.
Primer Yakutian Kazakh Whiteheaded Zeboid Tagil native Estonian Red Holstei-nized "Zarya" Ayrshire Holstei-nized MTAAV
(AG)9C 0.0502 0.0000 0.0690 0.2009 0.2518 0.2074 0.1173 0.1887
(GA)9C 0.0086 0.2070 0.1277 0.2123 0.0966 0.0000 0.1918 0.1028
(GAG)6C 0.0000 0.1869 0.1667 0.0610 0.2227 0.0845 0.0804 0.1021
(CTC)6C 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0922 0.0000
(AGC)6G 0.0000 0.0000 0.1419 0.0000 0.1575 0.0000 0.1080 0.0000
(ACC)6G 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000
Average 0.0697 0.0647 0.1277 0.0787 0.1871 0.1133 0.1290 0.0972
All the spectra could be arbitrarily divided into 3 groups - the light fragments (200-500 bp), the medium fragments (600-1400 bp), the heavy fragments (1700-2500 bp). In case of (AG)9C-derived spectra, the zeboid cattle was specific because of the presence of 200-300 bp fragments which were lacking in all other animals. In case of heavy and medium fragments, the difference between dairy and non-specialized cattle was not shown. In (GA)9C-derived spectra, there was no significant difference between two groups of breeds. In (GAG)6C-derived spectra, only the dairy cows had the 750 bp fragment as opposite to the non-specialized animals. In (CTC)6C-derived spectra, the difference between two groups of breeds also was not observed (except for the Ayrshire cattle). In (AGC)6G-derived spectra, the significant difference between two groups of breeds also was not observed, as in case of (CTC)6C-derived spectra; the only exception being two light fragments of 200 and 250 bp length which were present only in the zeboid cattle. In (ACC)6G-derived spectra, the dairy cattle group demonstrated the presence of light fragments of 200 and 300 bp length as opposite to the zeboid, the Kazakh and the Tagil cattle. Notably, the same fragment was present in the Yakutian cattle which fact is particularly interesting because Yakutian cow, being small, produces the more significant amount of milk with respect to body weight than many of the native breeds.
The unique composition of DNA amplification products obtained by PCR with different primers was also demonstrated by dendrogram building with the use of TPFGA program based on Nei genetic distances calculation (Nei, 1972) from frequency of DNA fragments of different lengths in the cattle groups under study (see Fig.). On figure the cluster divided into two subclusters is shown. The first subcluster consists of the Estonian Red cattle and the holsteinized cattle from Moscow region, the second subcluster consists of the Ayrshire cattle and the holsteinized cattle from "Zarya" farm. Thus, four groups of this cluster belong to specialized dairy breeds. Four other groups represent the less specialized breeds with higher adaptive potential such as Kazakh whitehead, Tagil native, Yakutian and zeboid breeds.
The analysis of genetic differentiation of dairy and non-specialized cattle breeds performed by ISSR-PCR method in this study has revealed that the dairy breeds differ from the native breeds in composition of amplicons in spectra rather than in presence of unique breed-specific fragments. The prevalence of the light fragments in (AG)9C- and (AGC)6G-derived spectra discriminated the zeboid cattle from all other breeds. The dairy cattle differed from the non-specialized cattle by presence of
750 bp fragment in (GAG)6C-derived spectra and from three native breeds (Tagil, Kazakh, Yakutian cattle) by presence of light fragments of 200 and 300 bp length in (ACC)6G-derived spectra. For holsteinized cattle the most polymorphic amplicon spectra could be achieved by the use of PCR primers based on dinucleotide microsatellite sequences such as (AG)9C and (GA)9C; for other breeds, (AGC)6G primer is also efficient, as is (CTC)6C primer for Ayrshire cattle.
.5 A .3 .2 .1 0.0UQ
- Estonian Red
- Hols temrce d Bla ck- and-White IVlTAAV
- Hol.stenuzed Black-and-White "Zarya"
- Ayrshire
- Yakutian cattle
- - Tagil. native cattle
--- Zeboul rattle
- Kazakh Whit ehe aie d
The dendrogram based on Nei genetic distances calculation from frequency of DNA fragments of different lengths in amplification products obtained from ISSR PCR with (AG)9C, (GA)9C, (GAG)6C, (CTC)6C, (AGC)6G and (ACC)6G primers. The cattle groups are the following: holsteinized Black-and-White cattle from "Zarya" farm; Estonian Red cattle (Dubrovy, Pskov region); Ayrshire cattle (Moscow region); Kazakh Whiteheaded cattle (Volgograd); Tagil native cattle; Yakutian cattle (Sakha Republic); Zeboid cattle (Snegiri, Moscow region); holsteinized Black-and-White cattle from Moscow Timiryazev Agricultural Academy vivarium (MTAAV).
The most polymorphic spectra were derived with (GAG)6C and (AGC)6G primers. It should be noted that GAG microsatellite belongs to polypurine sequences which participate in triplex formation involved in complex network processes of gene expression regulation and genomic instability (Buske et al., 2011). More important seems to be the involvement of the AGC microsatellite repeat in the polymorphism. The cattle genome sequencing revealed that this microsatellite is overrepresented in Bos taurus as opposed to other animals. Notably, the high frequency of AGC microsatellite is characteristic for cattle and sheep genomes compared to human, dog and rat genomes. Also, AGC tandem repeat is shown to be associated with 39% frequency with non-autonomous transposone Bov-A2 (SINE) in cattle genome (Tellam and Worley, 2009). It allows speculating that abundance of this microsatellite is associated with exogenous retroviral infection specific for ruminants and with its further proliferation due to transposition of the mobile genetic elements. Interbreed differences in AGC inverted repeat-flanked DNA fragments polymorphism might be expected to demonstrate specific for AGC pattern of participation in such transpositions.
Conclusions
The discovered breed differentiation evaluated by the microsatellite amplification spectra demonstrates the specific features of distribution of short inverted repeats in 2000 bp length range in the genomes of the studied cattle breeds. The dendrogram based on evaluation of ISSR-PCR polymorphism shows a close association between discovered polymorphism of DNA fragments under study and breed differentiation by the direction and level of specialization.
The presented data demonstrate that every cattle group has specific features in amplicon spectrum polymorphism revealed by the use of microsatellite sequences as PCR primers (ISSR-PCR
markers). This fact proves the necessity of preliminary studies for choice of these markers for specific cattle groups upon test system development for genome scanning efficiency which directly depends on the amount of the polymorphic loci and their polymorphism level.
The obtained data suggest the possibility to use the approach of ISSR-PCR merkers for development of breed-specific test systems. These would improve the selection and breeding of animals with desirable phenotypes and help consolidate the groups of cattle with respect to their breed affiliation.
REFERENCES
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2. Costa R., Pereira G., Garrido I., Tavares-de-Sousa M.M., Espinosa F. Comparison of RAPD, ISSR, and AFLP Molecular Markers to Reveal and Classify Orchardgrass (Dactylis glomerata L.) Germplasm Variations. PLoS One. 2016, 11(4):e0152972.
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11.Zietkiewicz, E. Rafalski, A. Labuda, D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics, 1994, 20(2):176-183.
УДК 636.2.082.13:575.22
ИСПОЛЬЗОВАНИЕ ISSR МАРКЕРОВ ДЛЯ ХАРАКТЕРИСТИКИ ГЕНЕТИЧЕСКОЙ ДИФФЕРЕНЦИАЦИИ ПОРОД КРУПНОГО РОГАТОГО СКОТА
Косовский Г.Ю., Глазко T.T., Архипов A.B., Хованкина A.B., Бабий A.B., Корниенко E.B., Ковальчук С.Н., Глазко B.^
Центр экспериментальной эмбриологии и репродуктивной биотехнологии, Москва, Российская Федерация
На сегодняшний день различные маркеры и методы ДНК-технологий широко используются для определения генетического полиморфизма и оценки генетической структуры популяций сельскохозяйственных животных, но несмотря на разнообразие разработанных методов, пока трудно выбрать самый надежный и наименее трудоемкий подход, позволяющий получить легко интерпретируемые результаты. Одним из перспективных и надежных методов, которые отвечают требованиям мультилокального генотипирования, является метод геноти-пирования по набору фрагментов ДНК, фланкированных инвертированными микросателлит-ными повторами (ISSR метод). B данной работе в качестве ISSR маркеров использовались 6 микросателлитов как праймеров в полимеразной цепной реакции (PCR) для оценки генетической дифференциации молочных и неспециализированных пород крупного рогатого скота (голштинизированная черно-пёстрая, эстонская красная, айрширская, казахская белоголовая, тагильская, якутская, зебувидный скот, по 10 голов от каждой породы). Наиболее полиморфные спектры были получены с использованием (GAG) 6C и (AGC) 6G праймеров. Дендро-граммы, построенные путем ISSR-PCR оценки различных пород коров, выявили тесную связь между обнаруженным полиморфизмом фрагментов ДНК и дифференциацией животных по типу продуктивности (молочный породы или неспециализированный скот). Полученные данные, свидетельствующие о том, что каждая группа крупного рогатого скота имеет специфические особенности полиморфизма изученных ампликонов, позволяют предположить возможность использования метода ISSR-PCR для совершенствования пород. Это позволит улучшить отбор и разведение животных с желательными фенотипами и поможет консолидировать группы скота в отношении их породной принадлежности.
Ключевые слова: породы крупного рогатого скота, генетическая дифференциация, ДНК-технологии, микросателлитные маркеры
Проблемы биологии продуктивных животных, 2016, 3: 91-97 ЛИТЕРАТУРА
1. Buske F.A., Mattick J.S., Bailey T.L. Potential in vivo roles of nucleic acid triple-helices // RNA Biol. -2011. - Vol. 8. - No. 3. - P. 427-439.
2. Costa R., Pereira G., Garrido I., Tavares-de-Sousa M.M., Espinosa F. Comparison of RAPD, ISSR, and AFLP molecular markers to reveal and classify orchardgrass (Dactylis glomerata L.) Germplasm Variations // PLoS One. - 2016. - Vol. 11. - No. 4. - P. e0152972.
3. Lenstra J.A., Groeneveld L.F., Eding H., Kantanen J., Williams J.L., Taberlet P., Nicolazzi E.L., Sölkner J., Simianer H., Ciani E., Garcia J.F., Bruford M.W., Ajmone-Marsan P., Weigend S. Molecular tools and analytical approaches for the characterization of farm animal genetic diversity // Animal Genetics. - 2012. - Vol. 43. - P. 483-502.
4. Miller M. Tools for population genetics analyses (TPFGA) 1.3: A Windows program for the analysis of allozyme and molecular population genetic data. 1997 // Computer software distributed by author.
5. Askari N., Abadi M.M., Baghizadeh A. ISSR markers for assessing DNA polymorphism and genetic characterization of cattle, goat and sheep populations // Iranian Journal of Biotechnology. - 2011. - Vol. 9. - No. 3. - P. 222-229.
6. Nei M. 1972. Genetic distance between populations // American Naturalist. - 1972. - Vol. 106. - P. 283292.
7. Stolpovskii Iu.A., Evsiukova A.N., Sulimova G.E. Genomic diversity in cattle breeds assessed using polymorphism of intermicrosatellite markers // Genetika. - 2013. - Vol. 49. - No. 5. - P. 641-648.
8. Tellam R.L., Worley K.C. The genome sequence of taurine cattle: a window to ruminant biology and evolution // Science. - 2009. - Vol. 24. - P. 522-528.
9. Utsunomiya Y.T., Bomba L., Lucente G., Colli L., Negrini R., Lenstra J.A., Erhardt G., Garcia J.F., Ajmone-Marsan P. European Cattle Genetic Diversity Consortium. Revisiting AFLP fingerprinting for an unbiased assessment of genetic structure and differentiation of taurine and zebu cattle // BMC Genetics. -2014. - Vol. 15. - P. 47.
10.Zhan B., Fadista J., Thomsen B., Hedegaard J., Panitz F., Bendixen C. Global assessment of genomic variation in cattle by genome resequencing and high-throughput genotyping // BMC Genomics. - 2011. -Vol. 12. - P. 557.
11.Zietkiewicz, E. Rafalski, A. Labuda, D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification // Genomics. - 1994. - Vol. 20. - No. 2. - P. 176-183.
Поступило в редакцию: 04.07.2016 Получено после доработки: 18.07.2016
Косовский Глеб Юрьевич, дир., 8(985)761-45-11, gkosovsky@mail.ru Глазко Татьяна Теодоровна, гл.н.с., info-ceerb@mail.ru Архипов Андрей Владимирович, н.с., batler51@yandex.ru Хованкина Анна Викторовна., м.н.с, khovankina@gmail.com Бабий Анна Владимировна, м.н.с., anna_1506@yahoo.com Корниенко Екатерина Валерьевна, н.с., kornienko.katerina@gmail.com Ковальчук Светлана Николаевна, зав. отд., s.n.kovalchuk@mail.ru Глазко Валерий Иванович гл.н.с., info-ceerb@mail.ru
