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Thousands years of animal production and controlled reproduction combined with the natural selection effects, has been gave place to the great genetic diversity among the world animals populations. The goats are among the most ancient animals in being domesticated, is one of the species more use to produce meat and milk, and it is estimated that there are more that 500 goat breeds in the planet, of which the 89 % live in African continent (FAO 2015). Skapetas and Bampidis (2016) reported that about 95 % of the world’s total of goats is in the tropical developing countries, mainly in Asia and Africa, while Arellano et al. (2020) refer that, for the ability of adapting to different environments, goats have great diversity as a result of natural selection.
In Mozambique Republic the animal and agricultural production is the main source of income and job for 85 % of the rural population. Goats are use for meat production, providing profits for small breeders, which have more than 95 % of the national census. In the country the goat raising plays an important socio-economic and cultural role because besides to satisfy the food needs of the rural sector, the surpluses are sell or exchange with other products, which allow to financing the health, school, parties and traditional ceremonies (Nhamcumbe et al. 2017).
The Landim goat is genetic heritage from Mozambique, and despite its high contribution to people feeding, this genetic resource has been little studied. There is not enough information related with the regularities of their traditional production systems, their morphologic structure and neither of their genetic diversity, aspects that limit the national strategic design effective for its conservation and improvement. These deficiencies determined that the objective of this research was to determine the genetic diversity of the breed using microsatellites molecular markers, as contribution to the conservation and genetic improvement of breed.
Materials and Methods
Animal material. For the molecular analysis hair samples of 60 Landim goats from six Mozambique provinces (Manica, Tete, Sofala, Nampula, Gaza and Inhambane) were taken, where there are the highest number of animals from the breed. The hairs were taken from the back of the nape and were individually placed in plastic bags. Origin, sex and hair color were identified for each animal. The samples were move to Laboratorio de Biología Molecular del Animal Breeding Consulting, S.L, en la Universidad de Córdoba, España. The ADN was taking from the capillary bulb, in accordance with the conditions collected in this protocol.
Used materials. TE: Tris-HCl 10 mM, EDTA 1 mM pH=8, Buffer K of extraction: 40 mL of Tris-HCI 1M (pH=8.5), 20 mL of NaCl, 2.5M, 10 mL of EDTA 0.5M, 10 mL of SDS at 10 % and H2O up to 200 mL; Sodium acetate 3M pH=5.2, Isopropanol, Ethanol at 70 % and hair simple with root
Methods. Wash 10 hairs with root from each animal with bidistilled water and then with ethanol at 100%, let dried, cut the hair roots with sterilized scissors in alcohol and put into micro tubes. Add 300 µL of extraction buffer, crush the roots with a plunger, add 150 µL of sodium acetate 3M pH=5.2, put the micro tubes in the freezer (-20 ºC) at least for 20 minutes, centrifugate during 10 minutes at 13.000 rpm. Pick up the supernatant and put it in another clean 1.5 mL micro tube. Throw away the precipitate. Add the same quantity to the extracted from isopropanol, put it in the freezer (-20 ºC) at least 20 minutes, centrifugate during 30 minutes at 13.000 rpm. Throw away the supernatant and dry the precipitate at air or with a vacuum pump. Redisolve the precipitate in 100 µL of TE pH=8 and eep at -20 ºC.
Analyzed microsatellites. A total of 33 microsatellites molecular markers were used, selected and recommended by the experts committee from FAO/ISAG genetic diversity studies in sheep and goats species (FAO 2011). The markers were: BM1329, BM1818, BM6506, BM6526, BM8125, CRSM60, CSRD247, CSSM66, ETH10, ETH225, HAUT27, HSC, ILSTS8, ILSTS011, ILSTS19, INRA5, INRA6, INRA172, INRA63, MAF65, MAF209, MM12, OarFCB11, OarFCB20, OarFCB48, OarFCB304, SPS115, SRCRSP5, SRCRSP8, SRCRSP23, SRCRSP24, TGLA53, and TGLA122.
Amplification and elctrophoresis of microsatellites sequences. The microsatellites were amplified by PCR, according to Martínez (2001) methodology in 25 µL of the final volume of a reaction: 5 µL of ADN (3 ng/µL), 2.5 Mm of magnesium chloride, 1unit of Taq Polymerase, 200 mM of dNTPs and 0.25 mM of primers. The reaction cycle was of 10 minutes at 94 °C, fallow by 35 cycles at 94 °C per 30'', 55 - 60 °C per 45'' and 72 °C per 30'', finally, after the 35 cycles, it was keep 10' at 72 °C.
To carry out the separation by size of the fragments obtaining by PCR, they were submitted to electrophoresis in poliacrilamide gel in an automatic sequencer ABI Prism® 377 XL (Applied Biosystems), marking the ADN primers with fluorochrome of three different colors (blue, green and yellow), applying other fluorochrome of a fourth color (red) to mark an standard of sizes. In this way the gel yield was optimized since various microsatellites of same size and marked with three different fluorochromes can be loaded in a same bowl. The used fluorochromes are described in table 1.
Table 1 Fluorochrome used to mark the primers and emission color with the set of filters D in the sequencer ABI 377 XL.
| Fluorochrome | Nomemclature | Emission color |
|---|---|---|
| TET | 4,7,2’,7’-tetrachlorine-6-carboxyfluoresceine | Green |
| 6-FAM | 6-carboxyfluoresceine | Blue |
| HEX | 4,7,2’,4’,5’7’-hexachloro-6carboxyfluoresceine | Yellow |
| TAMRA | N,N,N’,N’-tetramethyl-6-carboxyrodamine | Red |
For the gel preparation the kit Reprogel 377 (Amersham Pharmacia Biotech) was used in accordance with the manufacture recommendations. The fragments analysis and genotyped was performed with the informatics programs GENESCAN ANALYSIS (Genescan 672v.2.0.2 and GENOTYPER® version 2.5.2).
Statistical analysis. Calulation of allelic frequencies. The calculation of allelic frequencies was based on the direct count of the alleles found in each locus. The observations with at least an allele were considered homozygote. When assuming that there is an ideal equilibrium state Hardy-Weinberg (EHW), the variance of an allelic frequency can be described through the binomial expression:
where x is the allelic frequency and n the number of sampled individuals.
The allelic frequencies for each lotus turned into divide the number of the same alleles per the total number of alleles. For its determination the informatics program GENEPOP, version 3.1c (Raymond and Rousset 1996) was used.
Heterozygosity calculation. The heterozygosity observed (Ho) and the heterozygosity expected (He) were calculated. The H was obtained by direct recount of the heterozygotes individuals and the He was calculated from the gene frequencies, the supposed equilibrium Hardy-Weinberg, using the Nei (1973) formula:
where: xi is the allele frequency I and k is the number of alleles
For the set of markers and for each of them in the total of analyzed samples, the heterozygosities were calculated with the informatics program GENEPOP, version 4.2.
Calculation of the Polymorphic Information Content (PIC). Its determination, the basic data of each allele frequency were introduced in a worksheet EXCEL® (Microsoft), the formula proposed by (Botstein et al. 1980) was used:
where: k is the number of alleles, xi, xj : alleles frequency i y j respectively.
Results and Discussion
Detected alleles. Table 2 shows that all the analyzed microsatellites showed polymorphism and the Landim population have a total of 214 alleles, with an allelic population that varies between 2 alleles in the locus ETH225 and 13 in the locus HSC, while the majority have between 4 and 8 alleles, with an average value of 6.4 for the breed; aspects that constitutes the first evidence that the breed have genetic variability. The total of alleles founded is lower to the value reported by Silva et al. (2019) in the Black creole goat from Mexico, but exceeded the number founded by Bustamante (2019) in Peruvian goats from Lima and Piura which represented 172 y 165, respectively. The values of the Venezuelan creole goat (182) and the Cuban creole were lower too, according to Aranguren et al. (2013) and Chacón et al. (2018).
Table 2 Number of alleles per marker, heterozigosities and PIC in the population of Landim goats.
| Locus | No. of alleles | He | Ho | PIC |
|---|---|---|---|---|
| BM1329 | 5.00 | 0.568 | 0.644 | 0.511 |
| BM1818 | 8.00 | 0.717 | 0.744 | 0.677 |
| BM6506 | 7.00 | 0.743 | 0.577 | 0.694 |
| BM6526 | 8.00 | 0.716 | 0.704 | 0.665 |
| BM8125 | 8.00 | 0.724 | 0.822 | 0.671 |
| CRSM60 | 6.00 | 0.803 | 0.813 | 0.762 |
| CSRD247 | 9.00 | 0.716 | 0.511 | 0.670 |
| CSSM66 | 12.00 | 0.853 | 0.439 | 0.825 |
| ETH10 | 4.00 | 0.686 | 0.644 | 0.613 |
| ETH225 | 2.00 | 0.315 | 0.113 | 0.263 |
| HAUT27 | 8.00 | 0.775 | 0.622 | 0.730 |
| HSC | 13.00 | 0.837 | 0.681 | 0.808 |
| ILSTS8 | 4.00 | 0.536 | 0.555 | 0.421 |
| ILSTS11 | 5.00 | 0.590 | 0.488 | 0.498 |
| ILSTS19 | 4.00 | 0.611 | 0.704 | 0.548 |
| INRA5 | 6.00 | 0.700 | 0.777 | 0.636 |
| INRA6 | 9.00 | 0.749 | 0.777 | 0.703 |
| INRA172 | 5.00 | 0.598 | 0.636 | 0.508 |
| INRA63 | 3.00 | 0.643 | 0.644 | 0.560 |
| MAF65 | 8.00 | 0.598 | 0.522 | 0.567 |
| MAF209 | 3.00 | 0.455 | 0.444 | 0.405 |
| MM12 | 11.00 | 0.854 | 0.844 | 0.828 |
| OarFCB11 | 7.00 | 0.795 | 0.697 | 0.753 |
| OarFCB20 | 6.00 | 0.717 | 0.704 | 0.658 |
| OarFCB304 | 8.00 | 0.510 | 0.511 | 0.485 |
| OarFCB48 | 6.00 | 0.688 | 0.533 | 0.624 |
| SPS115 | 3.00 | 0.343 | 0.355 | 0.314 |
| SRCRSP5 | 6.00 | 0.761 | 0.755 | 0.715 |
| SRCRSP8 | 8.00 | 0.638 | 0.568 | 0.588 |
| SRCRSP23 | 8.00 | 0.696 | 0.688 | 0.640 |
| SRCRSP24 | 5.00 | 0.533 | 0.525 | 0.495 |
| TGLA053 | 5.00 | 0.515 | 0.395 | 0.476 |
| TGLA122 | 4.00 | 0.682 | 0.733 | 0.609 |
| Total | 214.00 | |||
| Average SD | 6.40 2.61 |
0.657 0.023 |
0.6117 0.0128 |
He= heterozygosity expected, Ho= heterozygosity observed.
PIC=polymorphic information content, SD= standard deviation.
In a previous study of this breed Garrine (2010) obtained an average of 5.5 alleles, figure that do not coincide with the 6.4 average alleles founded in this study, which can be due to the sampling performed by that author did not include animals from all the regions of the country, and lower number of microsatellites were analyzed (17), although 11 of the included in this analysis were used. The average value of 6.4 alleles founded in this research is similar to what some authors reported in different goat breeds of several latitudes. De Sousa et al. (2011) reported 6.9 alleles for six native goats from Portugal, Chacón et al. (2018) showed 6.38 in the Cuban creole goat and in this same environment are the Iranian and Saudi goats and from Kashmir in China, accordintg to Vahidi et al. (2014), Al-Atiyat et al. (2015) and Du et al. (2017), respectively.
In this indicator, the Landim breed exceeds the Santanderena goat studied by Jiménez et al. (2014) which showed an average of 4.23 alleles. On the other hand, Landim breed is exceeded by the Nigerian breeds Maradi, West African Dwarf and Sahel that averaged 7.9, 8.7 and 8.2 alleles, respectively according to Murital et al. (2015) and for the Apureña breed which show allelic average of 8.6 as Gómez (2013) point out. Also the Mexican Black creole goat (8.1alleles) studied by Silva et al. (2019) and the dairy goats from South Africa according to Bosman et al. (2015) have better performance.
There were four microsatellites (ETH225, INRA63, MAF209 and SPS115) that showed less than 4 alleles. Similar find was informed by Chacón et al. (2017) in the Cuban creole goat, where the locus ETH225 and MAF209 has 2 and 3 alleles, respectively. For this last marker, Murital et al. (2015) found 3 alleles in Nigerian goats and Gómez (2013) detected only 2 alleles in the Apurimeña goat from Peru. It also coincides with Aranguren-Mendez (2013) that showed little polymorphism for INRA63 marker in Brazilian and Venezuelan goats; also because of the lack of alleles of this locus has in the Mahabubnagar breed form India, Raghavendra (2016) propose eliminating it from goat diversity studies. These results suggest that those markers can be excluded in future diversity studies in Landim goat, in turn that the other 29 used to new studies of the breed, included those of the genetic distance between populations.
The microsatellites MM12, CCSM66 and HSC were the one with the higher number of alleles with 11, 12 and 13, respectively. In this same locus in the study with the Cuban creole goats there were respective values of 11, 9 and 9 alleles. Higher number were reported by Gómez (2013) in the Apurimeño goat that found 10, 18 and 13 for each of them, respectively, and higher values (19, 35 and 20) reported Murital et al. (2015) in Nigerian goats.
Heterozygosities and Polymorphic Information Content (PIC). Table 2 shows the values of the obtained He and Ho by each marker and it can see that both exceeded the 60%, with figures of 0.657 and 0.611, respectively, showing that the breed has a good diversity degree, because more than the 50 % of individuals are heterozygotes. These obtained diversity values are higher than the indicated averages for this breed by Garrine et al. (2010) which were 0.60 and 0.54 for He and Ho, respectively and for Pafuri breed, another Mozambican goat breed, the He reached 0.67 and the Ho 0.58.
In a study performed in the same laboratory were this study was carried out, and with the use of the same markers, were three Nigerian goat breeds were researched, Murital et al. (2015) obtained He of 0.67 in Maradi breed, 0.70 in the West African Dwarf breed and 0.68 in Sahel breed, all of them exceeded the Landim goat but, in the case of Ho do not occur in that way since 0.61, 0.59 and 0.60, were obtained, respectively. In the case of Saanen breed there were higher values for He and Ho (0.66 and 0.64, respectively) and more closer in Kalahari breed (0.67 and 0.63).
In American breeds as the native from the apurina region in Peru, Gómez (2013) reported higher values of d He (69 %) and Ho (66 %), respectively. Ginja et al. (2017) reported for the creole goat from Peru a value of He 0.64 similar to those founded in this study. In that environment is also the Colombian goat Santandereana for which Jiménez et al. (2014) reported 60 % of heterozygotes individuals. However, Chacón et al. (2018) founded 58 % of heterozygotes individuals in the Cuban creole goat, slightly lower to the percentage of heterozygotes of the Landim goat.
The evaluation of heterozygosities per marker showed that the He and the Ho had variation among minimum values of 0.315 and 0.113, respectively in the microsatellite ETH225 and maximum values of 0.854 and 0.844, respectively in the MM12, which show the correspondence of these values with the microsatellites that has the lower and higher number of alleles. This levels that the heterozygosities values in the Landim goat has similarity with the level 0.31 to 0.81 that Asian goats showed, according to Sulabda et al. (2012) and Hussain et al. (2013), but are lower to the levels of He (0.20 and 0.90) and Ho (0.18 and 0.93) that were obtained in the Mexican Black creole breed according to Silva et al. (2019) reported.
With the exception of the markers ETH225, MAF209 and SPS115 which has lower number of alleles, the resting has a good polymorphism, exceeding all, the 50% in both heterozygosities; which is evidence that this breed has an adequate genetic diversity.
From the 33 microsatellites analyzed, a total of 11 obtained Ho values higher to 70%, that is why they can be qualify as highly polymorphic for the breed, if it taken into account what Ott (1992) stated, that a marker has this category when exceeded the mentioned value. These markers are: BM1818, BM6526, BM8125, CRSM60, ILSTS019, OarFCB11, MM12, OarFCB20, SRCRSP05, INRA005 and INRA006. It coincide with Gómez (2013) in four microsatellites (BM1818, BM6526, CRSM60 and MM12) that reached more than 70 % of Ho in Apureña breed and with Chacón et al. (2017) in three (BM1818, MM12 and BM6526) in the Cuban creole breed.
Another indicator of interest showed in table 2 is the Polymorphic Information Content, which is used to determine the informative quality of the marker. It can be see that, from the 33 markers used in this study, a total of 25 were very informative, and only 8 (ETH225, ILSTS008, ILSTS011, MAF209, OarFCB304, SPS115, SRCRSP24 and TGLA053) did not reached such designation due they do not reached the PIC value higher to 50 %, as the international scientific community requires to identify the markers with higher informative quality. The markers CSSM66, HSC and MM12 by having the PIC above 80 % can be considered as exceptionally informative for Landim breed.
An aspect to highlight is that the values of PIC and hetrozygosities have similarity, or direct relation, the increase of one of them causes the increase of the other one. Regarding that relation, Vaiman et al. (1994) explained that the proximity between the values of both indicators show the marker quality for diversity studies in determine breeds, and it can also said that is the result of performing the correct sampling in the studied population.
Conclusions
All the microsatellites were polymorphic because the used battery is adequate to study the genetic diversity of Landim breed. It has the appropriate genetic diversity because more than the 60 % of the individuals are heterocygotes. Most of the markers were highly informative with PIC higher than 50 %, 11 of them reached the more than the 70 % of heterocygosity and PIC, and 3 exceeded 80 %, which make them exceptionally informatives for the breed. These finds should take into account to improve the national strategy of conservation and breed improvements, as well as for future molecular studies with the breed.
