Functional analysis of metagenomes and routine identification of plant polymers during fermentation in the rumen of free-living ruminants

Rastline, s katerimi se prehranjujejo prežvekovalci, ki naseljujejo visokogorska področja, imajo izjemno visoko vsebnost lignina, kar predstavlja dodaten izziv za prebavno mikrobioto. Prostoživeči prežvekovalci pa so kljub temu eni izmed najuspešnejših skupin rastlinojedih sesalcev na svetu. Zaradi števila rastlin, ki jih imajo na voljo, je njihova prehrana zelo slabo raziskana. Namen magistrskega dela je bil proučiti prehrano košute, srne, jelena in gamsa, ki živijo na področju LPNKK, z uporabo enostavnih in zelo poceni metod ter vzpostaviti podatkovno zbirko in platformo za rutinsko analizo vzorcev vampne vsebine in fecesa. Po končani ekstrakciji DNA in verižni reakciji s polimerazo regije ITS smo dobljenim DNA vzorcem ugotavljali dolžino s kapilarno elektroforezo. Na koncu smo izdelali podatkovno bazo z 79 rastlinskimi vrstami ter z uporabo konzolnega orodja PlantID uspešno identificirali rastline znotraj vzorcev vampne vsebine in fecesov. Ugotovili smo, da so posamezne živali zaužile različne rastline in da se njihova prehrana razlikuje. Hkrati smo primerjali metagenome ovce, goveda in krave na ravni vseh genov ter na ravni genov, ki so vključeni v metabolizem ogljikovih hidratov, saj se znotraj teh genov nahajajo funkcionalni geni za razgradnjo rastlinskih polisaharidov. Eksperimentalni parametri, ki so imeli največji vpliv na porazdeljenost teh podatkov so bili študija, metoda sekvenciranja, vrsta gostiteljske živali in eksperimentalne ponovitve.The wild ruminants which live in the high mountains feed on plants that have a very high lignin content. This represents an additional challenge for the intestinal microbiota. However, these wild ruminants are one of the most efficient animals in their digestion of this kind of plants. Due to the fact that they have basically an unlimited number of different plant species on which they can feed, their diet is poorly understood. The purpose of this master\u27s thesis was to determine the doe\u27s, deer\u27s, roe deer\u27s and chamois\u27 diet, all of which live in the State hunting reserve with the special purpose Kozorog Kamnik. By using very simple and inexpensive methods we managed to establish a database consisting of 79 plant species and a platform for the routine analysis of ruminal and fecal samples. With the use of the PlantID platform, we have successfully identified the plant species found in the ruminal and fecal samples. Furthermore, we have made a comparison of the sheep\u27s, bovine\u27s and cow\u27s metagenomes at the level of all genes and at the level of only those genes which are involved in the break down of carbohydrates. Additionally, we have identified the experimental parameters that had the greatest impact on the dispersion of these data and they were: the type of study, sequencing method, the type of the animal host and experimental replicates, respectively