Univerza na Primorskem Fakulteta za matematiko, naravoslovje in informacijske tehnologije
Več informacij o projektu / More info about the project
Vsebina projekta / Project content
Izhodišče: Kmetijska biotehnologija združuje znanstvene tehnike in orodja, ki pomagajo v
procesih izboljšanja genetskega materiala različnih organizmov. V rastlinski biotehnologiji
imajo zaradi izjemne aplikativne uporabe prav posebno mesto molekulski markerji DNK.
Merkerji DNK so postali nepogrešljivo orodje pri identifikaciji rastlin, žlahtnjenju, selekcijigenetskega materiala, kartiranju, itd. Med razpoložljivimi markerskimi sistemi, so
mikrosateliti izjemno uporabni zaradi visokega polimorfizma in kodominantnosti. V projektu
nameravamo orodja kmetijske biotehnologije uporabiti za dva konkretna aktualna primera
sredozemskega kmetijstva v Sloveniji in na Hrvaškem: (1) genotipizacija in identifikacija
jadranskih sort in ekotipov granatnega jabolka, (2) izvedba starševskih testov vodilnih
jadranskih sort oljk in identifikacija najpogostejših donorjev peloda. Genotipizacija
jadranskih sort granatnega jabolka z mikrosatelitskimi markerji: granatno jabolko pripada
družin granatovk Lythraceae. Rod Punica pa vključuje dve vrsti: P. granatum L. in P.
protopunica Balf. Nativno območje gojenja vrste P. granatum se nahaja med Iranom in
Himalajo na severu Indije. Granatno jabolko je prilagojeno na različna podnebja in tipe tal,
zato danes uspeva v številnih različnih geografskih območjih, kamor sodi Sredozemlje, Azija
in Kalifornija. V Sloveniji so nasadi granatnega jabolka izjemno redki, najdemo le
posamezna dreves, ki so pogosta zastopana na domačih vrtovih in v manjših sadovnjakih,
skupaj z oljkami, figami in drugimi sredozemskimi rastlinami. Med tem ko se na Hrvaškem
granatno jabolko tradicionalno goji v manjših nasadih, poleg tega pa tudi na domačih
vrtovih. Granatno jabolko odlikuje visoka vsebnost hranil, zato se proizvodnja granatnega
jabolka hitro povečuje. Granatno jabolko je tudi bogat vir biološko aktivnih molekul in
mineralov, ki lahko varujejo zdravje človeka. Dejstvo, da je granatno jabolko funkcionalno
živilo in potrebe po izboljšanju programov žlahtnjenja so spodbudili genetske raziskave
granatnega jabolka. Čeprav je granatno jabolko na območju Sredozemlja zapostavljena
sadna vrsta, so različne raziskovalne skupine po svetu razvile nove in zelo informativne
mikrosatelitske markerje (Curro in sod., 2010; Pirseyedi in sod., 2010; Ebrahimi in sod.,
2010; Hasnaoui in sod., 2010, 2012; Soriano in sod. 2011), s katerimi se je uspešno
genotipiziralo genske vire granatnega jabolka v mnogih državah. Z genotipizacijo slovenskih
in hrvaških granatnih jabolk bo tako mogoče analizirati genetsko raznolikost in razlikovati
med različnimi sortami in ekotipi. Poleg tega se bo v obeh državah vzpostavilo enoten
protokol za identifikacijo sort te sadne vrste. Poenoten protokol bo v kolekcijskih nasadih
olajšal identifikacijo pomanjkljivosti in hkrati predstavlja velik potencial za prihodnji razvoj
granatnega jabolka. Starševski test vodilnih oljčnih sort na območju Sredozemlja: čeprav je
večina oljčnih sort delno samooplodnih, sta uspešnost oploditve in posledično končni
pridelek bistveno večja v primeru tujega opraševanja. V zadnjih dvajsetih letih narašča
trend postavljanja novih oljčnih nasadov, katerih genetska struktura se spreminja, zaradi
vnosa tujih sort in širjenja domačih sort na nova pridelovalna območja. Pri postavljanju
novih nasadov je izrednega pomena izbor sort, ki vpliva na uspešnost oploditve in
posledično na končni pridelek, zato je pridelovalcem potrebno zagotoviti nove informacije o
reproduktivni kompatibilnosti sort v novih okoljskih razmerah. Predlagan projekt bi omogočil
proučevanje reproduktivne kompatibilnosti vodilnih oljčnih sort z molekularnimi metodami,
mikrosatelitskimi markerji in starševskim testom oljčnih embrijev. Za najprimernejše
opraševalne sorte se bo spremljalo tudi interakcijo pelod-brazda po izvedbi kontroliranega
opraševanja. Faze raziskovanja predlaganega projekta: Tema 1: Genotipizacija jadranskih
sort granatnega jabolka z mikrosatelitskimi markerji 1. Vzpostavitev popisa genotipov
granatnega jabolka v Sloveniji in na Hrvaškem 2. Identifikacija najbolj informativnih
mikrosatelitskih molekulskih markerjev 3. Celovita molekulska analiza: proučevanje
raznolikost in primerjava genotipov granatnega jabolka iz hrvaškega kolekcijskega nasada
in iz Slovenije Tema 2: Starševski test vodilnih oljčnih sort na območju Sredozemlja 1. Izbor
mešanih nasadov z najznačilnejšimi vodilnimi oljčnimi sortami 2. Genotipizacija potencialnih
donorjev peloda (opraševalnih sort) 3. Izolacija DNA iz oljčnih embrijev (semena) in izvedba
starševskega testa z mikrosatelitskimi markerji 4. Analiza rasti in razvoja pelodnega mešička
v in vivo pogojih po izvedbi kontroliranega opraševanja Vzorčenje rastlinskega materiala in
izolacija DNA bo opravljena s strani obeh partnerjev. Glede na razpoložljivo laboratorijsko
opremo bodo molekulske analize izveden na UP FAMNIT, proučevanje rasti pelodnega
mešička na Inštitutu za jadranske kulture in melioracijo Krasa. Cilji sodelovanja: –
vzpostavitev enotne metodologije in protokolov za raziskave genoma granatnega jabolka –
podatkovna baza genotipov granatnega jabolka, primerljiva med obema laboratorijema –
identifikacija najprimernejših donorjev peloda vodilnih oljčnih sort – skupne znanstvene
objave Cilj predlaganega sodelovanja je prav tako organizacija izobraževalnih aktivnosti za
raziskovalce, z namenom prenosa znanja o genomskih študijah med partnerji in
spoznavanja novih pristopov molekularne genetike. Dodana vrednost projekta: dodano
vrednost raziskovalnega projekta predstavlja možnost izmenjave genskih virov
sredozemskih sadnih vrst, izmenjava znanja o molekularnih tehnikah in o njihovi uporabi ter
izmenjava rastlinskega materiala. Predlagan raziskovalni projekt bo okrepil mednarodno
sodelovanje med raziskovalnima skupinama s skupnim raziskovalnim interesom, ki zadeva
sredozemske rastline, kot so granatna jabolka, fige in oljke.
Background: Agricultural biotechnology includes scientific techniques and tools that can
contribute to the improvement of the genetic material of various organisms. Molecular
markers have a special place in plant biotechnology due to their exceptional applicability.
DNA-based molecular markers became an indispensable tool for plant identification,
breeding programs, genomic selection, gene mapping, etc. Microsatellites are extremely
useful in comparison with other marker systems due to their high polymorphism and codominance.
In the project, we intend to use agricultural biotechnology tools for two
concrete actual cases in the Mediterranean agriculture in Slovenia and Croatia: (1)
genotyping and identification Adriatic pomegranate varieties and ecotypes, (2) paternity
tests of leading Adriatic olive varieties and identification of the most probable pollen donors.
Genotyping Adriatic pomegranate with microsatellites: Pomegranates (Punica granatum L.)
belong to the Lythraceae family. The Punica genus includes only two species: P. granatum L.
and P. protopunica Balf, with P. granatum believed to be autochthonous to an area between
Iran and the Himalayas in northern India. Due to its adaptability to a wide range of climate
and soil conditions, pomegranate is now grown in many different geographical regions
including the Mediterranean basin, Asia and California. In Slovenia, pomegranate orchards
are rare, although individual trees are frequently planted in home and market gardens
along with olives, figs, and other Mediterranean plants. On the other hand, in Croatia
pomegranate has been traditionally grown in smaller orchards and home gardens.
Production is rapidly increasing with the growing popularity of pomegranate as a highly
nutritional fruit. Pomegranate fruit represents a rich source of bioactive components and
minerals, which can have protective influences on human health. The functional properties of
this fruit and the need for breeding programs for pomegranates have encouraged genetic
investigations. Although the pomegranate is considered as an underutilized fruit species in
Adriatic region, new and highly informative microsatellite molecular markers have been
developed by researchers around the world (Curro et al., 2010; Pirseyedi et al., 2010;
Ebrahimi et al., 2010; Hasnaoui et al., 2010, 2012; Soriano et al. 2011), which have been
successfully used for genotyping pomegranate genetic resources in many countries.
Therefore, genotyping of Slovenian and Croatian pomegranates will allow analysis of
genetic diversity and differentiation of varieties and ecotypes, and establishment of unified
protocols for variety identification of this fruit species in both countries. This will facilitate
identifying deficiencies in the collection and the potential for future development of the
pomegranate crop. Paternity tests of leading Adriatic olive varieties: although the most of
olive varieties are partially self-incompatible, successful fertilization and fruit set are
significantly higher under cross-pollination. The area under olive cultivation has been
continuously growing in the last two decades and the genetic structure of olive orchards
has been significantly changed due to introduction of foreign varieties and planting
traditional varieties to new growing areas. The selection of appropriate varieties is
extremely important at the establishment of new plantations in order to guarantee good
fertilization, and consequently the productivity of olive orchards. Therefore, it is necessary to
provide new information for growers about reproductive compatibility of varieties in new
environmental conditions. The proposed project would allow the study of reproductive
compatibility of leading olive varieties with molecular methods, including microsatellite
markers and paternity test of olive embryos. For the most appropriate pollen donors, pollenstigma
interaction after control pollination will be studied. Research phases of the proposed
project are: Topic 1: Genotyping Adriatic pomegranate with microsatellites 1. Creating an
inventory of the genotypes of pomegranates in Slovenia and Croatia 2. Identification of the
most informative microsatellite molecular markers 3. Complete molecular analyses: diversity
study and comparison of pomegranate genotypes from Croatian collection and Slovenia
Topic 2: Paternity tests in leading Adriatic olive varieties 1. Selection of multivarietal olive
orchards, including the most characteristic leading olive varieties 2. Genotyping of potential
pollen donors 3. DNA isolation from olive embryos (seeds) and paternity analysis with
microsatellite markers 4. Analyses of pollen tube growth and development (in vivo) after
control pollination The sampling and DNA extraction of plant material will be performed by
both partners. According to available laboratory equipment, molecular analysis will be
performed at UP FAMNIT and pollen tube growth studies at Institute for Adriatic Crops and
Karst Reclamation. Aims of the collaboration: – established uniform methodology and
protocols for pomegranate genome investigations – comparable genotype DNA profiling data
for pomegranate between both laboratories – identified adequate pollen donors for leading
olive varieties – mutual research papers The aim of this collaboration is also to organize
training activities for consortium researchers in order to transfer knowledge on genomic
investigation between partners and facilitate the acquisition of new molecular genetics
approaches. Added value of the project: The added value of the project is the possibility to
share the genetic resources of the Mediterranean fruit species, to exchange the knowledge
and use of molecular techniques and exchange of plant material. The proposed research
project will contribute to the closer international cooperation between both institutions with
common research interests concerning Mediterranean plants such as pomegranates, figs
and olives.