| Abstract | U svijetu je grah (Phaseolus vulgaris L.) gospodarski najznačajnija i najraširenija vrsta iz
roda Phaseolus te je navažnija zrnata mahunarka za izravnu ljudsku prehranu. U Hrvatskoj
je ova kultura zapostavljena i hrvatski tradicijski kultivari graha su u opasnosti od genetske
erozije uzrokovane složenim društveno-gospodarskim promjenama u ruralnim
zajednicama. Kako bi se produbilo znanje o genetskoj i morfološkoj raznolikosti hrvatskih
tradicijskih kultivara graha, napravljena je procjena na temelju mikrosatelitnih biljega, tipa
fazeolina te morfoloških svojstava. Primke su razvrstane u 10 morfoloških skupina koje
predstavljaju najčešće tradicijske kultivare (morfotipove) u Hrvatskoj te u miješanu skupinu
manje zastupljenih morfotipova. Pomoću 26 mikrosatelitnih biljega utvrđeno je postojanje
245 jedinstvenih genotipova od ukupno 299 primki. Bayesovskom analizom populacijske
strukture na temelju mikrosatelitnih biljega utvrđeno je postojanje tri genetske skupine (A,
B1, B2) koje su se u većini slučajeva podudarale s rezultatima analize tipa fazeolina (H1,
H2, H3). Od 245 primki, 78 primki (31,84%) pripada skupini H1A (Srednjoameričko
podrijetlo), a 131 primka (53,47%) pripada skupini H2B1/H3B2 (Andsko podrijetlo), od čega
37 primki (15,10%) pripada H2B1 (Andsko podrijetlo; tip fazeolina 'H' ili 'C') te 94 primke
(38,37%) H3B2 skupini (Andsko podrijetlo, tip fazeolina 'T'). U skupinu atipičnih primki je
razvrstano ukupno 36 primki (14,69%) koje se mogu smatrati hibridnima između različitih
tipičnih skupina. Nakon usporedbe 209 tipičnih primki s tradicijskom podjelom na
morfotipove, imajući pritom na umu i habitus rasta, 192 primke su razvrstane u 14
morfogenetskih podskupina koje su razvrstane u tri morfogenetske skupine prema podrijetlu
i habitusu rasta: H1A (Srednjoamerička), H2B1 (Andska - visoki habitus rasta) i H3B2
(Andska - niski habitus rasta). Prilikom izrade sržnih kolekcija na temelju mikrosatelitnih
biljega pomoću standardne maksimizacijske (M) strategije utvrđen je podskup od 20
genotipova (Core20) koji je imao nesignifikantno manji prosječan broj alela u odnosu na
cjelokupnu kolekciju, dok je naprednom maksimizacijskom strategijom odabrano 40
genotipova (Core40) koji predstavljaju najmanji podskup primki u kojem su sadržani svi aleli
zapaženi u cjelokupnoj kolekciji. Sržne kolekcije će olakšati buduća istraživanja raznolikosti
graha upotrebom fenotipizacije visoke propusnosti kao i genotipizacije pomoću
sekvenciranja nove generacije. Time će se omogućiti učinkovitije očuvanje biljnih genetskih
izvora hrvatskih tradicijskih kultivara graha te potaknuti njihova upotreba u budućim
oplemenjivačkim programima. |
| Abstract (english) | Common bean (Phaseolus vulgaris L.) is the most economically important species of the
genus Phaseolus and the most important food legume for direct human consumption
worldwide. Before domestication wild P. vulgaris had already diverged into two major gene
pools, each with a proper geographical distribution. Domestication occurred independently
in Mesoamerica and Andean region and the two major gene pools have been defined also
within the cultivated forms. Both Mesoamerican and Andean cultivars were disseminated
all over the world and the distribution of each major gene pool varies from region to region.
In Croatia, common bean is traditional but neglected crop. Most of the common bean
production is based on local landraces, grown by small-scale farmers in low input production
systems. The long tradition of common bean cultivation in Croatia enabled the evolution of
many landraces with great morphological diversity adapted to the specific growing and
agronomic conditions and traditional agrotechnics. These traditional landraces are
commonly known by their traditional names based mostly on seed coat colour and pattern.
They are important sources of genetic resistance for plant breeders, owning considerable
genetic variation and extreme adaptation to different environmental conditions. However,
these landraces are in danger of genetic erosion caused by complex socio-economic
changes in rural communities. The major factors affecting genetic erosion are the low
profitability of farms and their small size, the advanced age of farmers, intensive agriculture
i.e. the replacement of traditional landraces with modern common bean cultivars and/or
other more profitable crops. Three hundred accessions belonging to most widely used
Croatian common bean landraces are maintained at the University of Zagreb, Faculty of
Agriculture, Department of Science and Technology as part of the project UIP-11-20133290
"Genetic basis of bioactive nutrient content in Croatian common bean landraces",
which is financed by Croatian Science Foundation. To assess the evolutionary origin,
genetic and morphological diversity and population structure of these landraces the
phaseolin genotyping, microsatellite marker analysis and morphological characterization
were evaluated. Also, the aim of these study was to construct core collections that represent
the Croatian common bean germplasm with minimum repetitiveness.
Seed samples of landraces were collected from diverse geographical regions of Croatia in
which this culture is cultivated. Common bean accessions were grown in unreplicated field
plots at the experiment field in Maksimir, Zagreb (45.8293 N, 16.0334 E) in year 2014. Data
on the morphological and agronomic traits of common bean landraces were obtained based
on eight qualitative and nine quantitative morphological characteristics based on the list of
descriptors for Phaseolus vulgaris L. DNA was isolated from 25mg of silica-gel dried leaves
using Plant DNeasy 96 kit according to the manufacturer’s instructions without any
additional clean-up. Twenty-six PCR primer pairs were used for microsatellite analysis. DNA
amplification was performed using multiplex PCR mix. Tailed PCR approach was used for
amplification of phaseolin sequences and the same two-step PCR protocol with an initial
touchdown cycle as in microsatellite analysis. Statistical data analysis was made, and
microsatellite markers were used to construct a set of core collections using two algorithms
(PowerCore and MSTRAT) based on maximization (M) strategy.
The information on phaseolin type and the results of population structure obtained by the
STRUCTURE analysis combined with the morphological characterization of the accessions
enabled the thorough classification of the accessions. These results were coinciding for the
most part. Microsatellite analysis revealed a total of 245 unique genotypes in the panel of
299 accessions. Accessions were classified into 10 groups representing the morphotypes
(landraces) most frequently found in Croatia and in a mixed group of less common
morphotypes. STRUCTURE analysis based on microsatellite markers revealed the
presence of two clusters at K = 2. Out of 245 accessions, 31.84% were of Mesoamerican
origin, 53.47% of Andean. Accession of Andean origin were further classified into two
clusters at K = 3 in congruence with the results of phaseolin type genotyping. Accessions
having the membership probabilities Q < 75% for all clusters at K = 3 and accessions that
did not show the correspondence between landrace/phaseolin type affiliation and cluster
membership could be classified as ‘offtype’ accessions (14.69%). These accessions could
be treated as putative hybrids between different gene pools. From these accessions 2.86%
of accessions represented putative hybrids between gene pools from the two original
centers of domestication in the Andes and Mesoamerica. Accessions of Mesoamerican
origin were further classified into phaseolin type H1 (‘S’) and accession of Andean origin
into phaseolin type H2 (‘H’ or ‘C’) and H3 (‘T’). It is determined that the 'T' type of phaseolin
prevails in Croatia (41.63%) over other phaseolin types (33.06% 'S' and 25.31% 'H' or 'C'
phaseolin type).
The results of the analysis of Croatian common bean landraces based on nine quantitative
traits were consistent with the results obtained by the Neighbor-joining tree and
STRUCTURE analysis, respectively, confirming the existence of three different genetic
groups. The biplot projecting the Croatian common bean accessions and the nine
quantitative morphological traits was constructed using the first four principal components
explaining 91.85% of the variance found among accessions. PC1 differentiates primarily
H2B1 (andean origin, phaseolin type 'H' or 'C') from H3B2 (andean origin, phaseolin type
'T'), with the main contributions of traits like Seed flatness and Flatness index. PC2
differentiates H1A from both H2B1 and H3B2, with the main contributions of trait Seed width
(mm). The offtype accessions were positioned among the true-types without any clear
distribution pattern. The stepwise discriminant analysis revealed that No. of days to
flowering, Duration of flowering, Seed elongation, 100 Seed weight (g), Flatness index,
Seed length (mm) and Seed height (mm) were the most useful traits in discriminating
accessions among the three true-types group (H1A, H2B1 and H3B2). These traits were
tested for their performance, and after Cross-validation analysis 94.40% of the accessions
were correctly classified into their respective true-type groups.
Out of 245 accessions, 192 were assigned to 14 newly formed morpho-genetic groups
originating form three distinct domestication events: H1A Mesoamerican, H2B1 Andean -
indeterminate types, and H3B2 Andean - determinate types. The rest of accessions could
represent putative hybrids among morpho-genetic groups including the hybrids among the
known gene pools.
The core collection obtained by the PowerCore was composed of 40 genotypes (Core40),
representing the smallest set of accessions with the full coverage of the alleles present in
the entire collection. By maximizing genetic diversity in a reduced set of genotypes, from
the entire collection, the subset of 20 accessions (Core20) were selected to form a core
collection that retains all the diversity within this germplasm, with minimum repetitiveness.
This core subset could facilitate future efforts in assessing common bean diversity by highthroughput
phenotyping and genotyping using next-generation sequencing approaches.
Furthermore, putative hybrids constitute representing a valuable resource for expanding the
genetic diversity that could be used in future breeding programmes. The detection of
accessions that were hybrids between gene pools implied that Croatia could be considered
as a secondary center of common bean diversity. |
