Origin and diversity of Croatian common bean (Phaseolus vulgaris L.) landraces

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.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