Revised descriptor list for the evaluation of genetic resources of European chestnut (Castanea sativa Mill.)
Milan Bolvansky1and L’ubomír Mendel2
1 Institute of Forest Ecology, Slovak Academy of Sciences, Branch of Woody Plant Biology, Akademická 2, SK-949 01 Nitra, Slovak Republic
nruebolv@savba.savba.sk
2 EBA Ltd., Mileticova 23, SK-82456 Bratislava, Slovak Republic
Abstract
Thirty-six morphological traits in a sample of 114 mature seedlings derived from intraspecific and interspecific crosses of European chestnut (Castanea sativa Mill.) were studied using analysis of variance, principal component and cluster analyses. The traits evaluated were selected on the basis of a preliminary study of the variability in half-sib and full-sib chestnut progenies and come from various official and unofficial descriptors used for the characterization of chestnut cultivars and
“wild” chestnut types. Thirty morphological and phenological descriptors with their priority status (I – III) were determined using discriminatory value of characteristics and their importance for fruit production. The naming of descriptors was clarified and the descriptor list modified to pro- vide a reduced, partially extended and complete list.
Keywords: seedlings, descriptors, morphometric study, priority, Castanea sativa
1 Introduction
The official descriptor list and guidelines for distinctness and characterization of cultivars of the European chestnut (Castanea sativa) and their interspecific hybrids was published by the International Union for the Protection of New Varieties of Plants (UPOV 1989). Another official descriptor list for chestnut varieties was distributed by FAO, Interregional Cooperative Research Network on Nuts – Chestnut Subnetwork (FAO 1997). It consists of nine descriptors, whereas the UPOV descriptor list contains 32 descriptors. However, a descriptor list for the evaluation of European chestnut which would also be suitable for wild chestnut types has not been published so far.
The characterization of registered chestnut cultivars in Italy and France generally involves fewer descriptors than the official 32, including some which do not occur in official descriptor lists (BERGOUGNOUXet al.1978, BOUNOUSet al. 1988, BREISCH1995). Descrip - tors used include tree characteristics (growth habit, vigour, flowering type or stamen type), fruit characteristics (shape, colour, double seed or polyspermia, period of ripening and in Italian descriptors also apex, strips and hymen) and utilisation (mechanical handling, candy- ing, confectionary). In countries with limited chestnut production or a limited number of registered cultivars, chestnut descriptors are used mostly in the selection of local chestnuts and sets of descriptors may differ. Although all usually include descriptors referring to yield and nut characteristics, the scoring of individual descriptors is either similar to the UPOV Guidelines (SOLARet al.1998; PODJAVORSEKet al. 1999) or to the weighted-ranked method, considering various quality groups – general quality, nut size, earliness, chestnut paste (SERDAR1999).
Key descriptors have been identified to accelerate and simplify the characterization of cultivars and/or accessions. The UPOV descriptor list (UPOV 1989) recommends six charac- teristics for subgrouping varieties, while the descriptor list for evaluating the genetic re -
sources of Japanese chestnut (KOTOBUKI1996) uses a twofold priority scale. Characteristics (descriptors) are ranked as being primary, secondary and tertiary according to biological and physiological criteria and then each of the three groups are divided according to whether they are essential and optional.
In English, a descriptor (and its various stages) can frequently have different names and synonyms. When comparing six different descriptor lists (BOLVANSKYand MENDEL 1999, BOUNOUSet al. 1988, FAO 1997, KOTOBUKI1996, SERDAR1999, UPOV 1989), ten descrip- tors were found to have between two to six different names. For instance, one had the fol- lowing synonyms: penetration of seed coat into embryo, penetration of pellicle into kernel, subsidence of pellicle into kernel, pellicle intrusions, nut wrinkles and testa entering the seed.
The objectives of this study were to compile a set of morphological descriptors of chest- nut which would: a) be eligible not only for classification and distinctness of cultivars and land races, but also for the description of chestnut types in pre-selection work, b) be easily recognisable and measurable, c) be highly discriminating , d) have clearly defined categories (stages), e) have exact instructions for data sampling, f) have the correct English name.
2 Material and methods
The testing of existing descriptors and selection of new descriptors for chestnut genetic resources was carried out on mature seedlings (17 years of age in 1999) growing on a permanent experimen- tal plot in the Arborétum Mlynany, Slovak Republic. The seedlings were derived from both open and controlled intraspecific (Castanea sativa x C. sativa)and interspecific (C. sativa x C. crenata) pollinations on selected old trees from several sites in Slovak Republic and two trees of C. sativa and C. crenata grown in Pontevedra, Spain. Parental trees were selected with contrasting morpho- logical traits (fruit size and shape, type of male flowers, fertility).
In 1994, preliminary observations of morphological variability in 262 mature seedlings, belong- ing to 18 full-sib and 19 half-sib progenies, revealed high variability in several important charac- teristics, including growth habit, phenology, type of male flowers, bur set and fruit size.
Consequently, 114 seedlings with the best fruit yields and broad morphological variability were selected for morphometrical studies over the next three years. Of the 36 characteristics evaluated, ten related to the tree, five to the vegetative parts, three to the male flowers, twelve to the fruit and five to the bur. Most were congruent with descriptors occurring in official descriptor lists (UPOV 1989, KOTOBUKI1996). Four were new and were selected during the preliminary investi- gation. These were proportion of fruits with three and more seeds, mean number of pellicle in - trusions, mean length of pellicle intrusions, and penetration index.
For each of the 114 seedlings, phenological and morphological characteristics were observed, from bud burst to fruit ripening. Some characteristics, especially those which are consistent over time (growth habit, vigour, time of bud break, time of male and female flowering, stamen type, glossiness and colour of fruit, seed coat peeling, sweetness of kernel and six bur characteristics), were observed only in one or two years. All these characteristics were evaluated visually using a three- or a five-degree scale as appropriate (3, 5, 7 or 1, 3, 5, 7, 9 in Table 1). Fruit quantitative characteristics were measured over three or four years. For 30 fruits per seedling, the following measurements were taken: Weight, height, width, thickness, length of hilum, width of hilum, num- ber of seeds per nut, number of pellicle penetrations and length of pellicle penetrations. Using some of these measurements three further characteristics were calculated: Fruit shape (width/
height), index of pellicle penetration and size of hilum.
The basic values for 10 fruit quantitative characteristics were evaluated by multi-way analysis of variance. Mean values of 18 characteristics were analysed by principal component analysis (PCA). In order to assess the discriminative function of individual characteristics, mean values of scores for 23 characteristics were taken.
Table 1. List of descriptors for European chestnut (Castanea sativa)with priority indication. Bold type indicates highest priority (I). NoName of descriptorStates (scores)Priority 1Tree: growth habiterect, semi-erect, spreading (3, 5, 7)II 2Tree: vigourlow, medium, high (3, 5, 7)III 3Tree: time of bud breakvery early, early, medium, late, very late (1, 3, 5, 7, 9)I 4Tree: time of start of male floweringvery early, early, medium, late, very late (1, 3, 5, 7, 9)I 5Tree: time of start of female floweringvery early, early, medium, late, very late (1, 3, 5, 7, 9)II 6Tree: time of start of fruit ripeningvery early, early, medium, late, very late (1, 3, 5, 7, 9)I 7Tree: ratio of multiple-seed fruits [%]none, low, medium, high, very high (1, 3, 5, 7, 9)I 8Tree: ratio of fruits with pellicle penetration [%]none, low, medium, high, very high (1, 3, 5, 7, 9)I 9Tree: yield of burslow, medium, high (3, 5, 7)II 10Annual shoot: length [mm]short, medium, long (3, 5, 7) III 11Annual shoot: thickness [mm]thin, medium, thick (3, 5, 7)III 12Fully developed leaf: size [cm2]small, medium, large (3, 5, 7)III 13Fully developed leaf: length/width ratiolow, medium, high (3, 5, 7)II 14Fully developed leaf: length of petiole [mm]short, medium, long (3, 5, 7)III 15Male flower: length of filament (stamen type) [mm]none, short, medium, long, very long (0, 1, 3, 5, 7)I 16Unisexual catkin: length [mm]short, medium, long (3, 5, 7)III 17Fruit: extent of pellicle penetrationvery low, low, medium, high, very high (1, 3, 5, 7, 9)I 18Fruit: size [g]very small, small, medium, medium large, large (1, 3, 5, 7, 9)I 19Fruit: shapetall triangular, ovoid, triangular, round, broad triangular, transverse ellipsoid, transverse broad ellipsoid (1, 2, 3, 4, 5, 6, 7)II 20Fruit: size of hilumsmall, medium, large (3, 5, 7)III 21Fruit: glossinessabsent, partial, high (3, 5, 7)II 22Fruit: colourlight brown, brown, dark brown, reddish brown and blackish brown (1, 2, 3, 4, 5 ) II 23Seed: seed coat peelingeasy, intermediate, difficult (3, 5, 7)II 24Seed: sweetness of kernellow, medium, high (3, 5, 7)II 25Bur: sizesmall, medium, large (3, 5, 7)III 26Bur: length of spines [mm]short, medium, long (3, 5, 7)II 27Bur: density of spineslow, medium, high (3, 5, 7)II 28Bur: length of peduncle [mm]short, medium, long (3, 5, 7)III 29Bur: cracking at the ripening timeabsent, partial, complete (3, 5, 7)II 30Bur: fertility (ratio of filled nuts) [%]low, medium, high (3, 5, 7)II
3 Results
The analysis of variance for ten fruit characteristics indicated highly significant effects (P <0.001) for all the assumed sources of variation (years, female parents, male parents, inter action of both parents and seedlings within families) on all characteristics. The calculated F-value for the seedlings within families varied from 13.1 for the number of seeds per nut to 60.3 for the fruit height.
When 18 variables were integrated by PCA, four principal components (PCs) with Eigen values higher than 1 were detected. These PCs explained 73.7% of the variability observed.
Following the correlations between original variables and four PCs, those exceeding 0.7 were assumed to be the most important in explaining the contribution of original variables integrated by individual PCs. Thus for PC1, which accounted for 47.2% of the total variation, the proportion of nuts with pellicle intrusions, length of filament, number of pellicle pen - etrations, length of pellicle penetrations, index of pellicle penetration and nut size, were shown to be the most important. For PC2, the proportion of fruits composed of three and more seeds, and for PC3, annual shoot thickness, length/width ratio of leaf blade, and length of leaf petiole were of the greatest importance.
By means of canonical discriminant analysis, the separation of seedlings derived from seven different female parents was tested. Five canonical axes (CAN) were shown to explain total variability among tested groups. Total canonical structure was calculated for the first three canonical axes. Correlation coefficients between original variables and individual canonical axes higher than 0.7 indicated, for CAN1, that the beginning of flowering, the beginning of fruit ripening, annual shoot length, the size of hilum, bur size, and the density of bur spines were the most important. For CAN2, however, the beginning of leaf bud burst, the pro portion of multiple-seed fruits, seed coat peeling, and the length of bur spines were the most important.
The results of the principal component analysis and discriminate analysis were used to reduce the original 36 characteristics to 30 by excluding and combining some fruit character- istics (the number and length of pellicle penetrations were combined to extent of pellicle penetrations; contrast of hilum to pericarp, pericarp stripes, fruit thickness, proportion of fruits with more than three seeds, thickness of leaf petiole were excluded). The results were also used to assign priority stages I, II, or III to the selected descriptors (Table 1). Priority I was given to the descriptors congruent or derived from the variables most important for PC1 as well as for some of CAN1 and CAN2 from the discriminant analysis, i.e. the pro - portion of fruit with pellicle penetration, index of pellicle penetration, nut size, length of filament, beginning of male flowering, beginning of fruit ripening, beginning of leaf bud burst and proportion of multiple-seed fruit. Time of bud break is also a priority I character - istic. Annual shoot length, size of hilum, bur size, density of bur spines, seed coat peeling, and length of bur spines were assigned a lower priority even though they have a high discriminant value.
4 Discussion
This study has shown that most of the characteristics used in describing large-fruited Mediterranean cultivars and cultivars of Japanese chestnut are also suitable for character - ising chestnut types grown at the limit distribution area for chestnut in Europe. The morpho-
logical variation observed in the progenies derived from crossing morphologically contrasting chestnut types is very similar to that observed among chestnut cultivars belonging to C. crenata, C. sativaor their hybrids grown under optimal cultivation conditions. For instance, the morphological variability of north-western Spanish chestnut cultivars showed statistically significant variation in the morphological characteristics of fruit among cultivars, with the exception of total weight of lateral nuts (PEREIRAet al.1996a). In our work, the character - istics with the higher discriminative value were found to be only partially congruent with those reported by PEREIRAet al. (1996a), where nut size, nut shape, male flowering and bur-spine length were determined as the most discriminatory characteristics. One reason for this may be the different material used in the study. For instance, while in Spanish cultivars nut size did not differ significantly, in our study nut size varied in chestnut seedlings from 4 to 10 grammes. Also while seed coat penetration is very rare in Spanish, and also in Italian or French cultivars, it is typical in chestnuts grown in Slovak Republic. In Slovak Republic vari- ation in fruit size and other quantitative morphological traits are markedly influenced by season, thus requiring evaluation over several consecutive years.
Following the results of this study and current official and unofficial descriptors lists, priority I descriptors should be used for the primary characterization and sub-grouping of chestnuts from wild populations and local cultivars and land races. Priority II descriptors also refer directly or indirectly to the quantity and the quality of nut production, and descriptors with priority III mostly belong to vegetative parts of tree. Some of the priority III descriptors are of high descriptive value (shoot thickness, length/width leaf ratio), but they are less important for nut production (Table 1).
Except for seven rather qualitative descriptors (No. 1, 2, 21, 22, 23, 24 and 29), all the remaining descriptors can be considered for quantitative traits. This also holds for pheno - logical characteristics, which can be quantified according to the number of days from the beginning of the year (Julian days). So in addition to the scores, it is recommended that the observed values for descriptors to characterize particular chestnut types and/or cultivars should be used. In all the descriptors except one, the three- and five-category scales are used for ranking values.
In recent years, increasing effort has been directed towards the molecular identification of chestnut clones and cultivars using both isoenzymes (PEREIRAet al.1996b) and DNA techniques (SEABRAet al.2000, BOTTAet al.2001). The use of morphological descriptors for cultivar identification is limited due to time constraints and the influence of environmental effects. As molecular markers are suitable for rapid cultivar identification and are most likely independent from environmental effects, they should be accepted as an official method for registering and protecting new cultivars in the future.
It has not been possible, due to space limitations, to present detailed explanations and methods for data sampling particular descriptors. Complete “Guidelines” will soon be avail- able on the internet. The web-site has not yet been selected but please contact the corre- sponding author for information.
Acknowledgments
This study was undertaken within the framework of the COST Action G4 “Multidisciplinary Chestnut Research”. The authors wish to thank Prof. Giancarlo Bounous, Dr. Anita Solar and Dr.
Karen Russell for their valuable advice.
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Accepted 29.1.02
