A method for obtaining flexible broccoli varieties for sustainable agriculture
The use of high inputs in agriculture resulted in few varieties (hybrids and pure lines) used in all agricultural systems. Also varieties of vegetables, including broccoli, for organic and lowinput agriculture, are almost exclusively hybrids, since there are very few specific breeding programs and varieties for sustainable agriculture systems. A strategy to overcome this issue is the adoption of specific breeding programs for developing heterogeneous varieties (i.e. synthetics, open pollinated
... s, open pollinated varieties, composite cross populations and mixtures). In fact, heterogeneous varieties are able to evolve and adapt to specific agro-climatic conditions. The aim of this study was to develop a method (an Evolutionary Breeding Program, EBP) for obtaining heterogeneous varieties of broccoli and test its efficiency in developing highly diverse varieties, as needed in sustainable agriculture. A synthetic variety originated from a landrace was multiplied in different environments for 3 cycles and morpho-phenological and genetic diversity of the derived populations were assessed. Results: The presented results are the first and unique indication about the efficiency of a short-time EBP for an allogamous species like broccoli. Few morphological changes were observed among varieties multiplied in different environments with different agro-climatic conditions. This could be probably due to the initial genetic diversity of the landrace from which the populations were selected and also to the great plasticity of the crop. However, SSR data highlighted a genetic differentiation among populations multiplied for two/three years across Europe and in Central Italy, that was not so evident when considering morphological data only. Conclusions: Few years of multiplication in different environments resulted in genetically differentiated broccoli populations that nonetheless preserved the original genetic diversity and productivity level and appear to evolve in relationship to different environments: the applied EBP is useful for developing heterogeneous materials for sustainable agriculture. Background 3 Brassica oleracea L. (2n=2x=18) is an economically important vegetable and its world annual commercial production (cauliflower and broccoli) is over 25 Mt, currently cultivated on about 1.4 Mha worldwide . China is the first country in producing broccoli and cauliflower (10 million tons), meanwhile, in Italy, the estimated production of both vegetables is about 390,000 tons  of which 10% is obtained in organic agriculture . Among the different B. oleracea subspecies, broccoli (B. oleracea L. var. italica, also identified as B. oleracea L. spp. capitata L. (DC.) convar. botrytis (L.) Alef. var. italica Plenck ) is an important source of vitamins, minerals and antioxidants. Currently, organic farmers largely depend on broccoli varieties bred for high external input in conventional farming systems [4, 5], and in particular, broccoli varieties are almost exclusively hybrids , due to lack of specific breeding programs and varieties for sustainable (organic and lowinput) agriculture. Suitable varieties for sustainable agriculture should have specific characteristics (as yield stability under different agro-climatic conditions, resistances to biotic and abiotic stresses, competition ability with weeds) in order to avoid the utilisation of external input like pesticides, herbicides and chemical fertilizers [5, 6]. In the context of sustainable agriculture, to allow varieties to evolve and adapt to specific agro-climatic conditions is also important . This is possible only when heterogeneous varieties (i.e. landraces, synthetics, open pollinated varieties, composite cross populations, mixtures) are used and reproduced on-farm for generations. As a consequence, breeding programs specifically adopted for sustainable agriculture should consider all the above. Among many breeding approaches, the Evolutionary plant Breeding Program (EBP), combining natural and artificial selection in target environments, is suggested to be effective in obtaining heterogeneous varieties for organic and low-input agriculture   . The success of an EBP, like that of any other breeding program, is mainly due the genetic diversity of the initial materials. For example, EBP in cereals has been based on composite cross populations (CCP) or mixtures [8, 10, 13, 14] that can be originated from old varieties or landraces. Also for allogamous species, genetically variable varieties, like open pollinated, synthetic varieties or landraces, which have advantages in terms of yield stability and resistance , have been Plant breeding procedure Aiming to develop materials suitable for organic agriculture (i.e. possessing the adaptation and the R Foundation for Statistical Computing, Vienna, Austria. Retrieved from http://www. R-project.org. 2013. 35. Husson F, Josse J, Le S, Mazet J. FactoMineR, multivariate exploratory data analysis and data mining with R. R package version 12 Retrieved from http,//CRANR project.org/package = FactoMineR. 2013. 36. Josse J, Husson F. missMDA: a package for handling missing values in multivariate data analysis. Journal of Statistical Software. 2016;70:1-31. 37. Lowe A, Moule C, Trick M, Edwards KJ. Efficient large-scale development of microsatellites for marker and mapping applications in Brassica crop species.