PROCEEDINGS OF THE LATVIAN ACADEMY OF SCIENCES. Section B, Vol. 73 (2019), No. 6 (723), pp. 487–493. DOI: 10.2478/prolas-2019-0075 GENETIC AND AGRONOMIC ANALYSIS OF LATVIAN FESCUE (FESTUCA SPP.), RYEGRASS (LOLIUM SPP.) ACCESSIONS AND THEIR HYBRIDS Pçteris Bçrziòð 1, Dainis Edgars Ruòìis2,#, Sarmîte Rancâne1, Vija Stesele1, Ivo Vçzis1, and Aldis Jansons1 1 Institute of Agriculture, Latvia University of Life Sciences and Technologies, Selekcija, Skrîveru novads, LV-5125, LATVIA 2 Latvian State Forest Research Institute “Silava”, 111 Rîgas Str., Salaspils, LV-2169, LATVIA # Corresponding author, [email protected] Contributed by Dainis Edgars Ruòìis The development of ecologically adaptable fodder crop varieties is of increasing importance, par- ticularly in the context of climate change. New varieties should be phenotypically and ecologically plastic and able to adapt to differing climactic and soil conditions, ensuring high yields and persis- tence. Combining Festuca and Lolium species and the development of hybrid (Festulolium) culti- vars can be a promising method of combining high yield, high feed quality, persistence, as well as cold, frost and drought tolerance. Breeders at the Institute of Agriculture of Latvia University of Life Sciences and Technologies have been utilizing Festulolium germplasm for several decades. Currently, in cooperation with the molecular genetics laboratory and Latvian gene bank at the Lat- vian State Forest Research Institute “Silava”, analysis of Festuca, Lolium and their hybrids with DNA markers has been initiated, in order to gain additional knowledge about the breeding mate- rial and to increase the efficiency of the breeding process. Results of the assessment of morpho- logical and agronomic traits in long-term field trials are combined with DNA markers analyses in order to determine the correlation of genetic and phenotypic traits. Key words: Festulolium, fodder crops, hybridization, SSR markers. INTRODUCTION winter hardiness and persistence (Bçrziòð et al., 2018a; Le- meþienë et al., 2004). Therefore, combining Festuca and The development of ecologically adaptable fodder crop va- Lolium species and the development of hybrid (Festulo- rieties is of increasing importance, particularly in the con- lium) cultivars can be a promising method of combining text of climate change. New varieties should be phenotypi- these desired agronomic properties. Festulolium hybrids cally and ecologically plastic and able to adapt to differing have high combining ability and recombination potential climactic and soil conditions, ensuring high yields and per- (Thomas and Humphreys, 1991; Casler et al., 2002), which sistence. In northern Europe, climate change models predict can be utilised for breeding, in order to combine the high a decrease of summer precipitation, increased autumn pre- yield and fodder quality of Lolium species (Wilkins and cipitation, and the increased incidence of freezing damages Humphreys, 2003) with the stress tolerance of Festuca spe- in winter (Becker et al., 2018). This means that breeders cies (Thomas et al., 2003; Berzins et al., 2015). Tall fescue must combine high yield, high feed quality, persistence, as (F. arundinacea) has high persistence (Berzins et al., well as cold, frost and drought tolerance. Fodder grass pro- 2018b), and is tolerant to both drought and waterlogging ductivity and persistence is mainly determined by the ge- (Cougnon et al., 2013). Therefore, in the context of climate netic potential of species and varieties (Moser et al., 1996). change, Festulolium cultivars may be of interest to forage One of the most widely utilised fodder grass species in tem- grass breeders due to their high regrowth capacity and nutri- perate climates is perennial ryegrass (Lolium perenne), tive value (Ostrem et al., 2013b). Breeders at the Institute of which has fast growth, high yields and high nutritive value. Agriculture of Latvia University of Life Sciences and Tech- L. perenne is particularly suited to clay soils and moist, nologies have been utilising Festulolium germplasm for coastal climates. In the Baltic region, L. perenne can lack several decades. Currently, in cooperation with the molecu- Proc. Latvian Acad. Sci., Section B, Vol. 73 (2019), No. 6. 487 lar genetics laboratory and Latvian gene bank at the Latvian florum, was initially registered in Latvia as a Festulolium State Forest Research Institute “Silava”, analysis of Fes- cultivar ‘Ape’, but subsequently was registered in the Euro- tuca, Lolium and their hybrids with DNA markers has been pean catalogue as the hybrid Lolium variety ‘Saikava’. In initiated, in order to gain additional knowledge about the addition, some germplasm from the gene bank was included breeding material and to increase the efficiency of the in the analysis, including some old varieties that are not cur- breeding process. Results of the assessment of morphologi- rently utilised, e.g. the F. pratensis variety ‘Priekuïu 519’. cal and agronomic traits in long-term field trials are com- In addition, some varieties were analysed, which have been bined with DNA markers analyses in order to determine the used to develop new cultivars, e.g. the L. perenne cultivar correlation of genetic and phenotypic traits. The aim of the ‘Priekuïu 59’, which was used to develop the new cultivar study was to assess the agronomic properties of Lolium, ‘Gunta’ (not included in the current analysis). Festuca and hybrid germplasm in the Latvian fodder grass breeding programme, as well as their genetic similarities in A total of 24 accessions were analysed, including Festuca, order to assess the possibilities for combining their desir- Lolium and hybrid varieties (Table 1). These accessions able properties. were selected to represent the range of species and hybrids within the Latvian fodder grass breeding programme. Sam- The use of DNA markers provides the opportunity to deter- ples of the analysed accessions were obtained from the gene mine the genetic diversity and relatedness of individuals, bank, breeding and cultivar trials, and genetic resource field populations and species. One of the fundamental parameters collections. Accessions were represented by both seed sam- of genetic diversity is allelic diversity (number of alleles). ples as well as plants collected from the field. Alleles are differing forms of one genomic location, which can be linked to functional loci and therefore be correlated To characterise the agro-morphological properties, expert with phenotypic or other morphological or biochemical assessment by grass breeders from the Institute of Agricul- variation, or they can be selectively neutral, and reflect the ture in Skrîveri was used based on long-term data and expe- neutral or background genetic variation. The allelic varia- rience. Morphological and agronomic properties were as- tion within individuals is determined by the genetic compo- sessed from accessions growing in long-term field trials and sition of the parents, mating type (outcrossing or selfing). In collections. In addition, observations were made at other diploid individuals, homozygous loci have two identical al- sites where the accessions were growing. Therefore, the leles, while heterozygous loci have two differing alleles. morphological assessments were made over several years The overall level of heterozygosity within a population is and from accessions growing in multiple locations. determined by the genetic diversity of the parents, phenol- Seeds were germinated on moistened filter paper in a ogy (the extent of flowering synchronisation) and mating growth cabinet (22 oC–20oC, 16 h light) for approxi- type. The calculation of the extent of allelic similarity be- mately two weeks. DNA was extracted from leaf tissue of tween individuals and populations determines their genetic 8–12 individuals using a CTAB-based method (Porebski et relatedness. In contrast to many field crops, forage grasses al., 1997). For samples collected from field trials, DNA was are outcrossing, and therefore have genetic diversity within extracted from a leaf sample from a single individual, using accessions, the extent of which is in part determined by the previously described method. Genotyping was done us- breeding techniques (selection intensity, isolation, choice of ing eight simple sequence repeat (SSR) markers — parent accessions etc.). This study is an initial survey of a G03_020, G05_033, G07_037, G01_053, G07_065, relatively small portion of the germplasm utilised in the G05_071, G05_088, and G05_099 (Studer et al., 2008). Latvian forage grass breeding programme, determining the Each forward primer was labelled with a different genetic diversity and relatedness, with the aim of increasing fluorophore (6-FAM, HEX or TMR) to facilitate visualisa- the efficiency of the breeding process. tion using capillary electrophoresis. The PCRs were carried out in a 10 µl solution containing 2 µL Hot FirePol Blend MATERIALS AND METHODS MasterMix with 10mM MgCl2 (Soltis BioDyne, Tartu, Es- tonia), 0.5 µl each primer (8 µM), and 2 µl DNA solution. Hybrid Festulolium cultivars and germplasm can have a The PCR cycling conditions were: 95 °C for 15 minutes, 40 wide range of agronomic properties and backgrounds, there- cycles of 95 °C for 20 seconds, 58 °C for 30 seconds, 72 °C fore, within this study, Festulolium material refers to Fes- for 45 seconds, and 72 °C for 5 minutes. The PCR products tuca and Lolium hybrids (mainly Lolium spp. × F. praten- were size separated on an ABI 3130xl Genetic Analyzer and