LITHUANIAN UNIVERSITY OF AGRICULTURE LITHUANIAN RESEARCH CENTRE FOR AGRICULTURE AND FORESTRY
Assessment of short-lived ryegrass (Lolium) genus for agro-biological traits and suitability for breeding
Summary of doctoral dissertation Biomedical sciences, Agronomy (06 B) Akademija, 2011
The doctoral dissertation was prepared during the period 2006 2010 at the Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry. Scientific supervisors: Dr. Pavelas Tarakanovas (Lithuanian institute of agriculture, biomedical sciences, agronomy 06 B), 10.2006 09.2008. Dr. Nijolė Lemeienė Research Centre for Agriculture and (Lithuanian Forestry, biomedical sciences, agronomy 06 B), 10.2008 09.2010. Scientific consultant: Dr. Juozas Kanapeckas (Lithuanian Research Centre for Agriculture and Forestry, biomedical sciences, agronomy 06 B) The dissertation will be defended at the Council of Agronomy Sciences at the Lithuanian University of Agriculture: Chairman: Prof. habil. dr. Rimantas Velička (Lithuanian university of agriculture, biomedical sciences, agronomy 06 B) Members: Dr. Rita Asakavičiūtė Research Centre for Agriculture and (Lithuanian Forestry, biomedical sciences, agronomy 06 B) Dr. Juozas Labokas (Nature Research Centre, biomedical sciences, botany 04 B) Prof. habil. dr. Izolda Paakinskienė(Vilnius University, biomedical sciences, biology 01 B) Dr. Skaidrė Supronienė (Lithuanian Research Centre for Agriculture and Forestry, biomedical sciences, agronomy 06 B) Opponents: Dr. Bronislava Butkutė (Lithuanian Research Centre for Agriculture and Forestry, biomedical sciences, agronomy 06 B) Doc. dr. Jolita Raduienė (Nature Research Centre, biomedical sciences, biology 01 B) Defence of the doctoral dissertation will take place at the public meeting of the Council of Agronomy Sciences on the 29th April, 2011 at 14.00 p.m. in room of 261, central building of the Lithuanian University of Agriculture The summary of the doctoral dissertation was distributed on the 29th of March, 2011. The doctoral dissertation is available in the libraries of the Lithuanian University of Agriculture and the Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry.
INTRODUCTION The ryegrass (Loliumspp.) genus comprises about 10 species (Terrell, 1968; Цвелев, 1976). Some species are very valuable forage grasses (L. perenneL., L. multiflorumLam.,L. x hybridumHausskn.), while others are noxious weeds of cereals (L. rigidumGaud., L. temulentumL.,L. remotumSchrank. etc.). The two most common forage ryegrass species areL. perenne andL. multiflorum (Humphreys et al., 2010). In Lithuania, the following 4 ryegrass species grow in natural habitats: perenne L., multiflorum L.subsp. italicum,L. temulentum and L. remotum(NatkevičaitėvIneiksuanaė, 1963). Italian ryegrass is biennialL. multiflorumLam. subsp.italicum annual and L. multiflorumLam. subsp. multiflorum (Reed et al., 2000). Some authors discriminate L. multiflorumsubspecies italicum or mruolfitlum(Lenuwiet und a., 2002; Humphreys et al., 2010), others indicate only speciesL. multiflorum (Lacefield et al., 2003). L. multiflorumLam. subsp.italicum rapidly, has a long growing grows season, produces high yield, exhibits good feeding value, therefore it is widely used not only for grazing but also for hay, haylage and silage production (Wilkins, 1991; Reheul et al., 2003 . This r e rass species is grown in pure stands or in mixtures with clover (Trifolium spp.L.) (Lenuweit und a., 2002). Lolium multiflorum subsp.multiflorum suited isfor growing in a pure stand or in various mixtures desi ned for reen forage. In annual mixtures, it is sown with oats (Avena sativa vetch ( L.),Vicia sativaL.) or annual clover (Trifolium spp.) (Dovydaitis, 1992; Cojocariu et al., 2008). It can also be sown together with perennial grasses. In this case, a higher, high - quality forage yield is produced already in the sowing year (Cosgrove et al., 1999 . It is suited for re-seeding of decayed perennial grasses and winter cereal crops (Humphreys et al., 1998). Seeking to improve resistance to adverse growing conditions,L. multiflorum was crossed withL. perenne, which producedL. x hybridum Hausskn. (Peeters, 2004). In some sources this species is also calledL. x boucheanumKunth. (The common EU catalogue, 2010; Humphreys et al., 2010).L. x hybridum higher- is yielding thanL. perenne and more resistant to adverse winter conditions than L. multifloruma, b; Reed et al., 2000).(Hannaway et al. 1999 The application of multiflorum L.Lam. subsp.italicum andL. x hybridum species in agriculture could be expanded if varieties with higher cold-tolerance (winter hardiness) were developed. In Lithuania, breeding work with L. multiflorumis rather limited, while no breeding work at all has been done with L. x hybridum. Investigation of short-lived ryegrass genetic collections enables selection of foreign varieties with the most valuable biological traits or agronomically valuable characteristics and their application as germplasm for the development of novel Lithuanian varieties. When developing new varieties, it is very important that they are not only high-yielding but also are characterised by yield stability, good nutrient uptake from the soil, and sufficient tolerance of
adverse weather conditions (cold and drought-tolerance), disease and pest resistance (Sliesaravičius, Nekroas, 1997; Humphreys et al., 2010). Currently, development of new short-lived ryegrass varieties employs not only conventional breeding methods (hybridization, selection, polyploidy and others), but also molecular technologies that are included in the breeding schemes with the aim of creating effective, breeding programmes based on molecular marker use (Barth et al., 2002, Busti et al., 2004). Short-lived ryegrass species are visually similar. Comparison of agro-morphological characteristics of the tested ryegrass species in field collections and analysis of morpho-anatomical traits and DNA laboratory assays facilitate assessment and identification of these species. Hypothesis. Employment of the molecular technique (ISSR) will enable visual identification of similar ryegrass species. Investigation of short-lived ryegrass species differing in ploidy will allow us to determine the prospects of diploid and teraploid varieties according to major agro-morphological traits and biological characteristics and to select valuable germplasm for breeding. Assessment ofL. multiflorumsubsp.multiflorumvarieties in different agroclimatic conditions according to the genotype x environment model will enable additional assessment of the varieties for one of the major traits DM yield stability. Having established the midparent heterosis ofL. multiflorum subsp.multiflorum parental varieties, promising hybrid plants will be developed. Investigation of L. multiflorum subsp.multiflorum with legumes ( mixturesVicia sativa,Trifolium resupinatumto estimate their cultivation possibilities in organic and) will enable us conventional production systems. Research objective. The study was designed to assessL. multiflorumsubsp. multiflorum,L. multiflorumsubsp.italicumandL.xhybridumspecies and varieties of different ploidy from the agro-biological and breeding viewpoint under Lithuanias conditions. Propositions to be defended: 1. Besides morpho-anatomical assessment, it is expedient to employ DNA elecrtophoretic analysis for the identification of short-lived ryegrass species. 2. The existing diversity of agromorphological traits and biological characteristics of ryegrass species and varieties enables comparison of short-lived ryegrass under Lithuanias conditions and selection of promising varieties for breeding. 3. The genotype x environment model complements assessment of L. multiflorumsubsp.multiflorumvarieties according to DM yield stability. 4. Assessment ofL. multiflorum subsp.multiflorum hybrid plants for agro-morphological traits and characteristics exhibits midparent heterosis of parental varieties. 5.L. multiflorumsubsp.multiflorummixtures with legumes can be grown in organic and conventional production systems.
Experimental tasks: 1. short-lived ryegrass species using the anatomical To identify morphological method. 2. To assess the suitability of the ISSR technique for the identification of short-lived ryegrass species and for determination of polymorphism. 3. To assess the collection material of short-lived ryegrass (L. multiflorum subsp.multiflorum,L. multiflorumsubsp.italicumandL.xhybridum) differing in origin and ploidy according to the most important argo-morphological traits (yielding capacity, seed set, plant height etc.) and biological characteristics (over winter survival, earliness etc.) and herbage quality parameters. To select valuable material for ryegrass breeding. 4. To investigateL. multiflorum subsp.multiflorum varieties in different agro-climatic conditions according to the genotype x environment model. To determine yield stability of each variety. 5. To determine the midparent heterosis of the most valuableL. multiflorum subsp.multiflorumparental varieties. 6. To assess two-three component mixtures (Trifolium resupinatum,Vicia sativa,Avena sativa) withL. multiflorumsubsp.multiflorumand to compare them in conventional and organic production systems. Originality of research. It has been established that morphoanatomical assessment and DNA electrophoregram analysis are suitable for the identification ofL. multiflorum subsp.multiflorum. It is the first time in Lithuania we have done comparative research on genetic collections of short-lived ryegrass species (L. multiflorum subsp.multiflorum,L. multiflorum subsp.italicum andL.x hybridum) agro-morphological traits, biological characteristics and feeding value. Dry matter yield stability ofL. multiflorum subsp.multiflorum has been established in varieties different agro-climatic conditions according to the genotype x environment model. Comparison and assessment ofL. multiflorum subsp.multiflorum cultivation possibilities in mixtures with legumes (Vicia sativa,Trifolium resupinatum) in conventional and organic production systems have been done. Practical application.The selected valuable short-lived ryegrass material will be used in breeding. Approval of the dissertation work. main research results have been The published in 3 scientific articles in refereed ISI Master List and CAB Abstracts data bases, 5 in conference proceedings. Scope and volume of the dissertation.The dissertation is written in the Lithuanian language. It comprises 110 pages and is composed of: an introduction, literature overview, experimental object, materials and methods, results and discussion, conclusions, list of publications on the dissertation topic, list of references and appendices. The dissertation includes 42 tables and 34 figures. A total of 291 literature references have been used.
EXPERIMENTAL MATERIAL AND METHODS Research object.L. multiflorum subsp.multiflorumvarieties (24) and breeding numbers (4),L. multiflorumsubsp.italicum(72) andL.xhybridum(24) varieties. Meteorological conditions Meteorological conditions in Dotnuva during the period 20062010.The spring of 2006 was cold and dry. Warm and dry weather prevailed in June and July. In August, the weather was warm and rainy. In September, the weather was warm, the first ten-day period was rainy, while later dry and cloudless weather prevailed. The winter of 2007 was changeable in terms of temperatures: the weather in January was windy, very mild and rainy during the 2 ten-day periods, while it was cold during the third ten-day period. The spring was very early and changeable. Dry weather prevailed. Like in spring, the weather in summer was changeable cool weather alternated with periods of warm weather, while dry weather alternated with rainy spells. In September, warm weather prevailed, in the third ten-day period it was dry. The winter of 2008 was short, with no permanent snow cover and no permanent frost. The spring was early, dry and, in terms of temperature, changeable weather prevailed. Warm and dry weather prevailed during the larger part of the summer. In September, the weather was changeable - unusually warm during the first ten-day period, cool during the second ten-day period and warmer again during the third-ten day period. The winter of 2009 was changeable in terms of temperatures, wintry cold weather alternated with warmer spells. February was the coldest month. The spring was extremely early with numerous frosts. Warm and dry weather was characteristic of April. In May, the weather was cool. The dry weather that started in April persisted during the first half of May. With the rainfall at the end of the month, the rainy period started (with short intervals), which persisted throughout the entire summer period. The winter of 2010 was cold, with permanent, not thick snow cover and permanent deep and long frost. Till mid March, the weather was cold and wintry. The spring was warm, with sufficient moisture quantity in the soil. The weather in June was cool. There were days in July-April months, when air temperature rose above 30° C. The whole growing season was characterized by abundant rainfall. Meteorological conditions in Dotnuva, Jonikėlis, and Perloja during the 20062007 period. The air temperature was similar in the three experimental sites. In 2006, more rainfall fell in Perloja, in 2007 in the first half of summer in Dotnuva, in the middle of summer (in June) in Perloja. Warmer and dryer weather prevailed in 2006. The lowest rainfall amount was recorded in June, while the highest in August (especially in the second ten-day period). In 2007, the average temperature of the growing season was several degrees lower than that in 2006. Although spring was dry, the rainy period started since the second ten-day period of June. July was the rainiest month. Much more rainfall occurred in 2006, compared with 2007.
General conditions of research, time and site Germplasm.The germplasm consisted of the varieties and breeding numbers of the Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry (IA, LRCAF) (before January 2010 the Lithuanian Institute of Agriculture) and the germplasm obtained from other countries gene banks, research and other institutions. Soil. The soil of the experimental fields of the Grass Breeding Department of the IA, LRCAF isEndocalcari Epihypogleyic Cambisols (CMg-p-w-can), characterised by a homogeneous texture, pHKCl7.3-7.0, humus content 1.9-2.2 %, available P2O5206270 mg kg-1and K2O 101-154 mg kg-1. The soil of the Jonikėlis Experimental Station of the LRCAF, is Endocalcari Endohypogleyic Cambisol (CMg-n-w-can)characterised by a pHKCl of 6.16.4, humus content 2.2-2.3 %, available P2O5 140156 mg kg-1 K and2O 210230 mg kg-1. The soil of the Perloja Experimental Station of the LRCAF, isHapli-Abic Luvisols (LVA-Ha)characterised by a pHKClof 4.46.4, humus content 1.11.9 %, available P2O5171294 mg kg-1and K2O 105180 mg kg-1. The soil of the organic agro-ecosystem of the Plant Nutrition and Agro-ecology Department of the IA, LRCAF isEndocalcari Endohypogleyic Cambisol (CMg-n-w-can Endocalcari)characterised by a pHKCl of 7.27, humus content 2.44 %, available P2O5170182 mg kg-1and K2O 150168 mg kg-1. Establishment of experiments Analysis of agro-biological characteristics and morphological traits of shortlived ryegrass species. A collection of shortlived ryegrass species was set up in the experimental fields of the IA, LRCAF. Eighteen plants were planted per plot (9 plants in 2 rows), at a distance of 50 x 50 cm, using a randomized complete block design with 3 replications. Nitrogenous fertilizers were applied: in spring N30(L. multiflorum subsp. italicumandL. x hybridum), and N45after cuts (all species). Analysis of midparent heterosis of L. multiflorum subsp.multiflorum varieties. The experiments were set up in the trial fields of the Grass Breeding Department of the IA, LRCAF. The crossings of varieties Rapid and Elunaria were made in 2007, of varieties Elunaria and Pollanum in 2008. The plants of the promising varieties Rapid, Elunaria and Pollanum selected for crossing were potted and transferred to greenhouses for wintering. In spring, the plants were cloned and planted in a hybrid nursery randomly, isolated from the plants of the same species, at 50 x 50 cm distances, 50 plants (clones) per variety for free cross-pollination. The seeds were collected from mother plants separately. The seeds collected fromL. multiflorum subsp.multiflorum were sown in the germination chambers in 2010. The emerged plants were transplanted in the breeding nursery by a nest method at 50 x 50 cm distances, one plant per nest. Eighteen plants were planted in 5 m2field plots. There were 2 rows per field plot. 7
Genotype x environment influence on dry matter yield stability of L. multiflorum subsp.multiflorum varieties. experiments were set up in three The different sites of Lithuania: Dotnuva (55°23´N, 23°57´E), Jonikėlis (56°07´N, 24°10´E) and Perloja (54°13´N, 24°25´E) during the period 2006 - 2007. In each location, genotypes were planted in 6.25 m2test plots using a randomized complete block design with four replications. N45 fertilizer was applied after cuts. The experimental material included 8L. multiflorumsubsp. multiflorumvarieties: Varpė, Rapid, Druva, Lifloria, Pollanum, Elunaria, Imperio and Lirasand. Cutting was performed at ryegrass inflorescence emergence stage. Investigation of mixtures with L. multiflorum subsp.multiflorum. The experiments were set up in two sites: in the fields of the Grass Breeding Department of the IA, LRCAF and organic agro-ecosystem of the Plant Nutrition and Agro-ecology Department of the IA, LRCAF during the period 20062008. The experiments were set up in 6.25 m2test plots using a randomized complete block design with four replications. The mixtures with legumes in the fields of the Grass Breeding Department were not fertilized. N45fertilizer was applied after cuts only forL. multiflorum subsp.multiflorum andA. sativa. The experimental material included the following varieties:L. multiflorum subsp.multiflorum Rapid,T. resupinatumArkadija ,A. sativaMiglaandV. sativaPilkiai. Experimental design: 1. Lolium multiflorumssp.multiflorum(25 kg ha-1) 2. Trifolium. resupinatum(12 kg ha-1) 3. L. multiflorumssp.multiflorum50 % +T. resupinatum50% (12.5+6 kg ha-1) 4. L. multiflorumssp.multiflorum25 % +T. resupinatum75% (6.25+9 kg ha-1) 5. L. multiflorumssp.multiflorum75 % +T. resupinatum25% (18.8+3 kg ha-1) 6. Avena sativa(180 kg ha-1) 7. Vicia sativa(180 kg ha-1) 8. V. sativa+A. sativa(70+130 kg ha-1) 9. L. multiflorumssp.multiflorum+V. sativa+A. sativa(20+60+120kg ha-1) Experimental site Structural (during 20062010) and morpho-anatomical (during 20092010) analyses of shortlived ryegrass species were made at the laboratory of Grass Breeding Department (IA, LRCAF). Molecular biology assays were done at IA, LRCAFs Laboratory of Genetics and Physiology during 20082010. Chemical analyses were carried out at the laboratory of Chemical Research during 2006 2010. Research methods and statistical analysis of data Analysis of morpho-anatomical traits of shortlived ryegrass species. For the analysis of morpho-anatomical traits, in the first (L. multiflorum subsp. multiflorum) and second years of growing (L. multiflorum subsp.italicum irL. x hybridum), the over-flowered plants were herbarized. The plant height (cm), leaf color (score), length and width of flag leaf (cm), inflorescence, glume, lemma, awn and spikelet length (mm) were measured, spikelets number per inflorescence, and 8
vascular bundle were calculated. For anatomical leaf analyses, the leaves were taken from the herbariums and cross-sectioned. Conjugation of sclerenchyma fibers was determined by a biological microscope. Genetic diversity assessment of short-lived ryegrass by ISSR fingerprinting. DNA was extracted from young leaves (510 mm) by a micro-method following the DNA extraction protocol of Doyle and Doyle (1990). Samples were made from 2530 plants of each species. UsingISSRmarkers, GeneRulerTM DNA Ladder Mix (Fermentas) was applied as the DNA fragment size marker. The reproducibility of the DNA profiles was tested by repeating the PCR amplifications twice. Only the robust and repeatable bands were considered in this study. The bands were scored for presence (1) or absence (0). Data analysis was performed with NTSYSpc v. 2.2. Analysis of agro-biological characteristics and morphological traits of shortlived ryegrass species. Morphological traits and agronomically valuable characteristics of the varieties and breeding numbers of shortlived ryegrass species were estimated in absolute values and points (according to the standards of visual assessment methods presented by the International Plant Genetic Resources Institute and adapted in the forage grasses working groups of European and Baltic countries) (UPOV, 2006). A 19 score scale was used, where 1 is very low...9 very high value of the trait. Diseases were assessed according to the methods described in the methodological publication Agricultural Pests and Diseases and Their Record (Dabkevičius, Brazauskienė, 2002). L. multiflorum subsp.multiflorum varieties were tested for 13 traits: inflorescence emergence date, leaf colour (score), plant height (cm), flag leaf length and width (cm), longest stem length (cm), damage byPuccinia coronnifera Kleb. (0 % very low damage... 50 % very high damage), damage byDrechlera spp.damage... 75 % very high damage), re-growth after cut % very low (0 (score), first third cut dry matter yield (DMY) (g), inflorescence length (cm), number of spikelets, seed yield per plant (g). L. multiflorum subsp.italicumandL. x hybridum were tested for varieties 17 traits: trend to form inflorescences in the year of sowing, leaf colour (score), winter damage (%), plant growth habit (score), growth in spring and after cut (score), inflorescence emergence date, plant height (cm), flag leaf length and width (cm), longest stem length (cm), damage byPuccinia coronniferaKleb. (0 % very low damage... 50 % very high damage), damage byDrechleraspp. % very (0 low damage... 75 % very high damage), first fourthly cut DMY (g), inflorescence length (cm), number of spikelets, seed yield per plant (g). Analysis of genotype x environment influence on dry matter yield stability ofL. multiflorumsubsp.multiflorumvarieties.Inflorescence emergence date, plant height (cm); first third cut DMY (t ha-1) and DM structure were estimated.
Analysis of twothree component mixtures withL. multiflorum subsp. multiflorum.Inflorescence emergence date, plant height (cm); first third cut DMY (t ha-1) and indicators of nutritive (feeding) value: CP, CF, WSC, DMD were estimated. Samples for herbage chemical composition determination were taken at inflorescence emergence stage for each cut. Indicators of nutritive (feeding) value: contents of crude protein (CP), crude fibre (CF), water soluble carbohydrates (WSC), dry matter digestibility (DMD) were measured using a near infrared spectrometer NIRS-6500 (Butkutėet al., 2003). Analysis of DMY structure shortlived ryegrass plants were assessed for stem diameter (mm), number of vegetative and generative stems, stem weight, inflorescence and leaf weight (g). Analysis of midparent heterosis ofL. multiflorum subsp.multiflorum varieties. In the breeding nursery established from freely cross-pollinated L. multiflorum subsp.multiflorum varieties assessments were done for 13 morphological traits and agronomically valuable characteristics (like in the L. multiflorumsubsp.multiflorumcollection nursery). Botanical-structural analysis of the DM yield was also done in the experiments Midparent heterosis (MPH) of the parental varieties Rapid, Elunaria and Pollanum was determined according to morphological traits and agronomically valuable characteristics of the hybrid plants. MPH was calculated by the formula (Falconer, MacKay, 1996): MPH=F1P, withP=P1+2P2(P1andP2 parental varieties) MPH percentage calculated in relation to parental varieties (100 %). The scale used to assess MPH: MPH % < 100 very low; MPH % = 100 low; MPH % > 100+110 average; MPH % > 100+1120 high; MPH % > 100+20 very high. Statistical analysisof the research data was done usingANOVA,STAT and DERLIUS software fromSELEKCIJApackage (F, R05,Sx, V%) (Tarakanovas, Raudonius, 2003), we also used YIELDSTAB (variance of stability (σ), M. S. Kang & R. Magari (1995)) and SigmaStat (coefficient of correlation) software. Polymorphism was estimated usingNTSYSpc 2.2software (Rohlf, 2005). EXPERIMENTAL RESULTS AND DISCUSION Morpho-anatomical analysis of short-lived ryegrass Analysis of leaf cross-section of short-lived ryegrass species revealed L. multiflorumsubsp.multiflorumdistinction, compared withL. multiflorumsubsp. italicumandL. x hybridum.L. multiflorumsubsp.multiflorumwas characterised by the conjugation of sclerenchyma fibers not only in the central but also in lateral bundles. This difference stands out only at inflorescence emergence stage (Fig. 1).
Fig. 1. Leaf cross-sections of short-lived ryegrass species, 20082009. Note. Letters denote different species: a L. multiflorumsubsp.italicum, b L. x hybridum, c L. multiflorumsubsp.multiflorum. The assessment of morpho-anatomical traits showed their average values to diverge, however, according to the values of individual plants, the results were very similar (Table 1). Table 1. Morpho-anatomical traits of short-lived ryegrass species, averaged over 20072009. Characteristics Plant height, cm Leaf color (points) Length of flag leaf, cm Width of flag leaf, cm Number of bundle Inflorescence length, cm Number of spikelets Spikelet length, cm Length of lower glume, cm Length of upper glume, cm Awn length, cm Number of flowers 1000 seed weight, g
L. multifloru subsp.multiflorumsubsp.itm L. x hybridumali um c x±Sx % Vx±Sx V %x±Sx V % 49.6±1.20 27.17 51.6±0.47 12.97 50.2±0.62 11.84 5.3±0.26 26.77 5.1±0.16 26.79 6.7±0.42 30.28 12.6±0.37 33.19 14.0±1.04 28.57 17.1±0.45 25.01 0.6±0.01 22.46 0.7±0.04 20.46 0.7±0.02 21.23 21.0±2.02 27.18 19.3±1.35 19.78 19.3±1.18 21.19 21.0±0.37 20.17 26.1±0.93 13.84 22.6±0.42 17.83 16.8±0.30 20.25 23.9±0.92 14.93 19.6±0.28 13.81 2.3±0.11 13.56 2.1±0.08 10.75 2.0±0.10 16.36 0.7±0.02 7.33 0.8±0.05 17.17 0.8±0.02 9.20 1.1±0.04 10.68 1.0±0.08 22.06 1.1±0.06 17.58 0.3±0.07 62.05 0.4±0.05 28.80 0.2±0.04 74.98 12.6±0.65 14.63 12.0±0.76 17.82 10.5±0.44 14.36 3.4±0.17 18.46 4.1±0.19 32.06 4.2±0.15 23.47