An Abstract of the Thesis Of

AN ABSTRACT OF THE THESIS OF

Elif Sahin for the degree of Master of Science in Crop Science presented on March 12, 2020.

Title: Breeding for Improved Forage Yield Potential and Digestibility in Tall Fescue (Schedonorus arundinaceus (Schreb.) Dumort.)

Abstract approved:

______David B. Hannaway

Tall fescue is a perennial forage grass widely used in areas not well-suited to perennial ryegrass due to climatic or edaphic stressors including drought and low or high pH. Although tall fescue has many agronomic attributes that make it well suited to a wide range of environments, it is lower in palatability and digestibility than Lolium species. To take full advantage of tall fescue, genetic improvements in digestibility are needed to increase animal performance. The goal of this study was to improve fiber digestibility and yield of early and medium maturing tall fescue genotypes through recurrent phenotypic selection. Improving fiber digestibility and yield concurrently is a challenge because yield and fiber content are positively correlated.

Sixteen plants were selected from both an early maturity source population of 1600 plants and from a medium maturity source population of 1700 plants. Selected plants from each maturity group were placed in separate polycross blocks. Using resulting half-sib seeds, two spaced-plant nurseries were established in Philomath, Oregon to evaluate forage and seed yield and agro-morphological characteristics including plant height, tiller number, and heading date. In 2 addition, small plot trials were established in Boyd, Kentucky in 2018 to quantify forage yield and estimate forage quality.

In the Oregon spaced-plant nurseries, forage yield, seed yield, heading date, tiller number, and plant height were significantly different (P<0.05) among genotypes. In the

Kentucky small plot trials, significant differences were found for forage yield, NDF, ADF,

TDN, RFV, and RFQ in the first cutting while only forage yields were significantly different for second and third cuttings. Thus, although there is a positive correlation between forage yield and fiber (NDF and ADF), concurrent improvements can be obtained for yield and digestibility through recurrent phenotypic selection. Since tall fescue does not produce culmed (jointed) vegetative shoots in regrowth, quality evaluation from the first cutting is most critical in selecting for improved digestibility.

Breeding for Improved Forage Yield Potential and Digestibility in Tall Fescue (Schedonorus arundinaceus (Schreb.) Dumort.)

by Elif Sahin

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Presented March 12, 2020 Commencement June 2020

Master of Science thesis of Elif Sahin presented on March 12, 2020

APPROVED:

Major Professor, Representing Crop Science

Head of the Department of Crop and Soil Science

Dean of the Graduate School

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request.

Elif Sahin, Author

ACKNOWLEDGEMENTS

First, I would like to express my sincere thanks to my supervisor Dr. David B.

Hannaway, for his invaluable guidance, wisdom, support, and patience during this research. I have been so lucky to have a supervisor who cared so much about my work, and who responded to my questions and queries all of the time. I will never forget the experiences that I have benefited from him throughout my life.

Thank you, my almost joint supervisor, Dr. Serkan Ates, and his lovely family members.

I always felt like they are my family.

I would also like to thank Dr. Sabry Elias for serving on my committee and for making the time to listen to me about all my concerns and questions about this project.

I would like to also thank Dr. Marcelo Moretti for his continuous encouragement, understanding, and motivation.

In addition, I would like to thank Steve Reid for teaching me all the aspects of the field breeding study. He always answered my questions with great patience, even when he had limited free time. I would like to thank Barbara Hints-Cook for helping me transfer the data that I collected from the field, and for answering my numerous questions for this project. I would like to thank DLF Pickseed USA for supplying all equipment, materials, and staff. Your kindness, wisdom, and free spirit humbles me.

Muhammet Sahin, who is my husband, was always there with all his love for me when I needed a warm-hearted hand. He always provided full support and helped me when I felt tired, upset, and depressed through my master’s program.

My special thanks go to my beloved parents; my mother Ayse, my father Yusuf, and my sisters Sevilay, Sabriye, Eda, Seyda, Gulseren, and Gulsah. I also would like to thank my second 7 parents, mother-in-law Gursun and father-in-law Recep for their endless support and encouragement in my every decision I have made throughout my whole life.

I would like to thank Ramazan Tuzen; I am sure that I would not have been at this point if you had not helped push me along. I learned that I should never give up on my way. I also thank Tuncay Guzel for his support and belief in me.

Last, but not least, I am thankful for the great two and a half years of my life here in

Oregon, where I have met the wonderful people in my life. I have always felt I am fortunate to have met these people.

Ayse and Merve were always there for me like sisters when I need a warm-hearted hand.

Also, Yunus, thank you for the countless ways you have helped me throughout this process.

I am genuinely grateful for the impact each of you has had on my life and career.

TABLE OF CONTENTS Page

Chapter 1: General Introduction and Literature Review ...... 1

1.1 Tall Fescue ...... 1

1.1.1 Origin and Distribution of Tall Fescue ...... 1

1.1.2 Systematics, Physiology, and Morphology ...... 2

1.1.3 Usage of Tall Fescue ...... 3

1.1.4 Endophytic Fungus Association ...... 5

1.1.5 Cultivars ...... 7

1.1.6 Climatic and Soil Tolerances ...... 8

1.1.7 Forage Yield and Quality ...... 9

1.1.8 Recurrent Phenotypic Selection ...... 12

1.2 Project Description ...... 14

1.3 Project Overview ...... 14

1.4 Anticipated Impact ...... 15

Chapter 2: Breeding for improved forage yield potential and digestibility of tall fescue (Schedonorus arundinaceus (Schreb.) Dumort.) ...... 16

Abstract ...... 16

2.1 Introduction ...... 17

2.2 Materials and Methods ...... 18

2.2.1 Development of Populations and Selection Criteria ...... 18

2.2.2 Field Planting and Evaluation ...... 22

2.2.2.1 Small Plot Trial (Experiments A1 and A2) ...... 22

2.2.2.2 Spaced-Plant Nurseries (Experiments B1 and B2) ...... 23

2.2.3 Measurements and Data Collection ...... 25 9 2.2.3.1 Dry Matter Yield and Forage Quality ...... 25

2.2.3.2 Agro-morphological Characteristics ...... 25

2.2.5 Experimental Design and Statistical Analysis ...... 27

2.3 Results ...... 28

2.3.1 Phenotypical Traits ...... 28

2.3.1.1 Dry Matter Yield (DM) and Spring Growth Indices (1st, 2nd, 3rd SGI) ...... 28

2.3.1.2 Heading Date, Seed Yield, Tiller Number and Plant Height ...... 30

2.3.1.6 Flag Leaf Length-Height-Width and Panicle Length ...... 32

2.3.2 Forage Yield and Quality ...... 33

2.3.2.1 First Cutting ...... 33

2.3.2.2 Second Cutting ...... 35

2.3.2.3 Third Cutting ...... 37

Correlations ...... 39

2.4 Discussion ...... 40

2.4.1 Yield and Agro-morphological Traits ...... 40

2.4.2 Dry Matter Yield and Forage Quality Traits ...... 43

Conclusions ...... 47

References ...... 49

10 LIST OF FIGURES

Figure Page

Figure 1. USDA PLANTS Database taxonomy of tall fescue in the USA (USDA-NRCS, 2020). 2

Figure 2. The distribution of tall fescue in the USA (source:https://www.invasiveplantatlas.org/ subject.html?sub=3037)...... 4

Figure 3. The US transition zone is located between the cool-season and warm-season zones (Lawn Care & Lawn Management Guide, 2020)...... 5

Figure 4. Suitability map of tall fescue for the USA based on tolerance to climatic and edaphic factors (Hannaway et al., 2009)...... 8

Figure 5. Establishment of a crossing block for harvest of half-sib seeds...... 21

Figure 6. Drone photograph of the small plot trial established in Boyd, Kentucky on 4 October 2018...... 22

Figure 7. Spaced-plant nursery established in Philomath, Oregon on 4 October 2018...... 24

11 LIST OF TABLES Table Page

Table 1. Dry matter yield (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and total digestible nutrients (TDN) quality results for some studies. .... 10

Table 2. USDA grass quality guidelines...... 10

Table 3. Equations used for calculation of forage quality parameters (digestible dry matter (DDM), dry matter intake (DMI), total digestible nutrients (TDN), net energy of lactation (NEL), relative feed value (RFV), relative forage quality (RFQ))...... 12

Table 4. Heading date and spring growth characteristics of the early maturity tall fescue parental genotypes selected for the polycross nursery...... 19

Table 5. Heading date and spring growth characteristics of the medium maturity tall fescue parental genotypes selected for the polycross nursery...... 20

Table 6. Agro-morphological characteristics used to evaluate the half-sib family seedlings in the spaced-plant nurseries in Philomath, Oregon...... 24

Table 7. Dry matter (DM) yield and three spring growth index (SGI) evaluations of 32 tall fescue genotypes in 2019 in Philomath, OR (sorted by dry matter yield)...... 29

Table 8. Heading date (HD), seed yield (SY) per plant, tiller number (TN) and plant height (PH) of 32 tall fescue genotypes in Philomath, OR (sorted by heading date)...... 31

Table 9. Flag leaf length (FLL), flag leaf height (FLH), flag leaf width (FLW) and panicle length (PNL) of 32 tall fescue genotypes in Philomath, OR (2019)...... 32

12 Table 13. Correlation coefficients between agro-morphological traits [dry matter yield (DM), seed yield (SY), tiller number (TN), plant height (PH), spring growth indices (SGI), panicle length (PNL), flag leaf height (FLH), flag leaf width (FLW), and flag leaf length (FLL)] for the 32 half-sib families grown in spaced-plant nurseries in Philomath, OR in 2019...... 39

Table 14. Correlation coefficients between dry matter yield (DM) and nutritive quality traits [crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), total digestible nutrients (TND), relative feed value (RFV), and relative feed quality (RFQ)] for the 32 half-sib families and a check cultivar (Kentucky 31 E+) grown in small-plot trials in Boyd, KY in 2019...... 40

Table 15. Quality standards for legume, grass, or grass-legume hay [crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), digestible dry matter (DDM), dry matter intake (DMI), and relative feed value (RFV)...... 44 13

LIST OF ABBREVIATIONS

ADF = Acid detergent fiber ADL = Acid detergent lignin CP = Crude protein DDM = Digestible dry matter DM = Dry matter DMI = Dry matter intake DMY = Dry matter yield FLH = Flag leaf height FLL = Flag leaf length FLW = Flag leaf width HD = Heading date NDF = Neutral detergent fiber NIRS = Near infrared reflectance spectroscopy PH = Plant height PNL= Panicle length RFQ = Relative forage quality RFV = Relative feed value SGI = Spring growth index SY = Seed yield TDN = Total digestible nutrients TN = Tiller number

I dedicate this thesis to my son Recep Omer Sahin

Chapter 1: General Introduction and Literature Review

1.1 Tall Fescue

1.1.1 Origin and Distribution of Tall Fescue

Tall fescue [Schedonorus arundinaceus (Schreb.) Dumort. = Lolium arundinaceum

(Schreb.) Darbysh.] is a perennial, cool-season grass native to Europe, Northern Africa, West and Central Asia, and Siberia (Gibson and Newman, 2001). Although tall fescue is considered a secondary forage species in Northwest Europe because of the presence of higher quality cool- season forages in terms of leaf texture and palatability, in the United States it has been one of the predominant cool-season perennial grass species since the 1970s. The planted area of tall fescue is estimated at approximately 14 million hectares (35 million acres) (Rogers and Locke, 2013).

Although the exact introduction date of tall fescue to the USA is unknown, it is thought to have entered the country by mixing with meadow fescue seeds imported from Europe in the

1800s (Hoveland, 2009). Extensive cultivation of tall fescue began following the release of

‘Alta’ and ‘Kentucky 31’ cultivars in the 1940s and 1950s (Hoveland, 2009). Cultivation and popularity of tall fescue were interrupted with the discovery of an endophytic fungus Epichloë coenophiala (Morgan-Jones & W. Gams) C.W. Bacon & Schardl (Helander et al., 2016), which causes animal health disorders (Hoveland, 2009), see section 1.1.4 “Endophytic Fungus

Association”. In the mid-1970’s, the value and usage of tall fescue decreased due to detection of the endophyte (Ball et al., 1991). Removing the endophytic fungi from seed was a way to avoid its negative effect, but that decreased its tolerance to heat, drought, pests, and grazing (Hoveland,

2009). The development and introduction of a novel endophyte in the 1990’s (Gunter and Beck, 2

2004; Hopkins et al., 2011; Hopkins et al., 2010), has resulted in renewed use and breeding to improve desired traits of tall fescue.

1.1.2 Systematics, Physiology, and Morphology

Tall fescue is a cool-season perennial bunchgrass species. It has C3 photosynthesis physiology and grows in a wide range of climate areas. It is an allohexaploid and outcrossing species, with 2n = 6x = 42 chromosome number. Tall fescue is moderately self-incompatible, so it does not readily accept pollen from plants that belong to the same family.

Based on its broad-leaved morphology and ovary structure, Schedonorus arundinaceus

(Schreb.) Dumort. is included in the Bovinae section along with Festuca pratensis Huds. and

Festuca gigantea (L.) Vill. (Craven et al., 2009).

Tall fescue is described as a member of the Festuca-Lolium complex because of its infertility relationships (Jauhar, 1993). The current taxonomic classification of tall fescue is shown in the Figure 1.

Rank Scientific Name and Common Name Kingdom Plantae - Plants Subkingdom Tracheobionta - Vascular plants Superdivision Spermatophyta - Seed plants Division Magnoliphyta - Flowering plants Class Liliopsida - Monocotyledons Subclass Commelinidae Order Cyperales Family Poaceae/Gramoneae - Grass family Genus Schedonorus P. Beauv. - fescue Species Schedonorus arundinaceus (Schreb.) Dumort.., nom. cons. - tall fescue Figure 1. USDA PLANTS Database taxonomy of tall fescue in the USA (USDA-NRCS, 2020). 3

1.1.3 Usage of Tall Fescue

While turf-type tall fescue is used as an amenity grass on golf courses, residential and commercial lawns, and athletic fields, forage types are used for pasture, hay, and silage. Forage types are also used for reducing soil erosion and recycling nutrients from manure and biosolids.

Turf-type tall fescues have mostly opposite characteristics with forage-types, including finer leaf texture, horizontal growth (dwarf or semi-dwarf growth habit), increased tiller density, desired endophytic fungus infection, and narrow root systems.

Tall fescue’s extensive use as forage is due to high yield, a long grazing season, long persistence, and high seed yield. Also, tall fescue has vigorous and competitive growth, and medium drought tolerance (Huang and Gao, 2000). It is often selected by farmers because of greater drought and heat tolerance than other cool-season grasses (Milne et al., 1997; Reed,

1996).

Although tall fescue is more often used by beef producers than by sheep or dairy farmers, newer, improved cultivars are being more widely used, even by dairy farmers, especially in summer and fall seasons. This is because perennial ryegrass has reduced growth and quality due to heat and moisture stress in these seasons (Milne et al., 1997). Tharmaraj et al. (2005) reported that tall fescue-based pastures, especially in summer and winter, have the potential to produce as much milk as ryegrass-based pastures. Therefore, tall fescue is growing in popularity as a forage species to feed dairy animals. Tall fescue’s extensive distribution in the USA is shown in Figure

2. 4

Figure 2. The distribution of tall fescue in the USA (source:https://www.invasiveplantatlas.org/ subject.html?sub=3037). In the USA, the transition zone (between the cool-season and warm-season zones) is where over 20% of the beef cows are raised (West and Waller, 2007) (Figure 3). The eastern portion of the transition zone is sometimes referred to as the ‘Fescue Belt’ due to extensive use of tall fescue for cattle production and its aforementioned heat and drought tolerance. Farther south, pastures rely on warm-season species like bermudagrass (Cynodon dactylon L.). 5

Figure 3. The US transition zone is located between the cool-season and warm-season zones (Lawn Care & Lawn Management Guide, 2020).

1.1.4 Endophytic Fungus Association

With respect to the endophytic fungus association, tall fescue cultivars can be divided into three groups: (1) toxic endophyte fescue, (2) endophyte-free fescue, and (3) novel endophyte fescue. Toxic endophyte fescue animal health problems by producing toxic alkaloids

(ergovaline), but neither of the latter groups pose an animal health risk (Ball et al., 2015). In plants, the endophytic fungi live between plant cells, and the fungus is transferred by seed to subsequent generations. Although the fungus can cause animal health problems, the suitability zones for tall fescue have been enlarged by the symbiotic relationship with the endophytic fungus [Epichloë coenophiala (Morgan-Jones & W. Gams) C.W. Bacon & Schardl] (Belesky and West, 2009; Strickland et al., 2011). The symbiotic relationship is based on the plant supplying water and nutrients to the fungus, and the fungus producing alkaloids which increase tolerance to climatic and pest stresses (Isleib, 2015). Higher levels of alkaloids produced by the 6 endophyte also reduce herbivory (Cheplick and Faeth, 2009). Other advantages of the grass- endophyte symbiosis are an increase in tillering and biomass production (Malinowski and

Belesky, 2000), and improved germination rates and seed production (Clay, 1987).

Although beneficial to the plant, when animals graze endophyte-infected plants, toxicoses often result (Burns, 2009). These include: increased respiration, foot and leg problems, elevated rectal temperature, hard hair coat, decreased grazing time, decreased feed intake, decreased milk production, and reduced body weight gains. High soil nitrogen or drought conditions increase the amount of toxins produced (Isleib, 2015); therefore, animals grazing endophyte-infected tall fescue in the summer can experience serious health problems. These result in decreased animal production and reduced income, with losses of $600 million to more than $1 billion annually for beef cattle producers (Ball et al., 2015; Isleib, 2015).

To address these issues, a non-toxic novel endophyte, identified in the late 1990s, has been found to provide plant benefits without causing animal health problems. That means animals can graze tall fescue without problems, and tall fescue can have drought and pest tolerance. Dr. Joe Bouton, from the University of Georgia and Dr. Gary Latch of AgResearch

Limited of New Zealand, released the first novel endophyte-infected tall fescue variety

(designated MaxQTM). They used “Jesup” and “GA 5” tall fescue cultivars for introducing the novel endophyte. These novel endophyte cultivars were sold under the names “Jesup MaxQTM” and “GA 5 MaxQTM” (Newsome, 2018). Developing novel endophyte-infected tall fescue cultivars continues to be important because of the benefits to tall fescue cultivars in terms of drought and heat tolerance and increased pest resistance without causing animal health problems. 7

1.1.5 Cultivars

Due to tall fescue’s extensive usage, numerous cultivars have been developed since its introduction. Historically, ‘Kentucky-31’ has been the predominant tall fescue cultivar in the mid-south region (Hoveland, 2019; Hoveland et al., 1980) even though ‘Alta’ was released in

Oregon in 1940 and ‘Kentucky 31’ was released in 1943. Additional tall fescue cultivars are described in the following paragraphs.

‘Fawn’ was developed by R. V. Frakes and J. R. Cowan in Oregon, and released in 1964

(Alderson and Sharp, 1994). ‘Kenhy’ was released in Kentucky in 1977 (Hannaway et al., 1999).

In 1985, Willamette Seed and Grain released the ‘Willamette’ forage tall fescue cultivar

(Alderson and Sharp, 1994). ‘Cajun’ was released by Auburn University and International Seeds

Inc. in 1987 (Alderson and Sharp, 1994). ‘Jesup’ was released by Dr. Joe Bouton at the

University of Georgia in 1995 (Bouton, 2019), and in the same year, Kentucky AES and USDA-

ARS released ‘Johnstone’, a forage type (Alderson and Sharp, 1994). Seed Research of Oregon released ‘Crewcut’ in 2002 (Hopkins et al., 2009). ‘Nanryo’ was developed by the Kyushu

Okinawa National Agricultural Research Station (KONARC), Kumamoto, Japan, and released in

2005, being promoted as providing earlier grazing potential.

The Samuel Roberts Noble Foundation with AgResearch Ltd. released a summer-active

‘Texoma’ MaxQ II tall fescue in 2009 (Hopkins et al., 2011) and in 2016, the Noble Research

Institute released ‘Chisholm’ as a summer-dormant forage tall fescue cultivar. This endophyte- free cultivar was promoted as producing high-quality forage, especially in the fall season

(Trammell et al., 2018).

Due to the extensive use of tall fescue in the USA and worldwide, the development and release of new tall fescue cultivars continues. 8

1.1.6 Climatic and Soil Tolerances

Tall fescue can be cultivated in a wide range of soil and climatic conditions

(Arachevaleta et al., 1989). Even though tall fescue gives satisfactory yield on low-pH soils, maximum productivity is obtained between pH 6.0 and 7.0. Mayland and Wilkinson (1996) reported the optimal soil pH for growth was from pH 6.5 to 8.0. Hannaway et al. (2009) provided quantitative climatic and edaphic tolerance values and utilized those values to create tall fescue suitability maps for the conterminous USA.

Figure 4. Suitability map of tall fescue for the USA based on tolerance to climatic and edaphic factors (Hannaway et al., 2009).

Abiotic stress factors such as drought, freezing, and salinity reduce plant growth and development, and seed production (Seki et al., 2002). Drought tolerance is an important research area to increase global food production. It is one of the critical limitations of crop productivity because it typically causes significant crop yield loss. Tall fescue is a cool-season forage species with moderate drought and high-temperature tolerance owing to its deep rooting potential that allows it to tolerate periods of water deficiency by extracting water from more of the soil profile 9

(Green et al., 1990; Zhang et al., 2012). Huang and Gao (2000) studied root physiological characteristics associated with drought resistance in tall fescue cultivars. They reported a positive correlation between root desiccation and root death of tall fescue cultivars. Under drought conditions, tall fescue’s total root length decreases (Narusaka et al., 2003). Fungal endophyte- infected tall fescue is more tolerant of drought, high temperatures, insects, and nematodes

(Marks and Clay, 1996), but, as previously described, endophyte-infected tall fescue negatively affects animal performance (Thompson and Stuedemann, 1993). Kobayashi et al. (2004) studied salt tolerance with six cool-season and six warm-season grasses and reported that tall fescue was the most salt-tolerant cool-season grass. Thus, tolerance to abiotic stress factors has made tall fescue a widely used forage species.

1.1.7 Forage Yield and Quality

Forage quality for animal performance depends on forage intake and digestibility (Wallau et al., 1993). To increase animal production, researchers have conducted numerous studies to increase forage feeding value. Because even a small enhancement of forage nutritive value can increase livestock gains, small improvements can significantly affect livestock economics

(Casler and Vogel, 1999). Forage nutritive value parameters typically include crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) (Burns, 2009). Calculated values include total digestible nutrients (TDN), relative feed value (RFV), and relative forage quality

(RFQ).

Forage yield and forage quality are the main factors used to determine forage value and, indirectly, animal production potential. Kanapeckas et al. (2011) evaluated 13 tall fescue accessions at the beginning of the heading stage for the first harvest and at the tillering stage for harvests two and three. First cutting yield of the seven most productive tall fescue accessions 10 ranged from 22 to 70 g plant-1 and from 62 to 95 g plant-1 for second and third harvests (Table 1).

Bohle et al. (2020) reported yields of tall fescue cultivars in their fourth production year. Yields averaged 5.88, 5.81, and 4.18 (t ha-1) for the three cuttings, with the range of yields shown in

Table 1.

Table 1. Dry matter yield (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and total digestible nutrients (TDN) quality results for some studies.

References DM CP (%) NDF (%) ADF (%) TDN (%) Kanapeckas et al. (2011) 22.0-95 (g/plant) 19.4-27.9 54.3-64.1 28.6-33.9 - Bohle et al. (2020) 2.82-7.29 (t/ha) 10.6-14.0 44.2-53.5 27.8-33.6 57.7-64.4

Riley 2015 - 11.7-24.8 43.5-61.2 21.1-32.9 - (2019) 2017 - 16.1-23.1 42.0-61.0 22.4-30.7 -

Crude protein is measured indirectly by determining the amount of N in the forage plant and multiplying that value by 6.25. The assumption is that N constitutes about 16% of protein in plant leaf and stem tissue (100/16 = 6.25). Grass hay quality classifications often include percent

CP as seen in the USDA grass quality guidelines (Table 2).

Table 2. USDA grass quality guidelines.

Quality Designation Crude Protein (%) Premium > 13 Good 9-13 Fair 5-9 Low < 5 Source: (USDA-LPGMN, 2020).

Riley (2019) studied forage quality in the transition zone and reported CP values of two tall fescue cultivars for two years. In 2015, 'Bronson' had CP values of 12.5, 18.1, and 24.8 % for

May, July, and October harvests in the vegetative stage. 'Cajun II' CP values were 11.7, 16.5, and 11

22.8 %. In 2017, 'Bronson' CP values were 21.4, 18.5, and 18.6 % and for 'Cajun II' were 20.7,

18.0, and 17.8 %. The range of CP values for these two genotypes are provided in Table 1.

The ruminant digestive system allows animals to obtain nutrients and energy from forage cell walls (Chen et al., 2003). Digestibility is how much forage passes from the animal’s digestive tract, and how much animal benefit from the consumed forage (Ball et al., 2001).

Decreasing fiber and lignin concentration in the cell wall can be beneficial because this helps to reduce rumen fill and enhance digestibility (Jung and Allen, 1995).

Several techniques are used to determine feed digestibility in ruminants. In-vitro techniques are expensive, time-consuming, and require accommodations for animal welfare

(Gosselink et al., 2004). For large numbers of samples, estimating forage quality by feeding animals is not practical; laboratory analyses are more often used. These methods include: visual appraisal, traditional laboratory chemistry, near infrared reflectance spectroscopy (NIRS), and in vitro disappearance (Blezinger, 2002). NIRS is a non-consumptive technique developed in the early 1970s (Marten et al., 1989). It is commonly used as a fast and inexpensive way to estimate fiber and other important plant constituents. NIRS was used to estimate forage quality in this study.

Neutral detergent fiber (NDF) includes lignin, cellulose, hemicellulose, and some fiber- bound protein. It is often used to estimate forage intake, being inversely related to dry matter intake (DMI); high NDF values predict low dry matter intake. Similarly, acid detergent fiber

(ADF) is inversely related to digestible dry matter (DDM) (Schroeder, 1994). Thus, measuring

NDF and ADF is a commonly used approach for estimating forage quality.

Time of day (am-pm) changes in NDF values were measured for two tall fescue cultivars

(Riley, 2019) because there is a decrease in fiber fraction concentrations in the afternoon due to 12 dilution from increased total nonstructural carbohydrates (TNC). Changes between am and pm were 8.3, 2.8, and 12 % in May, July, and October 2017 for 'Bronson', and 6.3, 2.8, and 13.4 % for 'Cajun II'. The range of values for CP, NDF, and ADF are shown in Table 1.

Bohle et al. (2020) reported TDN calculated values for tall fescue. Average TDN values ranged from 57.7 to 64.4 % (Table 1). Equations used for calculating forage quality parameters are provided in Table 3.

Table 3. Equations used for calculation of forage quality parameters [digestible dry matter (DDM), dry matter intake (DMI), total digestible nutrients (TDN), net energy of lactation (NEL), relative feed value (RFV), and relative forage quality (RFQ)]. Quality Parameters Equations DDM 88.90-(0.779*ADF) DMI 120/NDF TDN 4.898+(89.796*NEL) NEL 1.044-(0.0119*ADF) RFV (DMI*DDM)/1.29 RFQ (TDN*DMI)/ 1.23 Source: (Moore and Undersander, 2002).

Since grasses typically constitute a large part of the ruminant livestock diet, improving grass forage quality is important. Thus, both high yield potential and fiber digestibility were targeted for improvement in this study.

1.1.8 Recurrent Phenotypic Selection

Due to self-incompatibility, the recurrent phenotypic selection breeding method is appropriate for developing improved cultivars of cross-pollinated species including tall fescue, perennial ryegrass, red clover, and white clover. The method consists of: (1) selecting plants having desirable traits, (2) creating a polycross with selected plants by isolating them from undesirable pollen, (3) evaluating each population or genotype, (4) repeating these phases, and

(5) subsequently releasing superior cultivars (Cai et al., 2014). The purpose of this breeding 13 method is to advance the frequency of desirable alleles. These desired alleles come from chosen parents, so the base population is of primary importance in terms of having high performance for targeted alleles.

Both ‘Alta’ and ‘Kentucky 31’, were both developed through traditional breeding techniques (i.e. ecotype selection) (Hoveland, 2019). ‘Chisholm,’ a synthetic cultivar, was developed by phenotypic selection and released in 2016. Forage-type tall fescue cultivars that have been developed through recurrent phenotypic selection include ‘Cajun’, ‘Martin’,

‘Maximize’, and ‘Stef’. This technique was also used for developing turf-type tall fescue cultivars, including ‘Rebel’, ‘Crossfire’, ‘Falcon II’, and ‘Safari’ (Alderson and Sharp, 1994).

The recurrent phenotypic selection method continues to be used to improve tall fescue breeding populations for forage yield and quality and seed yield (Hopkins et al., 2009; Majidi et al., 2009).

The goal of this study was selecting tall fescue genotypes with high yield potential and increased digestibility. The recurrent phenotypic selection breeding technique was chosen since it is the most often used method for developing new tall fescue cultivars having high seed yield, high forage yield, and high forage quality (Posselt, 2010).

1.1.9 Near Infrared Reflectance Spectroscopy (NIRS)

Forage quality can be estimated through traditional wet chemistry or by NIRS. In this study, NIRS was chosen to predict forage quality parameters due to speed and convenience and its frequent use to analyze fiber content for ruminants in forage research (Corson et al., 1999).

NIRS uses light reflection to evaluate important organic (carbon-containing) forage quality components. NIRS can also be used for predicting the concentration of organic anti- quality compounds including ergovaline and other endophyte-related constituents (Andrés et al., 14

2005). The working principle of NIRS is related to the wavelength of light which allows rapid collection of compositional data of prepared samples. While wet chemistry takes approximately

15-16 hours to analyze a sample, samples can be analyzed in 2-3 minutes by NIRS. This makes nutritional analyses by NIRS five times cheaper than traditional methods (Corson et al., 1999).

Ground samples are used for forage analysis in NIRS and since the method is non-consumptive, samples may be reanalyzed. Another advantage is that it does not require chemicals. Overall, it is a fast and practical method for analyzing forage quality (Andrés et al., 2005).

1.2 Project Description

The purpose of this study was to develop superior tall fescue genotypes (with higher yield and reduced fiber concentration) by using recurrent phenotypic selection before pollination.

1.3 Project Overview

The hypothesis of this study was that sufficient genotypic variation exists among tall fescue lines to enable simultaneous improvement of yield potential and fiber digestibility of tall fescue genotypes by using recurrent phenotypic selection.

To test this hypothesis, two locations (Oregon and Kentucky) were used to evaluate annual forage yield potential and nutritive value of different tall fescue families. Based upon the hypothesis, the following research questions guided our work.

Q1. Can digestibility be improved by using recurrent phenotypic selection?

Q2. What is the relationship between yield and digestibility?

Q3. What are the agro-morphological characteristics that affect dry matter yield?

Q4. What are the interrelationships among agro-morphological characters?

Q5. What are the relationships between maturity and yield potential on fiber digestibility? 15

Q6. What is the relationship between maturity and seed yield?

Q7. What is the relationship between spring growth and yield potential?

The objective of this study was to select for early- and medium maturity tall fescue genotypes that have high forage yield potential and low fiber levels.

1.4 Anticipated Impact

The anticipated impact of this study is that it will lead to the development of improved tall fescue cultivars that have high yield and quality enabling livestock producers to meet their production goals. 16

Chapter 2: Breeding for improved forage yield potential and digestibility of tall fescue (Schedonorus arundinaceus (Schreb.) Dumort.)

Abstract

Tall fescue is a perennial forage grass widely used in areas not well-suited to perennial ryegrass due to climatic or edaphic stressors including drought and low or high pH. Although tall fescue has many agronomic attributes that make it well-suited to a wide range of environments, it is lower in palatability and digestibility than Lolium species. To take full advantage of tall fescue, genetic improvements in digestibility are needed to increase animal performance. The goal of this study was to improve yield and digestibility of early and medium maturing tall fescue genotypes through recurrent phenotypic selection. Concurrently improving yield and reducing fiber content is a challenge because yield and fiber are positively correlated.

Sixteen plants were selected from both an early maturity source population of 1600 plants and from a medium maturity source population of 1700 plants. Selected plants from each maturity group were placed in separate polycross blocks. Using resulting half-sib seeds, two spaced-plant nurseries were established in Philomath, Oregon to evaluate forage yield, seed yield, and agro-morphological characteristics including plant height, tiller number, and heading date. In addition, small plot trials were established in Boyd, Kentucky in 2018 to quantify forage yield and estimate forage quality.

In the Oregon spaced-plant nurseries, forage yield, seed yield, heading date, tiller number, and plant height were significantly different (P<0.05) among genotypes. In the

Kentucky small plot trials, significant differences were found for forage yield, NDF, ADF, TDN,

RFV, and RFQ in the first cutting while only forage yields were significantly different for second and third cuttings. Thus, although there is a positive correlation between forage yield and fiber 17

(NDF and ADF), concurrent improvements can be obtained for yield and digestibility through recurrent phenotypic selection. Since tall fescue does not produce culmed (jointed) vegetative shoots in regrowth, quality evaluation from the first cutting is most critical in selecting for improved digestibility.

2.1 Introduction

Grazing systems are typically based on two primary goals: 1) pastures having high yield, quality, and persistence, and 2) animals having high performance (Newman et al., 2006). For the first goal, selecting appropriate forage species and developing desirable cultivars for pastures is essential. Although grassland vegetation includes a wide diversity of plants, grasses are typically the dominant species (Blair et al., 2014). In temperate, improved grasslands, most pastures supporting animal production contain cool-season perennial grasses such as perennial ryegrass, orchardgrass, and tall fescue, and forage legumes such as red clover and white clover. Tall fescue is often preferred for pastures due to having higher yield than other cool-season perennial grasses

(Riley, 2019). This preference has resulted in a long breeding history for tall fescue.

Livestock production based primarily on pastures requires that the forage species used will produce a high quantity of palatable, digestible feed. Tall fescue is high yielding, has a high tolerance to grazing, and has a good tolerance of drought and salinity (Huang and Gao, 2000).

However, it has less-than-optimal forage quality; it is lower in palatability and digestibility than ryegrass (Lolium) species. Thus, improving tall fescue’s forage quality will assist in producing high average daily gains for grazing livestock.

Forage nutritive value is directly associated with total digestible nutrients (TDN) and fiber affects digestibility. Thus, fiber components are often used to predict forage digestibility.

Neutral detergent fiber (NDF) includes cellulose, hemicellulose, and lignin. Acid detergent fiber 18

(ADF) is composed of cellulose and lignin. NDF is inversely related to forage intake and ADF is inversely related to digestibility (Newman et al., 2006). Thus, low values of both NDF and ADF are associated with higher quality.

Tall fescue is an outcrossing species with a high degree of self-incompatibility. This makes recurrent phenotypic selection an appropriate breeding technique for improving tall fescue. Phenotypic selection is based on evaluating each population, family, or genotype by considering desirable traits to obtain improved genotypes and cultivars. High yield and digestibility are critical selection criteria for tall fescue. Phenotypic selection and evaluating forage quality estimators such as NDF, ADF, and crude protein (CP) can lead to improving the nutritional value of tall fescue. Even small changes may result in improvements in livestock production efficiency and economics.

This study was designed to select early and medium maturity tall fescue genotypes for (1) high yield potential and (2) improved forage digestibility for subsequent breeding studies.

2.2 Materials and Methods

2.2.1 Development of Populations and Selection Criteria

Two broad-based source population nurseries of early and medium maturity tall fescue genotypes were established in Oregon in 2015 and 2016 at the DLF Pickseed USA research farm in Philomath, OR. The early maturity nursery had 1600 plants (16 rows) and the medium- maturity nursery had 1700 plants (17 rows). These nurseries were used to select parental genotypes for crossing. Parental genotypes used for the two crossing block nurseries were selected from the early and medium maturity groups (Tables 4 and 5). At the end of 2017 and the beginning of 2018, initial observations were taken from both source population nurseries. 19

Selection of plants for the crossing blocks was based on desirable characteristics, including early heading, high-vigor spring growth, and freedom from stem rust.

Table 4. Heading date and spring growth characteristics of the early maturity tall fescue parental genotypes selected for the polycross nursery. Parental Genotypes Heading Date Spring Growth (1)* Spring Growth (2) Spring Growth (Ave.) FTF 108-2 M2- F(E)17 98 8 9 8.5 FTF 108-3 M2- F(E)14 100 9 9 9 FTF 108-4 M2- F(E)8 98 9 9 9 FTF 108-5 M2- F(E)5 101 8 9 8.5 FTF 108-6 M2- F(E)4 100 8 9 8.5 FTF 108-7 M2- F(E)2 101 8 9 8.5 FTF 108-7 M2- F(E)10 103 8 9 8.5 FTF 108-7 M2- F(E)11 104 7 9 8 FTF 108-9 M2- F(E)1 102 8 9 8.5 FTF 108-9 M2- F(E)6 99 8 8 8 FTF 108-9 M2- F(E)13 101 8 9 8.5 FTF 108-9 M2- F(E)18 100 6 9 7.5 FTF 108-10 M2- F(E)12 102 8 9 8.5 FTF 108-11 M2- F(E)3 105 8 8 8 FTF 108-11 M2- F(E)9 98 8 9 8.5 FTF 108-11 M2-16 99 8 8 8 *Spring growth rating on a scale of 1 (weak growing and light green color) to 9 (high vigorous and green color).

For selection, the phenotypic rating was done by visual evaluation. Plants having the best spring growth and earliest maturity prior to anthesis were selected, dug out of their initial field position, and transferred into isolated crossing blocks to allow all possible crosses for developing half-sib families. The best 16 parental genotypes were selected from the early maturity group spaced-plant nursery and were transferred to the first crossing block on 7 May 2018. The best 20 individual plants were selected from the medium maturity group spaced-plant nursery based on the same desirable traits and were transferred to the second crossing block on 9 May 2018. Rye grain (Secale cereale L.) was used to isolate each crossing block from contamination with other pollen. 20

Table 5. Heading date and spring growth characteristics of the medium maturity tall fescue parental genotypes selected for the polycross nursery. Parental Genotypes Heading Date Spring Growth (1)* Spring Growth (2) Spring Growth (Ave.) FTF 98-7- F(M)13 107 9 8 8.5 FTF 98-7- F(M)15 107 8 9 8.5 FTF 98-7- F(M)2 107 8 8 8 FTF 98-7- F(M)4 98 7 8 7.5 FTF 98-7- F(M)16 103 9 9 9 FTF 94-5- F(M)7 100 9 9 9 FTF 94-5- F(M)10 103 8 8 8 FTF 94-5- F(M)14 101 7 8 7.5 FTF 90-6- F(M)5 106 9 9 9 FTF 90-6- F(M)6 106 9 9 9 FTF 90-6- F(M)18 102 8 9 8.5 FTF 90-3- F(M)3 106 8 8 8 FTF 90-3- F(M)19 107 8 9 8.5 FTF 90-3- F(M)12 109 8 8 8 FTF 89 B- F(M)8 107 8 8 8 FTF 84 M2- F(M)1 110 7 9 8 FTF 84 M2- F(M)9 105 9 9 9 FTF 84 M2- F(M)17 104 7 8 7.5 FTF 84 M2- F(M)20 104 7 8 7.5 FTF 83-6- F(M)11 107 9 8 8.5 *Spring growth rating on a scale of 1 (weak growing and light green color) to 9 (high vigorous and green color).

Transferring selected plants involved digging plants with a shovel, ensuring that a substantial root system was preserved. Selected plants were planted into a hollowed-out area surrounded by elevated soil to allow for providing adequate water for the transplanting period

(Figure 1). In the crossing block, plants were spaced at approximately 50 cm. All possible crosses for both the 16 and the 20 parental genotypes were allowed among these desirable plants.

After plants set seeds, individual plants were harvested on 18 June 2018 and transferred to paper bags. 21

Figure 5. Establishment of a crossing block for harvest of half-sib seeds.

Bagged plants were placed in a hoop house to dry for two weeks until seeds were sufficiently dry for threshing and cleaning. All seeds were threshed manually to prevent the loss of seeds, cleaned by a seed cleaner (Westrup, Denmark), then separated into good and poor- quality seeds using a gravity table (Westrup, Denmark). Half-sib seeds from crossing blocks were divided into three groups for: (1) small plot trials, (2) spaced-plant nurseries, and (3) remnant seed. Trials and nurseries were established in 2018; small-plot trials [Experiments A1

(Early) and A2 (Medium)] in Kentucky and spaced-plant nurseries [Experiments B1 (Early) and

B2 (Medium)] in Oregon. The remnant seed was not used but kept for future evaluation of progeny (Darrah et al., 2019).

2.2.2 Field Planting and Evaluation

2.2.2.1 Small Plot Trials (Experiments A1 and A2)

Two forage yield trials (Experiments A1 and A2) were planted to evaluate forage yield and quality of the 32 half-sib families (Exp. A1=16, Exp. A2=16). These small plot trials were established with the residual seed of each plant obtained from the crossing block at the DLF

Pickseed USA farm in Boyd, Kentucky (38° 32'45” N 84° 24’03” W). Seeds of some genotypes in Experiment A1 did not come from crossing blocks because some selected plants did not produce enough seed. We chose to add other half-sib seed into the forage yield trials. Sowing was accomplished using a belt-cone plot seeder (Haldrup SB-25, Løgstør, Denmark). Plots were

5.5 m x 1.4 m (7.7 m2) and the seeding rate was 32 kg/ha (28 lb/a) with 15 cm (6 inch) row spacing.

Figure 6. Drone photograph of the small plot trial established in Boyd, Kentucky on 4 October 2018.

2.2.2.2 Spaced-Plant Nurseries (Experiments B1 and B2)

To obtain seedlings suitable for transplanting into the spaced-plant nurseries

(Experiments B1 and B2), seeds obtained from the crossing blocks were sown into vermiculite on 23 August 2018. After one week, individual small seedlings were transplanted into 50-cell plastic trays. Two weeks after transplanting, seedlings were cut from 13 cm (5 inches) to 5 cm (2 inches) to encourage root system development and transferred to a hoop house to allow for a gradual transition to field conditions. At about one month after planting the seedlings in the trays, they were transplanted to the spaced-plant nurseries for further evaluation and selection.

On 4 October 2018, seedlings were planted on 61 cm centers (25 inches), with 25 individual plants per row; 16 rows of each of the early- and medium maturity groups (Fig. 2.2). The early maturity group was established with 16 genotypes from the polycross nursery. Due to limited seeds from the medium maturity group, there were sufficient seeds to establish only 16 of the 20 parental genotypes. Experiments 1B and 2B were established separately since they were of different maturity groups (early vs. medium genotypes) and separation prevented pollen transfer between the groups.

Weed control of the experimental field was provided by using 2-4, D (a broadleaf herbicide). The transplanting fertilizer was 48 kg ha-1 (43 lbs a-1) of NPK applied in September, early spring fertilizer application was 48 kg N ha-1 (43 lbs of N a-1) applied in April, and 48 kg N ha-1 (43 lbs of N a-1) applied immediately following first, second, and third cuttings, in May,

July, and October. 24

Figure 7. Spaced-plant nursery established in Philomath, Oregon on 4 October 2018.

Agro-morphological characteristics (Table 6) were collected from both nurseries

(Experiments B1 and B2) that included 32 families. Tall fescue plants were harvested as bulk by family (total of 25 plants) on 26 June 2019, then threshed and cleaned to evaluate seed yield per plant.

Table 6. Agro-morphological characteristics used to evaluate the half-sib family seedlings in the spaced-plant nurseries in Philomath, Oregon.

Traits Abbreviation Measurement Plant height PH cm Flag leaf height FLH cm Flag leaf length FLL cm Flag leaf width FLW mm Panicle length PNL cm Spring growth index SGI Score index (1, no growth -9, best growth) Heading date HD Julian Day Tiller number TN - Seed yield SY g

2.2.3 Measurements and Data Collection

2.2.3.1 Dry Matter Yield and Forage Quality

Three Kentucky harvests were taken in 2019: 16 May, 25 July, and 17 October. Whole plots were harvested with a forage harvester (Haldrup, Løgstør, Denmark) equipped with a Zeiss

Corona 45 NIR diode array spectrometer (Carl Zeiss Jena GmbH, Jena, Germany). Plots were swathed to a residual height of 5 cm (2 inches). Means of single cuttings and total dry matter yields were determined (kg DM ha-1; lb DM a-1). Forage quality was assessed for CP, NDF and

ADF by NIRS, and calculating TDN, RFV, and RFQ.

2.2.3.2 Agro-morphological Characteristics

Agro-morphological traits were collected from the spaced-plant nurseries (Experiments

B1 and B2) (16 genotypes in each of the maturity groups). Data were collected from four plants per row; these four plants were used as replicates in Experiments 1B and 2B. The mean values of the four plants were calculated using a handheld computer equipped with the PlantBreeder software (Psion Teklogix Ikon 7505, Europe) used by DLF Pickseed personnel in the field and uploaded to their proprietary database. All genotypes were evaluated under field conditions for the following agronomic characteristics; plant height, tiller number, spring growth, heading date, forage dry matter yield per plant, panicle length, and flag leaf length-height-width (Table 6).

Measurements were made with a ruler for all characteristics except flag leaf width; it was measured with a caliper.

Dry matter yield (g/plant) of tall fescue genotypes was determined as the amount of dried biomass resulting from clipping plants with a grass hook after seed harvest. 26

Spring growth (1-9) of tall fescue families was scored as a visual rating from 1 to 9 (with

9 having the highest vigor, density, and color value, and 1 having the lowest value of the same parameters). To improve consistency of evaluation, all visual estimates were determined by one person, the thesis author.

Heading date (Julian day) of each plant in both spaced-plant nurseries was determined according to the Julian calendar (the number of days from January 1st to the full emergence of three heads). The beginning of heading was defined as the date when plants had three heads fully emerged from the flag leaf.

Tiller number of all fertile tillers per plant was determined by hand counting.

At full anthesis, data were collected for plant height, panicle length, flag leaf length, flag leaf height, and flag leaf width.

Plant height (cm) was measured with a ruler after panicle emergence from the ground level to the top of the highest panicle.

Panicle length (cm) was measured with a ruler after anthesis of a randomly chosen panicle from the panicle neck to the top of the plant.

Flag leaf length (cm) was measured with a ruler on 19 May 2019 of a randomly selected flag leaf from the leaf base to the tip.

Flag leaf height (cm) was measured with a ruler on 19 May 2019 of a randomly chosen flag leaf from the ground level to the base of the flag leaf.

Flag leaf width (mm) was measured with a caliper on 19 May 2019 of a randomly selected flag leaf at mid-point of the leaf. 27

Seed yield per plant (g) was measured after seeds were harvested, cleaned, and threshed.

Each row (25 individual plants) was harvested into a single paper bag, and total seed weight was divided by 25 to determine seed yield per plant.

2.2.5 Experimental Design and Statistical Analysis

Kentucky small-plot yield and quality trial data from Experiments A1 and A2 (32 half-sib families Exp. A1=16, Exp. A2=16) were analyzed as single experiment and subjected to analysis of variance (ANOVA) using a complete block design with one factor (genotype) and two replications. [Using two replications for evaluating yield and quality is standard procedure of the

DLF Pickseed breeding program.] ‘Kentucky 31’ E+ (endophyte infected) was used as a comparator cultivar. Fisher’s protected LSD at the 0.05 probability level was used to separate means whenever the effect of the treatment was significant according to the ANOVA. A coefficient of covariance analysis was performed to determine relationships among the variables

[dry matter yield (DM), CP, NDF, ADF, TDN, RFV, and RFQ].

Data from the Oregon spaced-plant nurseries [Experiments B1 and B2 (32 half-sib families Exp. B1=16, Exp. B2=16)] were subjected to ANOVA with a randomized complete block design with four replications. Single plants were used as replicates. Data were analyzed as a single experiment because all treatments and soil types were the same; including planting date and fertilizer. Plants were evaluated for plant height (PH), tiller number (TN), spring growth index (SGI), heading date (HD), forage dry matter yield per plant (DM), panicle length (PNH), and flag leaf length, height, and width (FLL, FLH, FLW). A correlation coefficient analysis was performed to determine relationships among the variables. The statistical package RStudio was used to analyze all data. 28

2.3 Results

2.3.1 Phenotypical Traits

In Experiments B1 and B2, agro-morphological traits were collected from 32 half-sib family seedlings in Oregon (Table 7). In the spaced-plant nurseries, all data were taken from four individual plants, and the means of these data were used for statistical analysis. Dry matter yield

(DM), heading date (HD), seed yield (SY), tiller number (TN) and plant height (PH) had significant differences, first spring growth indices (1st SGI) had a tendency (P = 0.068), and second and third spring growth indices were not significant.

2.3.1.1 Dry Matter Yield (DM) and Spring Growth Indices (1st, 2nd, 3rd SGI)

Dry matter yield (g/plant) differences among genotypes were highly significant (P <

0.01) (Table 7). The early maturity F(E)5 tall fescue genotype had the greatest dry matter yield with 161.86 g plant-1, approximately 4.5 times more than that of F(E)2 (35.01 g plant-1). Highest dry matter yields were found in the F(E)5, F(M)9, F(E)9, F(E)3, and F(M)11 tall fescue genotypes with 161.86, 143.91, 140.75, 139.00, and 138.74 g plant-1 in 2019 whereas genotypes

F(E)18, F(M)13, F(M)6, F(E)8 and F(E)2 had the lowest yields with 74.10, 73.96, 62.68, 61.09, and 35.01 g plant-1, respectively.

First spring growth index (SGI) values were almost significant (P = 0.06), whereas the second and third spring growth indices had no significant differences among the 32 tall fescue genotypes. Genotypes F(E)4 and F(E)12 had the highest values. The F(E)5 genotype had 6.25,

5.75, and 4.75 spring growth indices, respectively, and the highest dry matter yield (161.86 g plant-1). F(E)2 had the lowest value of the three-spring growth indices and the lowest dry matter yield (35.01 g plant-1). 29

Table 7. Dry matter (DM) yield and three spring growth index (SGI) evaluations of 32 tall fescue genotypes in 2019 in Philomath, OR (sorted by dry matter yield).

Genotypes DM Yield (g plant-1) 1st SGI (1-9)** 2nd SGI (1-9) 3rt SGI (1-9) Ave. SGI (1-9) F(E)5 161.86 a* 6.25 cde 5.75 bcde 4.75 bcd 5.58 cdef F(M)9 143.91 ab 6.75 bcde 6.50 abcde 6.00 abcd 6.42 abcdef F(E)9 140.75 ab 7.75 abcd 6.50 abcde 6.75 ab 7.00 abcde F(E)3 139.00 abc 8.25 abc 7.00 abcd 7.25 ab 7.50 abc F(M)11 138.74 abc 7.75 abcd 7.25 abc 7.00 ab 7.33 abcd F(E)17 126.78 abcd 8.00 abc 6.25 bcde 5.25 abcd 6.50 abcdef F(E)11 125.33 abcd 7.75 abcd 7.25 abc 6.75 ab 7.25 abcd F(E)4 123.54 abcd 9.00 a 8.75 a 8.00 a 8.58 a F(E)1 113.04 abcde 5.50 e 4.75 de 4.75 bcd 5.00 def F(M)18 110.75 abcde 6.50 cde 6.75 abcde 5.75 abcd 6.34 abcdef F(M)10 109.95 abcde 8.00 abc 7.75 ab 7.25 ab 7.67 abc F(M)1 106.46 bcde 5.75 de 5.75 bcde 5.25 abcd 5.59 cdef F(E)12 103.68 bcde 8.75 ab 7.75 ab 7.50 ab 8.00 ab F(E)6 102.20 bcde 7.25 abcde 7.00 abcd 6.75 ab 7.00 abcde F(M)4 101.65 bcde 7.00 abcde 7.50 abc 5.75 abcd 6.75 abcdef F(M)20 100.96 bcde 6.75 bcde 6.50 abcde 5.50 abcd 6.25 abcdef F(M)7 100.86 bcde 8.00 abc 7.25 abc 6.50 abc 7.25 abcd F(M)5 98.90 bcde 7.75 abcd 7.50 abc 7.00 ab 7.42 abc F(E)10 98.67 bcde 7.25 abcde 5.75 bcde 5.25 abcd 6.08 bcdef F(E)16 91.41 bcde 7.00 abcde 5.75 bcde 5.25 abcd 6.00 bcdef F(E)13 90.20 bcde 8.25 abc 7.25 abc 7.00 ab 7.50 abc F(M)14 85.54 cdef 7.00 abcde 5.75 bcde 5.00 bcd 5.75 bcdef F(M)17 83.96 def 6.25 cde 5.75 bcde 4.75 bcd 5.59 cdef F(M)16 82.54 def 5.50 e 5.25 cde 3.75 cd 4.83 ef F(E)14 81.04 def 6.50 cde 7.00 abcd 6.00 abcd 6.67 abcdef F(M)12 79.81 def 7.00 abcde 7.00 abcd 6.00 abcd 6.67 abcdef F(M)3 75.50 def 7.25 abcde 6.00 bcde 5.00 bcd 6.08 bcdef F(E)18 74.10 def 7.00 abcde 6.50 abcde 5.75 abcd 6.42 abcdef F(M)13 73.96 def 6.75 bcde 6.25 bcde 5.50 abcd 6.17 bcdef F(M)6 62.68 ef 7.25 abcde 7.25 abc 6.50 abc 7.00 abcde F(E)8 61.09 ef 7.25 abcde 6.75 abcde 5.75 abcd 6.58 abcdef F(E)2 35.01 f 5.25 e 4.50 e 3.50 d 4.42 f

Mean 100.75 7.13 6.58 5.90 6.54 LSD 54.088 2.098 2.323 2.784 2.338 Prob. > F 0.004 0.068 0.214 0.292 0.212 * Genotype dry matter yields and spring growth indices within rows followed by a different letter are significantly different according to Fisher’s protected least significant difference test, P < 0.05. **Spring growth rating on a scale of 1 (weak growing and light green color) to 9 (high vigorous and green color).

2.3.1.2 Heading Date, Seed Yield, Tiller Number and Plant Height

In Experiments B1 and B2, there was a highly significant (P < 0.01) difference among heading dates of the 32 tall fescue genotypes (Table 8). Heading dates ranged from F(M)1=119 to F(E)4=100.25 Julian days, with mean heading dates extending from 10 April to 29 April for the 32 early and medium tall fescue genotypes. Seventeen genotypes had heading dates greater than 110 Julian days. Even though F(E)2 and F(E)1 were identified as early maturity tall fescue genotypes, they had heading dates similar to the medium maturity tall fescue genotypes, with

118 and 115 Julian days, respectively. F(M)10 and F(M)7 were identified as medium maturity tall fescue genotypes, but they were similar to early maturity genotypes, having heading dates of

107.25 and 109.50 Julian days.

There was a highly significant (P < 0.01) difference for seed yield per plant of the 32 tall fescue genotypes (Table 8). The F(E)9 genotype had the earliest heading date and the highest seed yield, with 71.13 g plant-1, and F(E)1 had the lowest seed yield with 10.99 g plant-1.

There was a highly significant (P < 0.01) difference among genotypes for tiller number

(Table 8). The F(E)12 genotype had the highest tiller number with 113, while the F(M)9 genotype had the lowest with 28.75 tillers. Ninety percent of the genotypes that had high tiller numbers were early maturity tall fescue genotypes, ranging from 66.25 to 113 tillers per plant.

There was a highly significant (P < 0.01) difference for plant height among the 32 genotypes. F(M)7, a medium maturity genotype, had the tallest plant height (141 cm), whereas

F(E)2, an early maturity genotype, had the shortest plant height (70.25 cm). The highest fourteen plant height values were from medium maturity tall fescue genotypes. 31

Table 8. Heading date (HD), seed yield (SY) per plant, tiller number (TN) and plant height (PH) of 32 tall fescue genotypes in Philomath, OR (sorted by heading date). Genotypes HD (Julian days) SY (g plant-1) TN (count per plant) PH (cm) F(M)1 119.00 a* 21.31 fgh 38.25 fghi 107.25 defghi F(M)18 118.75 ab 23.19 efgh 43.50 efghi 120.00 abcdefg F(M)20 118.50 abc 30.01 cdefgh 45.75 defghi 119.25 abcdefgh F(E)2 118.00 abcd 19.04 gh 39.75 fghi 70.25 k F(M)17 117.25 abcde 30.96 cdefgh 33.00 hi 118.75 abcdefgh F(M)16 117.00 abcde 36.86 bcdefg 38.25 fghi 112.00 bcdefgh F(M)9 116.00 abcdef 32.83 cdefg 28.75 i 125.75 abcdef F(E)1 115.00 abcdefg 10.99 h 35.50 ghi 82.00 jk F(M)3 114.75 abcdefgh 34.58 bcdefg 66.00 bcdefgh 118.00 abcdefgh F(M)12 114.00 abcdefgh 37.50 bcdefg 49.25 defghi 134.75 abc F(M)14 114.00 abcdefgh 32.61 cdefg 49.00 defghi 132.75 abc F(M)6 114.00 abcdefgh 32.33 cdefg 52.25 cdefghi 127.50 abcde F(M)13 112.00 abcdefghi 44.87 bcd 45.00 efghi 130.50 abcd F(M)4 111.75 abcdefghi 54.52 ab 56.50 cdefghi 122.50 abcdef F(M)11 110.50 bcdefghij 42.63 bcde 49.50 defghi 127.75 abcde F(E)10 110.25 cdefghij 29.05 cdefgh 59.50 cdefghi 94.50 hijk F(E)14 110.00 defghij 30.88 cdefgh 56.75 cdefghi 96.25 ghij F(M)5 109.75 defdghij 42.75 bcde 81.75 abcd 136.00 ab F(E)6 109.50 efghij 18.32 gh 67.00 bcdefgh 86.75 ijk F(M)7 109.50 efghij 37.56 bcdefg 54.50 cdefghi 141.00 a F(E)16 109.00 efghij 37.60 bcdefg 70.25 bcdefg 96.75 ghij F(E)5 108.25 fghijk 24.89 defgh 42.75 efghi 102.00 fghij F(E)17 107.75 fghijk 40.91 bcdef 78.25 abcde 106.00 defghij F(E)18 107.25 ghijk 19.21 gh 40.50 fghi 97.00 ghij F(M)10 107.25 ghijk 38.50 bcdefg 46.25 defghi 137.50 a F(E)9 106.75 ghijk 71.13 a 102.00 ab 101.00 fghij F(E)8 106.50 hijk 23.24 efgh 59.50 cdefghi 105.00 efghij F(E)11 104.25 ijk 33.15 cdefg 81.50 abcd 103.50 efghij F(E)12 103.00 jk 41.25 bcdef 113.00 a 107.50 defghi F(E)3 102.25 jk 29.33 cdefgh 66.25 bcdefgh 110.75 cdefghi F(E)13 100.25 k 33.78 bcdefg 71.75 bcdef 117.00 abcdefgh F(E)4 100.25 k 47.88 bc 88.00 abc 116.25 abcdefgh

Mean 110.70 33.860 57.800 112.62 LSD 8.3150 20.744 36.048 25.213 Prob. > F ----- 0.0004 0.0005 ------= Prob. > F value is very high. * Genotype heading date, seed yield, tiller number, and plant height within rows followed by a different letter are significant according to Fisher’s protected least significant difference test, P < 0.05.

2.3.1.6 Flag Leaf Length-Height-Width and Panicle Length

Flag leaf length, flag leaf height, flag leaf width, and panicle length values of 32 tall fescue genotypes are presented in Table 9. Flag leaf length varied considerably (P < 0.1) among the 32 genotypes investigated; F(M)14 had the highest value with 28.25 cm, whereas F(M)10 was 15.25 cm.

Table 9. Flag leaf length (FLL), flag leaf height (FLH), flag leaf width (FLW) and panicle length (PNL) of 32 tall fescue genotypes in Philomath, OR (2019). Genotypes FLL (cm) FLH (cm) FLW (mm) PNL (cm) F(E)1 19.50 74.00 7.78 26.50 F(E)2 17.50 62.00 6.74 23.00 F(E)3 18.38 74.25 8.04 37.25 F(E)4 18.50 77.75 8.93 36.25 F(E)5 16.38 73.75 7.47 28.63 F(E)6 23.25 81.50 9.06 35.50 F(E)8 21.50 77.50 9.03 39.75 F(E)9 18.63 76.50 7.94 34.13 F(E)10 21.13 76.75 8.26 34.50 F(E)11 21.13 74.00 8.55 33.88 F(E)12 21.25 75.50 8.84 32.00 F(E)13 18.75 79.75 7.30 30.00 F(E)14 18.75 75.75 8.19 34.50 F(E)16 23.25 67.50 8.82 33.75 F(E)17 25.25 74.75 8.51 32.25 F(E)18 19.00 65.75 8.73 34.00 F(M)1 21.50 69.75 9.40 30.00 F(M)3 22.38 72.50 8.95 30.50 F(M)4 21.25 64.75 8.40 33.25 F(M)5 24.75 69.00 10.14 34.13 F(M)6 17.50 78.25 7.48 30.00 F(M)7 20.75 65.00 8.38 32.50 F(M)9 20.50 78.00 8.58 31.50 F(M)10 15.25 77.25 6.32 29.50 F(M)11 18.50 77.75 7.86 40.50 F(M)12 18.25 81.00 8.09 33.00 F(M)13 21.00 66.25 7.86 43.00 F(M)14 28.25 72.25 10.34 31.25 F(M)16 19.25 62.50 7.56 32.75 F(M)17 19.25 67.25 9.11 31.75 F(M)18 19.25 70.75 8.74 32.13 F(M)20 17.00 72.75 9.05 28.38

Mean 20.21 72.88 8.39 32.81 LSD 6.1133 NS NS NS Prob. > F 0.0507 NS NS NS NS= Not significant

Flag leaf height, flag leaf width, and panicle length were not statistically different (P > 0.1) among genotypes. The highest value for flag leaf height was 81.50 cm [F(E)5], and the lowest value was 62.50 cm [F(M)16]. For flag leaf width, F(M)14 and F(M)10 had the highest and lowest values with 10.34 and 6.32 mm, respectively. For panicle length, F(M)13 and F(E) had the highest and lowest values of 243.00 and 23.00 cm.

2.3.2 Forage Yield and Quality

2019 forage yield and forage quality data from Experiments A1 and A2 in Boyd, Kentucky are provided in Tables 10, 11, and 12. Three cuttings were harvested in May, July, and October.

Samples were analyzed with the on-board NIRS for DM, N (subsequently converted to CP),

NDF, ADF, and ADL. Small plot dry matter data were converted to t ha-1, and TDN, RFV, and

RFQ values were calculated from NDF and ADF values using equations previously provided in

Table 2.

2.3.2.1 First Cutting

The first cutting was taken on 16 May 2019. CP, NDF, ADF, and ADL were estimated via the on-board NIRS, and DM, TDN, RFV, and RFQ values were calculated as previously described. Data are provided in Table 10.

Significant differences (P < 0.001) among genotypes were found for DM yields among the

32 tall fescue genotypes and the check cultivar (Kentucky 31 E+). The mean DM yield for all tall fescue genotypes was 3.93 t ha-1. F(E1)4, F(E1)3, and F(E1)1 genotypes had 4.8 t ha-1 DM yield for the first cutting, almost 50 % more than the Kentucky 31 E+ yield of 3.17 t ha-1. F(M)20 had the lowest DM yield of 2.73 t ha-1, 18 % lower than the check.

Table 10. First cutting (16 May 2019) dry matter (DM) yield, crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), total digestible nutrients (TDN), relative feed value (RFV) and relative forage quality (RFQ) results for 32 tall fescue genotypes and a check variety (Kentucky 31 E+) from the Boyd, Kentucky small-plot trial (sorted by yield). Genotypes DM Yield (t ha-1) CP (%) NDF (%) ADF (%) TDN (%) RFV RFQ F(E1)4 4.79 16.2 45.0 27.5 69.3 139.5 150.2 F(E1)3 4.77 15.8 44.3 26.6 70.2 143.1 154.5 F(E1)1 4.75 15.4 46.8 28.7 68.0 132.3 141.8 F(E1)7 4.74 14.8 43.5 28.0 68.7 143.4 154.1 F(E)17 4.73 15.1 44.5 27.0 69.8 141.7 152.8 F(E1)2 4.58 14.6 43.6 26.9 69.9 145.0 156.5 F(E1)6 4.54 15.0 44.4 26.5 70.3 142.9 154.4 F(M)10 4.51 16.6 44.7 26.4 70.5 142.4 154.0 F(M)7 4.51 16.2 44.9 26.9 69.9 140.7 151.8 F(E1)8 4.32 15.6 43.9 26.5 70.3 144.7 156.3 F(M)11 4.30 15.8 44.4 26.4 70.5 143.3 155.0 F(M)4 4.25 17.2 44.8 25.9 71.0 142.6 154.5 F(M)5 4.21 16.1 45.0 27.1 69.7 140.1 151.1 F(M)1 4.20 17.1 44.8 26.4 70.4 141.8 153.3 F(E)5 4.16 16.2 44.2 26.5 70.4 143.7 155.3 F(M)16 4.07 16.5 42.9 24.0 73.0 152.2 165.9 F(E)3 4.06 16.3 44.1 26.1 70.8 144.8 156.7 F(M)13 4.00 16.6 44.3 25.6 71.3 145.0 157.2 F(E)10 3.93 16.3 43.8 25.7 71.2 146.2 158.4 F(M)17 3.91 15.9 44.5 26.5 70.3 142.7 154.2 F(M)6 3.89 16.5 43.7 26.6 70.3 145.3 157.0 F(M)9 3.76 16.5 43.9 26.4 70.4 144.7 156.4 F(M)12 3.60 16.4 44.2 25.7 71.2 145.1 157.2 F(E1)5 3.54 16.7 43.7 26.1 70.8 145.9 157.9 F(E)11 3.48 14.9 43.3 25.0 71.9 149.2 162.1 F(M)18 3.40 16.2 43.8 25.7 71.2 146.3 158.5 F(M)14 3.19 15.8 43.7 25.5 71.4 147.0 159.5 KY 31 E+ 3.17 16.5 42.8 23.9 73.1 152.9 166.8 F(E)14 3.02 16.7 42.9 24.3 72.6 151.6 165.2 F(E)1 2.98 15.5 44.3 27.0 69.8 142.7 154.0 F(E)13 2.81 15.8 42.6 24.9 72.1 151.8 165.1 F(M)3 2.81 15.6 43.9 25.9 71.0 145.6 157.7 F(M)20 2.73 16.8 42.8 23.5 73.5 153.5 167.7

Mean 3.93 16.0 44.1 26.1 70.7 144.8 156.8 LSD 872.446 NS 1.422 1.946 2.055 NS 8.386 Prob. > F ----- NS 0.002 0.003 0.002 NS 0.0006

----- = Prob. > F value is very high NS= Not significant

There were no significant differences among genotypes for crude protein (CP %). The highest CP % was 17.2 % for F(M)4, and the lowest value was 14.6 % for F(E1)2. Based on the

USDA grass quality guidelines (Table 1), all tall fescue genotypes evaluated in this trial would be classified as premium quality (> 13% CP). 35

There were significant differences in fiber levels among the 32 tall fescue genotypes and the check. F(M)20, Kentucky 31 E+, F(M)16, F(E)14, F(E)13, and F(E)11 had significantly lower NDF and ADF values, and highest TDN, RFV, and RFQ values. Even though F(M)20 had the lowest ADF value (23.5 %), it also had the lowest first cutting yield. The F(M)16 genotype had a similar ADF value (24.0 %) and had a yield (4.07 t ha-1) that was greater than the mean value (3.93 t ha-1). The other five genotypes with lower ADF values had lower yields than the mean.

2.3.2.2 Second Cutting

The second cutting was taken on 25 July 2019. CP, NDF, ADF, and ADL were estimated via the on-board NIRS, and DM, TDN, RFV, and RFQ values were calculated as previously described. Data are provided in Table 11.

Yields among the 32 tall fescue genotypes and the check cultivar were not significantly different (P > 0.1). The mean DM yield of all tall fescue genotypes was 5.3 t ha-1. F(M)12,

F(M)10, and F(M)11 had an average of 5.9 t ha-1. This is almost 7 % more than the check cultivar yield of 5.5 t ha-1. F(E)11 had the lowest DM yield (4.39 t ha-1), 28 % lower than the check.

There were no significant differences among investigated genotypes and the check for

CP. The highest CP value was found for F(M)9 with 13.2 % and the lowest value was 9.9 % for

F(M)9. Based on the USDA grass quality guidelines (Table 1), the forage from all of investigated tall fescue genotypes (except F(M)9) would be classified as good quality (9-13%

CP).

Table 11. Second cutting (16 May 2019) dry matter (DM) yield, crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), total digestible nutrients (TDN), relative feed value (RFV), and relative forage quality (RFQ) values for 32 tall fescue genotypes and a check variety (Kentucky 31 E+) from the Boyd, Kentucky small-plot trial (sorted by yield). Genotypes DM Yield (t ha-1) CP (%) NDF (%) ADF (%) TDN (%) RFV RFQ F(M)12 5.98 10.7 43.5 26.9 69.9 146.0 157.6 F(M)10 5.90 10.2 44.8 27.3 69.5 143.3 154.6 F(M)11 5.86 10.4 46.7 28.2 68.5 134.6 144.7 F(M)4 5.82 11.3 45.1 25.4 71.5 144.7 157.3 F(E)10 5.69 10.6 41.5 22.1 75.1 160.9 176.6 F(M)5 5.61 12.3 50.3 30.4 66.2 120.9 128.6 F(M)3 5.57 10.0 44.7 26.7 70.1 142.0 153.4 F(E1)2 5.56 11.7 43.3 26.9 69.9 146.5 158.1 KY 31 E+ 5.55 10.3 38.7 22.2 74.9 172.2 189.0 F(E)17 5.54 10.6 39.5 22.0 75.1 169.1 185.6 F(E)5 5.48 10.6 40.7 21.7 75.5 164.5 180.8 F(E1)3 5.39 10.5 38.8 24.7 72.3 167.4 182.2 F(E1)4 5.30 11.0 38.3 22.7 74.4 172.8 189.3 F(M)20 5.29 9.9 45.1 27.2 69.6 139.7 150.5 F(E)1 5.27 9.9 44.4 25.6 71.3 144.8 157.1 F(E)13 5.25 11.2 40.3 23.1 74.0 163.6 179.0 F(E1)5 5.21 10.4 50.0 28.5 68.2 124.9 134.0 F(E1)1 5.21 10.9 44.2 28.3 68.4 140.6 150.9 F(E1)7 5.18 11.3 43.5 26.2 70.6 148.0 160.3 F(M)9 5.17 13.2 47.9 27.8 69.0 131.7 141.7 F(E1)8 5.15 11.2 40.0 25.0 71.9 161.5 175.5 F(M)13 5.15 11.3 46.1 27.7 69.1 138.3 149.0 F(M)7 5.14 10.8 43.6 27.5 69.3 144.1 155.1 F(M)6 5.08 12.6 44.3 28.7 68.0 140.1 150.1 F(M)18 5.07 12.0 42.7 27.4 69.4 148.3 159.8 F(E)3 4.92 12.5 40.1 25.9 71.0 160.2 173.6 F(M)17 4.90 10.8 45.0 26.5 70.3 143.0 154.8 F(M)14 4.87 11.2 46.7 28.6 68.1 133.0 142.6 F(M)1 4.83 11.1 39.0 23.6 73.4 168.5 183.9 F(E)14 4.77 10.5 40.3 23.0 74.1 164.8 180.3 F(E1)6 4.75 11.0 42.3 23.7 73.3 155.3 169.6 F(M)16 4.56 12.2 40.0 23.7 73.4 163.7 178.8 F(E)11 4.39 10.5 42.0 23.0 74.1 157.2 172.1

Mean 5.26 11.1 43.1 25.7 71.2 150.2 162.9 LSD 830.160 NS NS NS NS NS NS Prob. > F 0.051 NS NS NS NS NS NS NS= Not significant.

There were no significant differences among the genotypes for NDF or ADF. F(E1)4,

Kentucky 31 E+, and F(E)17 had the lowest NDF and ADF values. Correspondingly, they had the highest TDN and the highest RFV and RFQ values. Even though F(M)17 had a low ADF value, it had a lower DM yield (4.90 t ha-1) than the mean value (5.26 t ha-1). The other two genotypes which had the lower NDF and ADF values had higher DM yields than the mean.

2.3.2.3 Third Cutting

The third cutting was taken on 17 October 2019. CP, NDF, ADF, and ADL were estimated by the on-board NIRS, and DM, TDN, RFV, and RFQ values was calculated as previously described. Data are provided in Table 12.

There were highly significant differences (P < 0.001) among genotypes for DM yields among the 32 tall fescue genotypes and check variety. The mean DM yield of all tall fescue genotypes was 2.83 t ha-1. F(E1)1, F(E1)2, and F(M)4 had 4.12, 3.89, and 3.39 t ha-1 yields, respectively, which were 71, 62.5, and 41.5 %, more than the yield of the check (2.41 t ha-1).

F(E1)6 had the lowest yield with 1.95 t ha-1, 20 % lower than the check.

There were no significant differences among genotypes for percent CP. The two highest

CP values were observed in Kentucky 31 E+, and F(E1)4; 21.1 and 20.8 %, respectively. The lowest value was 16.6 % for F(M)16. Based on the USDA grass quality guidelines (Table 1), the forage from all genotypes would be classified as premium quality (> 13 % CP).

There were no significant quality differences among the 32 tall fescue genotypes and

Kentucky 31 E+. F(E)1, F(E)10, F(E)14, F(E)11, F(E)13, F(E1)3, F(E1)6, F(E1)8, F(M)3,

F(M)4, F(M)14, F(M)17, and F(M)20 had lower NDF and ADF values than the check cultivar.

Correspondingly, they had the highest TDN and the highest RFV and RFQ values. Even though 38

F(M)20 had a low percentage ADF value, it had a lower DM yield (2.70 t ha-1) than the mean value (2.83 t ha-1). The other two genotypes with the highest quality indicators had higher DM yields than the mean value.

Table 12. Third cutting (16 May 2019) dry matter (DM) yield, crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), total digestible nutrients (TDN), relative feed value (RFV) and relative forage quality (RFQ) results for 32 tall fescue genotypes and a check cultivar (Kentucky 31 E+) from the Boyd, Kentucky small-plot trial (sorted by yield). Genotypes DM Yield (t ha-1) CP (%) NDF (%) ADF (%) TDN (%) RFV RFQ F(E1)1 4.12 19.4 40.9 21.3 75.9 166.6 183.5 F(E1)2 3.89 20.3 38.7 22.9 74.2 171.8 188.1 F(M)4 3.39 20.3 36.9 21.6 75.6 181.8 200.0 F(E)10 3.36 18.6 35.7 21.6 75.5 188.0 206.7 F(M)11 3.33 20.0 39.9 22.5 74.6 166.8 182.9 F(M)14 3.25 20.8 37.6 21.7 75.5 178.1 195.8 F(M)5 3.19 19.4 40.0 23.0 74.1 165.4 181.0 F(M)10 3.16 17.6 38.5 23.0 74.1 173.5 190.0 F(M)3 3.08 18.9 37.9 21.1 76.1 179.0 197.3 F(E)13 3.07 19.4 36.6 20.8 76.4 185.2 204.2 F(E1)7 2.94 19.5 39.0 22.1 75.0 171.4 188.2 F(E)17 2.91 19.7 37.7 22.5 74.6 176.3 193.3 F(E)11 2.88 18.5 36.4 19.8 77.5 187.6 207.5 F(M)13 2.82 19.3 38.8 22.2 74.9 172.2 189.0 F(M)7 2.76 19.3 40.1 21.4 75.8 169.0 186.0 F(E)3 2.74 19.6 39.1 23.1 74.0 169.8 185.9 F(M)6 2.73 18.6 38.0 21.2 76.0 178.0 196.0 F(M)18 2.73 19.4 38.6 21.0 76.2 175.6 193.4 F(M)9 2.73 18.7 39.8 21.1 76.1 170.0 187.3 F(M)20 2.70 20.5 35.9 19.7 77.6 191.1 211.4 F(M)12 2.69 20.2 41.0 22.2 75.0 162.6 178.4 F(E)5 2.67 19.6 39.7 22.6 74.5 167.9 184.1 F(E1)8 2.63 19.9 37.8 21.0 76.2 178.8 197.0 F(E1)3 2.58 19.7 37.4 21.7 75.4 179.2 197.0 F(M)16 2.56 16.6 38.5 21.5 75.6 175.6 193.2 F(E)14 2.55 19.9 37.0 21.3 75.9 182.0 200.4 F(E1)4 2.47 20.8 38.0 22.4 74.7 175.1 192.0 KY 31 E+ 2.41 21.1 37.9 22.3 74.8 175.5 192.6 F(M)1 2.37 19.6 39.2 20.7 76.6 172.7 190.4 F(E)1 2.31 20.8 36.0 21.7 75.4 186.2 204.7 F(E1)5 2.28 17.8 38.3 21.0 76.2 177.6 195.8 F(M)17 2.23 19.8 37.2 19.5 77.8 184.1 203.8 F(E1)6 1.95 20.4 37.3 21.7 75.4 179.7 197.5 Mean 2.83 19.5 38.2 21.6 75.6 176.2 193.8 LSD 639.320 NS NS NS NS NS NS Prob. > F ----- NS NS NS NS NS NS ----- = Prob. > F value is very high. NS= Not significant. 39

Correlations

Correlation coefficients among dry matter yield, seed yield, tiller number, plant height, spring growth indices, panicle length, flag leaf height, flag leaf width, and flag leaf length are shown in Table 13.

Table 13. Correlation coefficients between agro-morphological traits [dry matter yield (DM), seed yield (SY), tiller number (TN), plant height (PH), spring growth indices (SGI), panicle length (PNL), flag leaf height (FLH), flag leaf width (FLW), and flag leaf length (FLL)] for the 32 half-sib families grown in spaced-plant nurseries in Philomath, OR in 2019.

2019 DM HD SY TN PH SGA PNL FLH FLW FLL DM 1 -0.27** 0.30*** 0.24** 0.19 0.31*** 0.12 0.20 0.17* 0.01 HD 1 -0.45*** -0.65*** -0.32*** -0.72*** -0.41*** -0.27** -0.09 -0.03 SY 1 0.78*** 0.52*** 0.66*** 0.24** 0.22* 0.15 0.06 TN 1 0.30*** 0.75*** 0.25** 0.30*** 0.25** 0.17* PH 1 0.67*** 0.39*** 0.37*** 0.26** 0.14 SGA 1 0.45*** 0.50*** 0.25** 0.08 PNL 1 0.23** 0.39*** 0.26** FLH 1 0 -0.01 FLW 1 0.63*** FLL 1

*, **, *** Significant at the 0.05, 0.01, and 0.001 probability levels, respectively.

Dry matter yield was positively correlated (P < 0.001) with seed yield and negatively correlated (P < 0.01) with heading date. The associations of dry matter with tiller number, spring growth, and flag leaf width were positive and significant. Significant and positive correlations (P

< 0.001) were observed among seed yield and spring growth, plant height, and tiller number. The highest positive correlation (0.78) was observed between seed yield and tiller number, whereas the lowest positive correlation (0) was observed between flag leaf width and flag leaf height. The associations of heading date with seed yield, tiller number, plant height, and spring growth were negative and highly significant. 40

The correlations of dry matter yield and nutritive quality indicators are shown in Table

14. Dry matter yield had a highly significant positive correlation with NDF and ADF (P <

0.001), whereas the association of dry matter yield with TDN, RFV, and RFQ were negative and highly significant. There was no significant association between dry matter yield and CP (r=

0.21, P > 0.1). A positive correlation was observed between NDF and ADF (r=0.71, P < 0.001).

There were also highly significant and negative correlations between NDF and TDN, NDF and

RFV, and NDF and RFQ (r=0.71, r=0.95, r=0.92, P < 0.001, respectively).

2019 DM CP NDF ADF TDN RFV RFQ

DM 1 -0.21 0.54*** 0.59*** -0.60*** -0.61*** -0.62***

CP 1 -0.15 -0.41*** 0.41*** 0.28* 0.30*

NDF 1 0.71*** -0.71*** -0.95*** -0.92***

ADF 1 -0.99*** -0.90*** -0.92***

TDN 1 0.90*** 0.92***

RFV 1 0.99***

RFQ 1 *, **, *** Significant at the 0.05, 0.01, and 0.001 probability levels, respectively.

2.4 Discussion

2.4.1 Yield and Agro-morphological Traits

Yield is an essential factor for forage and livestock systems and it can be enhanced in tall fescue by recurrent phenotypical selection breeding methods (Piano et al., 2007). In this study, yield varied from 35.01 to 161.9 g plant-1 among the 32 tall fescue genotypes. Seven of the ten 41 genotypes having the highest yield values were from early maturity tall fescue genotypes.

Kanapeckas et al. (2011) reported 198 g plant-1 as the highest yield from three harvests, while in this study, 162 g plant-1 was obtained from only one harvest taken at the plant tillering stage

(after regrowth of the aftermath). The improved yield of the 32 tall fescue genotypes in this study may be due to phenotypic selection based on spring growth and heading date. This confirms our hypothesis that there is sufficient genetic variation in tall fescue genotypes to improve yield potential by recurrent phenotypical selection. Thus, currently highest yielding selections can be used for further selections and evaluations in the breeding program to improve dry matter yield in forage-type tall fescue.

Agro-morphological characteristics of 32 early and medium maturity tall fescue genotypes were determined on plants obtained from half-sib seeds from polycross nurseries in Oregon.

Based on correlation analysis, traits positively related to yield potential were seed yield, tiller number, and average spring growth index values. Heading date was negatively associated with yield potential (Table 13). There was a positive correlation between DM yield and seed yield.

Majidi et al. (2009) and (Amini et al., 2011) also reported a significant positive correlation between these traits in tall fescue.

For DM yield and tiller number, there was a slightly positive correlation (r =0.24). A similar result was reported between yield and tiller number in orchardgrass by Jafari and Naseri (2007) and Majidi et al. (2015), and in tall fescue by Amini et al. (2013).

Correlations between the DM yield and spring growth indices were positively significant in the tall fescue genotypes. Previously, Majidi et al. (2015) reported a positive correlation between these characteristics in tall fescue. 42

Another important association was the negative correlation between DM yield and heading date (r = -0.27). This is consistent with the results reported by Majidi et al. (2015).

No significant correlation (r = 0.19) was found between DM yield and plant height. A positive correlation between DM yield and plant height was expected for the current study because it would seem logical that height is related to yield and it has been reported in other studies; (Amini et al., 2013; Khayam-Nekouei et al., 2000; Majidi et al., 2015; Majidi et al.,

2009) for tall fescue and(Araghi et al., 2014) for smooth bromegrass. However, it may be that canopy structure is more important than height.

There was a negative correlation between heading date and seed yield for these tall fescue genotypes. Nguyen and Sleper (1983) also found a negative correlation between heading date and seed yield in tall fescue; early maturing tall fescue plants had higher seed yield potential.

Fang et al. (2004) also reported an inverse relationship between heading date and seed yield in meadow fescue.

Seed yield and tiller number had a positive correlation (r =0.78). Amini et al. (2013) also reported a positive correlation in tall fescue, and Fang et al. (2004) reported a similar correlation in meadow fescue.

In the present study, the correlation between seed yield and plant height was positive and significant. Majidi et al. (2009) had similar results for tall fescue. This positive relationship was explained by Fang et al. (2004) as taller plants being able to capture more pollen than shorter plants. 43

In addition, the correlation between seed yield and panicle length was positive (r =0.24).

Amini et al. (2013) found a positive correlation for these traits in tall fescue, and Fang et al.

(2004) reported a positive correlation for see yield and panicle length in meadow fescue.

2.4.2 Dry Matter Yield and Forage Quality Traits

Dry matter yield and forage quality were evaluated for 32 early and medium maturity tall fescue genotypes. Genotypes were selected based on vigorous growth and disease-free conditions in two polycross blocks for early and medium maturity genotypes. Seeds obtained from these crosses were planted and evaluated for yield and quality.

Dry matter yields were measured from small plot trials by cutting samples from plots and converting yield to kg ha-1. The average DM yields for the three cuttings were 3.93, 5.26, and

2.83 t ha-1. These values are similar to a first year study reported by Burns et al. (2002), with yields between 3.8 and 5.6 t ha-1 DM. However, for 4th year harvests, Bohle et al. (2020) reported 5.88, 5.80, and 4.17 t ha-1 DM for three cuttings. Decreasing dry matter yields for later harvests is expected due to the reduced stems in later cuttings and the negative correlation between dry matter yield and reduced fiber concentration (Araújo and Coulman, 2004; Vogel and Pedersen, 1993).

High dry matter yield does not always mean high animal production because digestibility is an essential component. Therefore, forage quality parameters were analyzed in the current study.

Protein and amino acids are important components for animals feeding for maintenance, growth, lactation, and reproduction. Crude protein is typically estimated from percent N, multiplying %N by 6.25, since plant proteins are approximately 16% N. In a breeding study on 44 forage species, genotypes having high N (expressed as CP) are desirable for making continuous progress through selection.

In the present study, there were no significant differences among the 32 tall fescue genotypes for CP; values averaged 16, 11.1, and 19.5 % for the three cuttings. These values would be classified as premium, good, and premium quality according to the USDA grass quality guidelines, while they are classified as ‘2’, ‘3’, and ‘prime’ quality in the American Forage &

Grassland Council (AFGC) guidelines (Marsalis et al., 2009) (Table 15).

Table 15. Quality standards for legume, grass, or grass-legume hay based on crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), digestible dry matter (DDM), dry matter intake (DMI), and relative feed value (RFV). % Quality Standard CP ADF NDF DDM DMI RFV Prime >19 <31 <40 >65 >3.0 >151 1 17-19 31-35 40-46 62-65 3.0-2.6 151-125 2 14-16 36-40 47-53 58-61 2.5-2.3 124-103 3 11-13 41-42 54-60 56-57 2.2-2.0 102-87 4 8-10 43-45 61-65 53-55 1.9-1.8 86-75 5 <8 >45 >65 <53 <1.8 <75 Source: Hay Market Task Force, American Forage and Grassland Council (Ball et al., 2015).

Riley (2019) conducted a study in Kentucky on tall fescue forage quality. He reported CP values of 12.5 %, 18.1 %, and 24.8 % for ‘Bronson’, and 11.7 %, 16.5 %, and 22.8 % for ‘Cajun

II’ tall fescue cultivars harvested in May, July, and October of 2015.

Although the mean CP level of our second cutting was lower than the first and third cutting mean values, this value would still be classified as good quality according to the USDA grass quality guidelines (USDA-LPGMN, 2020).

Neutral detergent fiber (NDF) is used as a predictor of forage intake, with lower values associated with higher intake. In the current experiment, NDF values averaged 44.1 %, 43.1 %, 45 and 38.2 % for the three cuttings. Bohle et al. (2020) reported ‘2’, ‘3’, and ‘1’ quality classifications for NDF values of 49.3, 53.5, and 44.2 %. Interrante et al. (2012) reported NDF values between 43.5 % and 60.2 % for tall fescue. Riley (2019) reported NDF values of 58.9,

58.4, and 45.1 % for ‘Bronson’, and 61.2, 60.7, and 47.7 % for ‘Cajun II’ tall fescue cultivars harvested in vegetative stage in May, July, and October.

Acid detergent fiber (ADF) is used as an estimator of forage digestibility, in which lower

ADF values are associated with higher digestibility. We observed mean ADF values of 26.1,

25.7, and 21.6 % in first, second, and third cuttings, respectively. These values would all be categorized in the prime quality level according to the AFGC guidelines. Similarly, Bohle et al.

(2020) reported prime and ‘1’ level quality classifications based on ADF values for three cuttings.

Fulkerson et al. (2007), in a study on the nutritive value of forage species in Australia, reported 28.4, 30.5, 26.3, and 23.3 % ADF for tall fescue harvested at the 3 leaf stage in the spring, summer, fall, and winter, respectively. Interrante et al. (2012) indicated that ADF values ranged from 23.7 to 36.5 %. In the current study, although the ADF value was somewhat higher

(26.1 % in the spring cutting), it is still within the ‘prime’ level according to AFGC guidelines.

Thus, the ADF values of this study were sufficiently low to indicate high digestibility levels.

TDN, RFV, and RFQ values were calculated with equations (Table 3) using NDF and ADF values (Moore and Undersander, 2002). High values for TDN, RFV, and RFQ are desirable since they are positively associated with animal performance. Bohle et al. (2020) reported TDN values of 62.2, 57.7, and 64.4 % for tall fescue in three cuttings. In this study, TDN values were 70.7,

71.2, and 75.6 %, and RFV values were found to be higher;156.8, 162.9, and 193.8 for the three cuttings, respectively. 46

Thus, the low NDF values (inversely related with animal intake), and low ADF values

(inversely related with digestibility) and the high TDN, RFV, and RFQ values indicate high quality. High quality estimates were expected and support our hypothesis that sufficient genetic variability exists to improve fiber digestibility and yield potential of tall fescue by using recurrent phenotypic selection.

The correlations between dry matter yield and NDF and ADF were positive and significant in this study. This is in agreement with Casler (1999) and (Vogel and Pedersen, 1993). However, this is undesirable because low NDF and ADF values are associated with high fiber digestibility.

The other significant (negative) correlations found were between NDF and ADF and TDN, RFV, and RFQ (Table 13). In parallel with these results, dry matter yield was negatively correlated with TDN, RFV, and RFQ. This is expected because the components of equations used to calculate these values are NDF and ADF. In contrast, the relationships between dry matter yield and CP for tall fescue were not significant (r = -0.21) , although Araghi et al. (2014) reported a negative correlation (r = -0.44) between yield and percent CP in smooth bromegrass.

These results of the negative correlation between dry matter and digestibility underscore the challenge of enhancing fiber digestibility and yield potential concurrently for these tall fescue genotypes. Due to the non-significant correlation between yield and CP, improving these desirable characteristics together may be easier.

Based on the results, we conclude that it is possible to develop superior tall fescue cultivars having moderately high DM yield, high CP, and moderately low fiber (NDF and ADF) content from tall fescue genotypes using recurrent phenotypic selection. 47

Summary and Conclusions

Tall fescue is a predominant forage species in the USA. However, it is low in digestibility due to the fact that high lignification of stem tissues limits forage digestibility directly, and animal performance indirectly. To take full advantage of tall fescue as a forage, superior cultivars having low NDF and ADF percentages are needed. Therefore, developing superior tall fescue genotypes with high digestibility and yield potential has become a goal for breeders to increase animal productivity. Since tall fescue has self-incompatibility, recurrent phenotypic selection is often used as the breeding method.

In this study, several characters were studied in 32 tall fescue genotypes in Oregon and

Kentucky. The agro-morphological and quality characteristics investigated were dry matter yield, seed yield, tiller number, plant height, heading date, spring growth in spaced-plant nurseries, and

CP, NDF, ADF, TDN, RFV, and RFQ in small plot trials.

In the spaced-plant nurseries, the correlation between dry matter yield and seed yield was positive and significant, indicating that selection for high dry matter yield and seed yield together in tall fescue is possible. Heading date was found to be negatively correlated with dry matter yield and seed yield, suggesting that early maturing tall fescue would be more reliable to select for high dry matter and seed yielding tall fescue genotypes. Finding a positive correlation of dry matter yield with tiller number, plant height, and spring growth indices suggests that these agro- morphological characters could be used in the recurrent phenotypic selection process to improve forage yield and perhaps forage quality.

In small plot trials, the positive correlation between DM yield and cell wall components

(NDF and ADF) suggests that focusing only on digestibility could cause some decline in dry matter yield. 48

Since yield and digestibility are inversely related, the concurrent improvement of yield and digestibility is challenging. However, the required characteristics of improved tall fescue cultivars (with respect to high yield and low fiber levels) depends on the production system. For example, while high fiber digestibility is essential for dairy cattle, higher forage yield may be a higher priority for beef cattle pastures.

In summary, tall fescue genotypes having high yield and acceptable digestibility levels should be chosen for further evaluation. F(E1)4, F(M)12, and F(E1)1 genotypes were the highest in dry matter yields, whereas F(E)13, F(E1)4, and F(E)10 had the lowest NDF percentage in the first, second, and third cutting. Also, F(M)4, F(M)9, and F(E1)4 were the genotypes having highest CP in the first, second, and third cuttings. Further evaluation is needed for the most promising tall fescue genotypes since these genotypes have been evaluated only through one selection cycle. For stable characteristics, at least four or five cycles are required.

Based on this study, F(E1)1, F(E1)4, F(E)10, F(E)13, F(M)4, F(M)9, and F(M)12 are recommended as the genotypes for further evaluation using recurrent phenotypical selection. One research question to evaluate is whether they display similar digestibility, yield, and CP levels in other environments.

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