1. Introduction
Quinoa (
Chenopodium quinoa Willd.) is an important grain crop belonging to the Amaranthaceae family, and has been cultivated for centuries [
1,
2]. Quinoa has remarkable adaptability to different agro-ecological zones and can withstand temperatures from −4 °C to 38 °C, while many varieties are able to tolerate cold, salinity, and dry desert climates and can grow at relative humidities of 40% to 88%. It produces satisfactory yields with rainfall of 100 to 200 mm and is a highly water efficient plant that is tolerant of low soil moisture [
3] and salinity, all of which signifies its importance in the development of sustainable agricultural systems [
4,
5].
Quinoa originated in the Andean region of South America and was later introduced to Asia, Europe, Africa and North America during the 20th century [
6,
7]. However, even though quinoa was introduced to China in the 1960s, germplasm was restricted to gene banks and was not put to any practical application. Official cultivation started in 1988 by the Tibet Agriculture and Animal Husbandry University and three quinoa varieties from Bolivia were introduced to China [
8]. A range of studies was conducted relating to breeding, plant diseases, cultivation and biological trait assessments in the following decade [
9,
10]. The cultivation of quinoa began in 2013 in northeastern China (Changchun and Baicheng cities), which produced about 3000 kg ha
−1 of grain yield in Changchun city in 2014 alone, while this cultivation area increased to 600 ha in Jilin province in 2018 [
11]. Since that time, quinoa has rapidly gained popularity and become a well-known crop in China due to its superior nutritional quality, with the total cultivation area having increased by nearly 17,000 ha in 2019 [
12].
Global demand for quinoa has risen sharply over a short period of time due to its tremendous nutritional qualities. Quinoa has the potential to contribute to food security in various regions of the world and has the advantage of both its nutritional properties and its agriculture versatility to produce quality food in abundance in countries that have poor access to protein sources [
3]. However, quinoa breeding programs have mainly focused on cultivars that produce high grain yields despite the complex interactions between genotypic traits and environmental factors [
13,
14,
15]. While it is undoubtedly a multipurpose crop, there is little literature concerning the non-grain-producing parts of the plant [
16,
17,
18].
Quinoa grain is an amylaceous raw material that has a high carbohydrate content, mainly consisting of starch and a small percentage of sugars [
19,
20,
21]. It has a higher level of protein than cereal grains such as barley, maize, rice and wheat and contains many of the essential amino acids including leucine, isoleucine, methionine, lysine, threonine, tryptophan, valine and histidine [
21,
22]. It is a plant food that is rich in vitamins (E, C and B complex), minerals (calcium, copper, magnesium, manganese, iron, phosphorus, potassium, sodium and zinc) and fiber, and a large diversity of antioxidant compounds while containing no gluten [
3,
19]. Its protein value is similar to casein from milk, which is essential for growth in children [
23]. Essential amino acids are found in the nucleus of the grain, unlike cereal grains such as rice or wheat, where they are instead located in the exosperm or hull [
3].
Quinoa is fit for both human and animal consumption [
24,
25]. Palatability can be affected by the presence of saponins in the grain, which give it a characteristic bitter taste, and these should be removed before feeding poultry and pigs [
26]. Saponins have been studied widely. Based on sapogenin content, grain containing 4.7–11.3 g/kg of dry matter is classed as “bitter” quinoa, 0.2–0.4 g/kg is classed as “sweet,” and grain with values between these two ranges are considered as “intermediate” [
27]. In mammals, negative effects of saponin have been associated with consumption, digestibility and productivity, lessening its feasibility as forage [
28].
Quinoa has been cultivated widely for its seeds, but it can be utilized as a forage that is highly preferred by ruminants and monogastric animals [
16,
29]. In particular, fresh harvested leaves and chaff are fairly well favored by Camelidae, bovines, goats, sheep and fishes [
26]. Studies have been conducted on quinoa forage and silage to maximize its production including the use of harvest remnants (leaves and stalks) in animal diets, where dry matter yields are acceptable for consumption due to their digestibility and high protein content, making quinoa an excellent quality forage [
16]. However, for better forage quality, anti-quality traits like neutral detergent fiber (NDF) and acid detergent fiber (ADF) should be kept to the lowest levels to improve digestibility. Both NDF and ADF decrease the digestibility of forage when the contents increase. These fibers (NDF and ADF) are cell wall components in the forage, with NDF comprised of ADF plus hemicellulose, and ADF is composed of cellulose and lignin. Further, various other factors are involved to determine the quality of forage, for instance genotype, growth stage, and management practices [
30].
At present, forage corn (
Zea mays L.), alfalfa (
Medicago sativa L.) and oats (
Avena sativa L.) are the three forage species most widely adopted as silage for feeding ruminants in northeastern China. However, it is well known that forage corn is a large production species with very low nutrition values that needs to be mixed with alfalfa to increase its CP content and oats to increase carbohydrates for improved palatability [
31]. There has been limited research on the forage quality of quinoa relative to the forage species mentioned above, despite its popularity across the globe [
32]. Alfalfa has been grown for centuries to feed livestock due to its high palatability and high nutritional properties, and is an excellent source of amino acids, essential vitamins and minerals [
33,
34]. Oat cultivation has been practiced for centuries to feed livestock. It is a fast-growing dual crop grown for both fodder and grain purposes, known for high levels of carbohydrates and essential minerals [
35,
36], producing significant amounts of fresh fodder within a short period (60–70 days) [
37]. Sheepgrass (
Leymus chinensis (Trin.) Tzvel) is one of the most important perennial forage grasses producing high yields, with superior nutritive and forage value in the grasslands of northeastern China [
38,
39]. It has a high forage value and good palatability with tender leaves and stems producing forage yields about 3000 to 4500 kg/ha without irrigation, while the yield reaches 6000 kg/ha with irrigation [
40].
The aim of this study was to identify agronomic traits to facilitate selection of superior high-yielding genotypes that are well adapted to the region. However, benchmarks for desirable forage traits and the best harvest time were not available for the quinoa genotypes currently grown in China. For this reason, varieties developed in recent years via modern breeding and advanced accessions from China were grown alongside quinoa control varieties with wide global distribution to identify agronomic traits associated with grain yield. At the same time the nutritive values of quinoa were compared both intra-specifically and inter-specifically. To our knowledge, no other studies have investigated the yield and nutritive value of quinoa forage harvested during the flowering and grain filing stages and compared these qualities to other well-known forages (alfalfa, oats and sheepgrass). The respective alfalfa and oat genotypes Dongmu 1 and Baiyan 16 were selected because they were bred in the local region and are among the most widely distributed in northeastern China. The corresponding forage quality of Dongmu 1 has been studied previously, and it exhibited higher CP values and lower NDF and ADF values than the mean of 20 tested genotypes [
41]. Similarly, the forage quality of the oat genotype Baiyan 16 represented the highest forage quality in a set of 20 oat genotypes [
35,
40]. The
L. chinensis material used in this experiment represented the most widely used forage type in northeastern China, but at present there are no selected genotypes cultivated in the field. Therefore, this experiment was conducted to examine whether there are differences in the forage yield and nutritive value of quinoa varieties harvested at different stages, and we correlated these properties with other forages to determine the best fodder quality.
2. Materials and Methods
2.1. Planting Material and Growth Conditions
In the middle of spring (12th of May) 2018, 15 quinoa genotypes, including three grain-producing commercial varieties (Titicaca, Rainbow and Illpa), six accessions selected locally from mutations of different varieties and six varieties (accessions) bred in other parts of China, were planted in the ground of the greenhouse at the Songnen Grassland Ecological Research Station of Northeast Normal University (NENU, 44°34′25.5″ north latitude, 123°31′5.9″ east longitude). The origin of the genotypes is described in
Table S1. The soil was sandy clay, with 6.75 g/kg of organic matter, 56.83 mg/g available phosphorous, 0.08% total nitrogen, pH 7.25 and 104.87 us/cm electronic conductivity (more properties of soil see
Figure S1). During tillage, 200 kg/ha nitrogen/phosphate/potash (NPK) complex fertilizer, 15-15-15 (Qingdao Sonef Chemical Company Limited, Qingdao, China), was applied. The experiment was carried out in a randomized complete block design with three replicates for each genotype. Each replicate occupied a 1 × 1 m area, with row spacing of 50 cm and plant spacing of 20 cm. Next to the quinoa, the locally bred alfalfa cultivar Dongmu 1 (one of the most widely planted cultivars with high nutritional values) and local provenance wild
L. chinensis (Trin.) Tzvelev were transplanted into the greenhouse in three replicates with a row spacing of 30 cm and a plant spacing distance of 3 cm. A forage-type oat cultivar Baiyan 16 (bred locally for high forage quality) was planted with 20 cm row spacing at a density of 350 seeds/m
2. Natural sunlight was used as illumination and a ventilation system regulated temperature under 23 °C/18 °C (day/night) until grain maturation of all plants, and drip irrigation facilities were installed to ensure adequate soil moisture.
2.2. Quinoa Crop Phenology and Leaf Chlorophyll
In this experiment, quinoa crop phenology was recorded during the course of growth. Due to crop phenology differences between genotypes, the squaring stage was recorded when more than 50 percent of plants exhibited a fully expanded inflorescence, anthesis was recorded when more than 50 percent of plants produced pollen, the grain filling stage was defined as twenty days after anthesis, and maturity was determined when the majority of leaves had turned yellow. During grain filling, five fully expanded leaves from mid-height on the plant stem were measured for chlorophyll content using a Soil Plant Analysis Development (SPAD) meter (Minolta SPAD 502, Plainfield, IL, USA) and the means calculated.
2.3. Sampling, Yield and Yield Components
During the squaring, anthesis and grain filling stages, two plants from each stage were sampled from each replicate, split along the stem, and oven dried 30 min at 105 °C, then kept for 48 h at 65 °C until constant weight. After that, the samples were combined and ground to a fine powder and stored for later forage quality analysis. During grain filling, the shoots of two plants were harvested 50 mm above the ground to measure fresh weight, water content and dry biomass weight per plant. After maturity, plant height, tiller number, main inflorescence length, culm thickness, 1000-grain weight and grain yield were measured from the remaining plants in each replicate. In the case of alfalfa, samples were harvested at anthesis by using the whole above ground shoot, while whole shoots of Leymus chinensis and oats were harvested during grain filling.
2.4. Fodder Nutritional Quality Analysis
In order to evaluate the nutritional quality of quinoa at different stages and compare with other species, the NDF and ADF were analyzed using standard procedures according to previous methods [
42,
43] developed at the Institute of Grassland Science, NENU. NDF and ADF were measured using a FOSS automatic fiber analyzer (Fibertec™ 8000, FOSS, Hilleroed, Denmark), while CP was measured using a FOSS Kjeldahl analyzer (Kjeltec 8400, FOSS, Hilleroed, Denmark). The phenol-sulfuric acid method was used for the determination of water-soluble carbohydrates (WSCs) as follows. Ten milliliters of 80% ethanol was added to 60 mg of plant material and was kept overnight. Centrifugation was carried out at 5073×
g for 15 min. The supernatant was transferred to a 50 mL volumetric flask. The remaining residue was supplemented with 5 mL of 80% ethanol and centrifuged at 5073×
g for 5 min. The second supernatant was transferred to the same volumetric flask and the combined supernatants made up to 50 mL with 80% ethanol for soluble sugar analysis. For the determination of WSCs, 1 mL of extract, 1 mL of phenol solution and 5 mL of concentrated sulfuric acid were mixed, shaken for 1 min, allowed to stand for 15 min, and the absorbance was measured at 490 nm with a microplate reader. WSC content was calculated using absorbance and standard curve:
where
C is the curve for determining the concentration of the tested sample;
V is the volume of the tested sample;
n is the dilution ratio; and
W the weight of the quinoa sample.
Total saponin content was determined through spectrophotometry, as described by [
44]. Four milliliters of anhydrous methanol was added to 0.2 g of the plant sample and was shaken at 50 °C for 2 h, and centrifuged at 4193×
g for 10 min. The supernatant was taken for subsequent analysis. To analyze the saponin content, 1 mL of extract supernatant and 4 mL of 5% vanillin glacial acetic acid solution were mixed. The mixture was then heated in a water bath for 30 min at 60 °C then cooled in water. The absorbance of the sample was measured at a wavelength of 527 nm using a spectrophotometer (Multiskan GO, Thermo Fisher Scientific Co., Waltham, MA, USA). Oleanolic acid was used as a standard (0–1000 μg mL
−1). Total saponin content was expressed as 100 g
−1 of oleanolic acid equivalents.
The equation used to calculate the relative feeding value was:
, where the terms DMI (dry matter intake) and DDM (digestible dry matter) in the prediction model are: DMI = 120/NDF and DDM = 88.9 − 0.779 × ADF [
42,
43].
2.5. Statistical Analysis
The effect of different genotypes on the agronomic and forage parameters was tested with analysis of variance (ANOVA), performed using the general linear model (GLM) procedure. Mean separation of genotypes for the measured parameters was undertaken with a Tukey’s b multiple comparison test (p < 0.05). Pearson correlation coefficients were calculated for the different genotype yields, and agronomic and forage traits. The data were statistically analyzed using IBM SPSS Statistics 25.0 (IBM Corporation, Chicago, IL, USA). Figures were created by using Sigma-Plot for Windows version 12.5 (Systat Software, Inc., San Jose, CA, USA).