Molecular markers have been widely utilized in population genetics, diagnostic taxonomy, and genetic mapping, and can be applied to cultivar discrimination during field selection processes for alfalfa. In this study, whole-genome sequencing information was obtained for seven alfalfa lines and cultivars developed in Korea, including ‘Alfaone (MS001)’, using Next-Generation Sequencing (NGS). Single nucleotide polymorphism (SNP) analysis revealed that ‘Alfaone (MS001)’ could be distinguished from other lines and cultivars using six SNP loci. Specifically, only two SNP loci were sufficient to differentiate ‘Alfaone (MS001)’ from major lines and cultivars such as ‘MS002’ and ‘Alfaking (MSCB07)’. This set of SNP barcodes provides a reliable standard for alfalfa cultivar discrimination, contributing to domestic cultivar protection and the advancement of the Korea forage industry. Furthermore, the development of distinguishing markers across alfalfa cultivars will enhance genetic resource identification and support the breeding of high-quality new cultivars.

Italian ryegrass (Lolium multiflorum Lam.) is a major winter forage crop requiring improved drought tolerance under climate change. This study evaluated 42 accessions, consisting of 20 Korean cultivars and 22 exotic germplasm under four polyethylene glycol (PEG) concentrations (0, 10, 15, and 20%) to assess germination and early seedling responses to drought stress. Increasing PEG levels significantly reduced germination percentage (GP), germination energy (GE), germination index (GI), and seedling vigor index (SVI), while mean germination time (MGT) increased. At 20% PEG, GP decreased by approximately 74% compared with the control. Early seedling vigor, expressed as SVI (integrating fresh weight, shoot length, and root length) also declined depending on concentration. Germination traits (GE and GI) showed strong positive correlations with SVI, indicating that early germination performance was closely associated with subsequent growth under drought stress. Fuzzy comprehensive evaluation and principal component analysis consistently identified several accessions (Earlybird, IR602, IR901, Florida 98, Maximus, Orlando, and Moscovsky 74) as relatively drought tolerant. Overall, the integration of germination and seedling vigor index provides a reliable phenotypic screening approach for identifying drought-tolerant Italian ryegrass.

Molecular markers have been widely applied in population genetics, diagnostic taxonomy, and genetic mapping, and they can also be used for classifying varieties of Italian ryegrass during field selection. In this study, genome-wide sequence information was generated for 10 Italian ryegrass cultivars (40 samples), including ‘Kowinearly’ (KW), using next-generation sequencing (NGS). Single nucleotide polymorphism (SNP) analysis revealed that only three SNP loci were sufficient to distinguish KW from the other cultivars. Furthermore, 21 alternative barcode sets, each consisting of three SNPs, were identified. These SNP barcode sets provide a reliable criterion for cultivar discrimination in Italian ryegrass and can contribute to the protection of domestic varieties and the advancement of the forage industry in Korea. More broadly, the development of distinguishing markers across Italian ryegrass cultivars will enhance genetic resource identification and support the breeding of high-quality new varieties.

This study reports the development of a new alfalfa (Medicago sativa L.) variety, ‘Alfaone’, at the Forage Production Systems Division, National Institute of Animal Science, Rural Development Administration, Korea, from 2015 to 2023. The variety originated from an artificial cross between Xun Lu (maternal parent) and RadarⅡ Brand (paternal parent), followed by pedigree selection and performance testing. The elite line ‘MsCB01’ was subsequently released as ‘Alfaone’. Regional adaptability trials were conducted for two years (2022–2023) across four representative sites in Korea (Cheonan, Pyeongchang, Jeongeup, and Jinju) to evaluate agronomic traits, forage yield, and quality. Evaluated characteristics included plant height, regrowth ability, winter survival, and lodging resistance. The average dry matter yield of ‘Alfaone’ was 20,811 kg/ha, approximately by about 3% higher than that of the standard cultivar ‘Vernal’ (20,236 kg/ha). Yield superiority was particularly evident in Pyeongchang, suggesting excellent cold tolerance and winter hardiness. Assessment of forage nutritive traits indicated that ‘Alfaone’ was comparable to ‘Vernal’, demonstrating that its yield advantage did not come at the expense of quality. Overall, ‘Alfaone’ is a promising cultivar that combines high productivity with strong adaptability to unfavorable environments, particularly cold-prone regions. Its release is expected to promote the expansion of alfalfa cultivation, enhance forage self-sufficiency, and reduce dependence on imported hay in Korea.

This study evaluated the growth characteristics, forage productivity, and feed value of three Italian ryegrass (Lolium multiflorum Lam.) cultivars ‘Earlybird’, ‘Green call’, and ‘Greenfarm2ho’ over two consecutive growing seasons. Growth traits such as cold tolerance, lodging resistance, disease resistance, and insect resistance were assessed, along with plant height. Forage yield was measured as both fresh and dry matter yields, and feed value was analyzed in terms of CP, NDF, ADF, DMI, DDM, RFV and CA content. Among the tested cultivars, ‘Earlybird’ demonstrated superior performance in several key traits. It showed the greatest lodging resistance and plant height, along with the highest fresh and dry matter yields, although differences in yield were not statistically significant. In terms of feed quality, ‘Earlybird’ had the highest CP (10.8%), lowest NDF (54.7%), and highest RFV (109), indicating excellent palatability and digestibility. ‘Greenfarm2ho’ showed intermediate values across all parameters, suggesting balanced productivity and quality. In contrast, ‘Green call’ showed relatively lower lodging resistance and feed value. Overall, ‘Earlybird’ was identified as a promising cultivar for forage production in temperate climates, combining stable yield performance with high nutritional value. These findings offer valuable guidance for cultivar selection in livestock forage systems aiming to improve both productivity and feed efficiency.

Italian ryegrass (Lolium multiflorum L.) is an annual forage crop widely cultivated across the globe for its excellent nutritional value and high productivity. Despite its importance, studies on differentially expressed genes (DEGs) involved in its morphogenesis remain limited. In this study, we employed a high-yielding mutant line developed through radiation mutagenesis to identify candidate genes associated with morphogenesis, focusing on Expansins (EXPs), GRFs (Growth regulating factors), GIFs (GRF-interacting factors), and growth hormone-related genes. RT-qPCR primers were designed, and differential gene expression analysis was performed. Gene expression was assessed in the leaves of seedlings at one to three weeks of age, comparing the control cultivar 'Kowinearly' with the mutant line. Expression patterns fell into four distinct categories: (1) genes consistently exhibiting lower expression in the mutant line across all developmental stages; (2) genes showing persistently low expression in the mutant, while the control displayed a sharp increase at early stages followed by a decline; (3) genes with low expression in the control but a marked early-stage increase in the mutant; and (4) genes with decreasing expression over time in the mutant, contrasting with gradually increasing expression in the control. These expression profiles highlight Actin and GRF6 as Group 1 genes, AXR1 (Auxin-resistant protein 1) and EXPB6 (c) as Group 2 genes, PEVMPP1 (Pyrophosphate-energized vacuolar membrane proton pump 1) as a Group 3 gene, and EF1A (Elongation factor 1-alpha) as a Group 4 gene, all of which are key candidate genes for the development of high-yielding Italian ryegrass cultivars.

This study was conducted from 2022 to 2024 at the Grassland and Forage Crops Division, National Institute of Animal Science (RDA), in Cheonan, Korea, to develop a medium-maturing variety of Italian ryegrass (Lolium multiflorum Lam.). The newly developed tetraploid cultivar, named ‘Spider’, is characterized by its green leaves, semi-erect growth habit in late autumn, and erect growth habit in mid-spring. With a heading date of May 16, ‘Spider’ is classified as a medium-maturing variety. Compared to the control cultivar ‘Kowinmaster’, ‘Spider’ has a 1.0 mm wider leaf blade, a 1.6 cm longer leaf blade, and is 5 cm taller in plant height. Its dry matter yield (10,169 kg/ha) is significantly higher than that of ‘Kowinmaster’ (p<0.05). The crude protein content of ‘Spider’ is 10.4%, which is 0.2% higher than that of the control. Additionally, ‘Spider’ has a neutral detergent fiber (NDF) content of 49.5% and an acid detergent fiber (ADF) content of 26.6%, showing a 2.2% lower NDF and a 0.2% higher ADF compared to ‘Kowinearly’.

Iron (Fe) is a vital element for plants and other organisms, involving in several physiological processes including respiration, chlorophyll biosynthesis, and photosynthesis. Unfortunately, how Fe accumulation regulates in response to light quality has not been well established in plants. Therefore, the aim of the study was to explore the mechanism of Fe homeostasis by light quality. In this study, we found morpho-physiological attributes were significantly improved in response to blue (λmax: 450) compared to white (λ max: 500) and red (λmax: 660) light. The root-shoot length, plant biomass, photosynthesis efficiency (Fv/Fm) and leafgreen (SPAD) significantly declined in response to white and red light. However, these parameters were improved and iron deficiency was substantially alleviated by blue light exposure in alfalfa seedlings. This study might be useful to the forage breeders and farmers for improving alfalfa yield and nutritional benefits.

Silicon (Si) has the potential to improve plant growth and stress tolerance. The study aimed to explore Si-involving plant responses and molecular characterization of different Si-responsive genes in alfalfa. In this study, the exogenous supplementation of Si enhanced plant growth, and biomass yield. Si-acquisition in alfalfa root and shoot was higher in Si-supplemented compared to silicon deficient (-Si) plants, implying Si-acquisition has beneficial on alfalfa plants. As a consequence, the quantum efficiency of photosystem II (Fv/Fm) was significantly increased in silicon-sufficient (+Si) plants. The quantitative gene expression analysis exhibited a significant upregulation of the Lsi1, Lsi2, Lsi3, NIP5;1, and NIP6;1 genes in alfalfa roots, while BOR1, BOR4, NIP2, and NIP3 showed no significant variation in their expression. The MEME results further noticed the association of four motifs related to the major intrinsic protein (MIP). The interaction analysis revealed that NIP5;1 and Lsi1 showed a shared gene network with NIP2, BOR1, and BOR4, and Lsi2, Lsi3 and NIP3-1, respectively. These results suggest that members of the major intrinsic proteins (MIPs) family especially Lsi1, Lsi2, Lsi3, NIP5;1, and NIP6;1 genes helped to pass water and other neutral solutes through the cell membrane and those played significant roles in Si uptake and transport in plants. Together, these insights might be useful for alfalfa breeding and genome editing approaches for alfalfa improvement.

Drought stress is a condition that occurs frequently in the field, it reduces of the agricultural yield of field crops. The aim of the study was to screen drought-adapted genotype of Italian rye grass. The experiments were conducted between the two Italian ryegrass (Lolium multiflorum L.) cultivars viz. Hwasan (H) and Kowinearly (KE). The plants were exposed to drought for 14 days. The results suggest that the morphological traits and biomass yield of KE significantly affected by drought stress-induced oxidative stress as the hydrogen peroxide (H2O2) level was induced, while these parameters were unchanged or less affected in H. Furthermore, the cultivar H showed better adaptation by maintaining several physiological parameter including photosystem-II (Fv/Fm), water use efficiency (WUE) and relative water content (RWC%) level in response to drought stress. These results indicate that the cultivar H shows improved drought tolerance by generic variation, improving photosynthetic efficiency and reducing oxidative stress damages under drought stress. These findings can be useful to the breeder and farmer for improving drought tolerance in Italian rye grass through breeding programs.

Fescues, which are widely cultivated as grasses and forages around the world, are often naturally infected with the endophyte, Epichloë. This fungus, transmitted through seeds, imparts resistance to drying and herbivorous insects in its host without causing any external damage, thereby contributing to the adaptation of the host to the environment and maintaining a symbiosis. However, some endophytes, such as E. coenophialum synthesize ergovaline or lolitrem B, which accumulate in the plant and impart anti-mammalian properties. For example, when livestock consume excessive amounts of grass containing toxic endophytes, problems associated with neuromuscular abnormalities, such as convulsions, paralysis, high fever, decreased milk production, reproductive disorders, and even death, can occur. Therefore, pre-inoculation with non-toxic endogenous fungi or management with endophyte-free grass is important in preventing damage to livestock and producing high-quality forage. To date, the diagnosis of endophytes has been mainly performed by observation under a microscope following staining, or by performing an immune blot assay using a monoclonal antibody. Recently, the polymerase chain reaction (PCR)-based molecular diagnostic method is gaining importance in the fields of agriculture, livestock, and healthcare given the method’s advantages. These include faster results, with greater accuracy and sensitivity than those obtained using conventional diagnostic methods. For the diagnosis of endophytes, the nested PCR method is the only available option developed; however, it is limited by the fact that the level of toxic alkaloid synthesis cannot be estimated. Therefore, in this study, we aimed to develop a triplex real-time PCR diagnostic method that can determine the presence or absence of endophyte infection using DNA extracted from seeds within 1 h, while simultaneously detecting easD and LtmC genes, which are related to toxic alkaloid synthesis. This new method was then also applied to real field samples.

This study was conducted to investigate the forage characteristics of very early-maturing Italian ryegrass (IRG) three cultivars within September, 2021 to July, 2022 in Cheonan, Korea. We used three different cultivars of Italian ryegrass in this study “Greenfarm” “Greenfarm2ho” and “Greenfarm3ho” The results of heading date in this study, “Greenfarm” was recorded 3 days earlier than the other two cultivars, and its lodge tolerance score was the highest among the three. However, in case of plant length, there was no significantly difference in whole cultivars, in addition disease resistance, insect resistance and cold tolerance were not significantly different in three cultivars. The forage productivity of “Greenfarm2ho” was recorded the highest, especially the 1st harvesting of “Greenfarm2ho” yielded significantly the highest and superior to other varieties. As a result of feed value analysis, three cultivars were generally superior to overseas varieties and in particular, “Greenfarm2ho” was recorded to have excellent value because of the lowest NDF and ADF content. On the other hand, there was no significant difference in crude protein content among three varieties. In case of the monosaccharides content "Greenfarm3ho” had significantly higher glucose content than other two varieties, therefore the “Greenfarm” has advantage for preparing high quality of silage. In contrast, there was no significant difference among three varieties in fructose content.

Pollution of agricultural soil by alkaline salts, such as Na2CO3, is a critical and long-lasting problem in cultivable land. The aim of the study was to examine the putative role of citric acid (CA) in alleviating Na2CO3-stress in alfalfa. In this study, Na2CO3 significantly induced leaf chlorosis, inhibited plant growth and photosynthesis related parameters, increased hydrogen peroxide (H2O2) and reduced major antioxidant enzymes (SOD ,CAD, APX) in alfalfa. However, the presence of CA these negative effects of Na2CO3-stress largely recovered. Interestingly, expression of antioxidant and ion transporter genes (Fe-SOD, CAT, APX, DHAR and NHX1) involved in Reactive oxygen species (ROS) homeostasis and oxidative stress tolerance in alfalfa. These findings suggest that CA-mediated Na2CO3 stress alleviation is an ecofriendly approach that would be useful to local farmer for alfalfa and other forage crop cultivation in alkaline soils.

Intercropping cereals with legumes is known to improve forage production and crude protein yield. Sorghum × sudangrass hybrids (SSH) have excellent dry matter content and high cultivation temperatures. In this study, we investigated the growth characteristics, forage productivity, and feed value of intercropping SSH with different legumes in rice paddy fields. We used five treatments in this study SSH monocropping and four intercropping treatments of SSH with, lablab, cowpea, sesbania, and two cultivars of soybean (Chookdu 1 and 2). SSH plant height was not significantly different between the monocropping and intercropping treatments. However, the plant heights of lablab, cowpea, and sesbania were significantly higher than those of the two soybean cultivars. The total dry matter yield (kg/ha) was significantly higher in SSH monocropping than in intercropping; among the intercropping treatments, the one with SSH and Chookdu 2 yielded the highest total dry matter yield. The SSH feed value was significantly different between the monocropping and intercropping treatments, although there were no differences between the intercropping treatments. Among the intercropped legumes, lablab showed the highest neutral detergent fiber and acid detergent fiber contents, and cowpea had the highest crude protein content. These results reveal that intercropping SSH with legumes in paddy fields could be a promising cultivation technique to maintain stable forage productivity.

The aim of this study was to investigate the feasibility of discrimination 12 different cultivar of sorghum × sudangrass hybrid (Sorghum genus) seed through near infrared spectroscopy (NIRS). The amount of samples for develop to the best discriminant equation was 360. Whole samples were applied different three spectra range (visible, NIR and full range) within 680-2500 nm wavelength and the spectrastar 2500 Near near infrared was used to measure spectra. The calibration equation for discriminant analysis was developed partial least square (PLS) regression and discrimination equation (DE) analysis. The PLS discriminant analysis model for three spectra range developed with mathematic pretreatment 1,8,8,1 successfully discriminated 12 different sorghum genus. External validation indicated that all samples were discriminated correctly. The whole discriminant accuracy shown 82 ~ 100 % in NIR full range spectra. The results demonstrated the usefulness of NIRS combined with chemometrics as a rapid method for discrimination of sorghum × sudangrass hybrid cultivar through seed.

Cadmium (Cd) toxicity is a serious limitation for agricultural production. In this study, we explored tolerance mechanism associated with Cd toxicity tolerance in alfalfa plants. We used three distinct alfalfa cultivars M. sativa cv. Vernal, M. sativa cv. Zhung Mu, and M. sativa cv. Xing Jiang Daye in this study. Cd showed declined chlorophyll score in Xing Jiang Daye compared with Zhung Mu and Vernal. No significant change observed among the cultivars for root and shoot length. Atomic absorption spectroscopy analysis demonstrated a significant accumulation of Cd, Fe, S and PC in distinct alfalfa cultivars. However, Zhung Mu and Xing Jiang Daye declined Cd accumulation in root, where Fe, S and PC incremented only in Zhung Mu. It suggests that excess Cd in Zhung Mu possibly inhibited in root by the increased accumulation of Fe, S and PC. This was further confirmed by the response of Fe (MsIRT1) and S transporters (MsSULTR1;2 and MsSULTR1;3), and MsPCS1 genes associated with Fe, S and PC availability and translocation in roots and shoots. It suggests that specially the transcript signal inducing the responses to adjust Cd especially in Zhung Mu. This finding provides the essential background for further molecular breeding program for forage crops.

Salicylic acid (SA) is an essential plant growth regulator that functions as a signaling molecule in plants. The purpose of this study was to clarify how the exogenous application of SA counteracts aluminum stress-induced growth and biomass yield reduction in alfalfa exposed to aluminum (Al) stress. Two-week-old alfalfa seedlings were exposed to a combination of AlCl3 (0 μM, 50 μM and 100 μM, respectively) and SA (0.1 mM) for 72 hours. We observed, Al stress-induced plant growth inhibition and forage yield reduction are Al stress-dependent manner. A significant reduction of plant height (42.0-52.9%), leaf relative water content (13.0-21.4%), root length (35.4-48.7%), shoot fresh weight (31.2-25.9%), root fresh weight (15.4-23.3%), shoot dry weight (12.7-22.2%), roots dry weight (47.3 -53.5%), were observed in alfalfa. In contrast, SA alleviated the Al-stress and enhanced growth and biomass yield in alfalfa. This study provides useful information concerning the role of SA that counteracts aluminum stress-induced growth and yield reduction in alfalfa.

Soil acidity or alkalinity are serious limitations for crop production. The purpose of this study was to clarify the negative effects of extreme pH stress (low and high) on alfalfa vegetative growth (VG) and biomass accumulation (BA). Two-week-old alfalfa seedlings were exposed to different pH (4.0, 4.5, 7.0, 8.0 and 8.5, respectively) levels for 72 hours. Alfalfa grown at pH 4.0 and 8.5 significantly reduced VG and BA, wherein as neutral pH (7.0) comparably exhibited better growth and biomass yield. These results indicate that extreme acidic or alkaline level are critical limiting factors for growth and biomass yield in alfalfa.

본 시험은 사료가치가 우수하고 엔도파이트에 감염되지 않은 톨 페스큐 신품종을 개발하기 위하여 2010년부터 2017년까지 농촌진흥청 국립축산과학원 초지사료과에서 수행되었다. 톨 페스큐 신품종 ‘그린마스터4호’는 6배체 품종으로서 잎은 녹색이며, 출수기가 5월 16일경으로서 대비품종인 ‘Fawn’ 보다 5일 늦은 중생품종이고, 출수기에 초장은 74.6cm 정도이다. 지역적응시험 4개 지역평균 건물수량은 ‘그린마스터4호’가 16.2톤/ha로서 대비품종인 ‘Fawn’보다 5% 많았으며, 지역별 생산성이 4개 지역 모두에서 ‘Fawn’보다 우수하였다. ‘그린마스터4호’의 사료가치는 조단백질(CP) 함량이 15.2%, 건물소화율(IVDMD)이 70.0%, 가소화양분총량(TDN)이 63.6%, 중성세제불용섬유(NDF)가 58.7%, 산성세제불용섬유(ADF)는 32.0%로서 Fawn보다 높거나 대등하였다. ‘그린마스터4호’는 엔도파이트에 감염되지 않는 품종이면서 ‘Fawn’보다 국내 기후환경 적응성과 영속성이 우수하여 초지조성에 알맞은 품종으로서 우리나라 전국에서 재배 가능하다.

Cold, salt and heat are the most critical factors that restrict full genetic potential, growth and development of crops globally. However, clarification of genes expression and regulation is a fundamental approach to understanding the adaptive response of plants under unfavorable environments. In this study, we applied an annealing control primer (ACP) based on the GeneFishing approach to identify differentially expressed genes (DEGs) in Italian ryegrass (cv. Kowinearly) leaves under cold, salt and heat stresses. Two-week-old seedlings were exposed to cold (4°C), salt (NaCl 200 mM) and heat (42°C) treatments for six hours. A total 8 differentially expressed genes were isolated from ryegrass leaves. These genes were sequenced then identified and validated using the National Center for Biotechnology Information (NCBI) database. We identified several promising genes encoding light harvesting chlorophyll a/b binding protein, alpha-glactosidase b, chromosome 3B, elongation factor 1-alpha, FLbaf106f03, Lolium multiflorum plastid, complete genome, translation initiation factor SUI1, and glyceraldehyde-3-phosphate dehydrogenase. These genes were potentially involved in photosynthesis, plant development, protein synthesis and abiotic stress tolerance in plants. However, this study provides new insight regarding molecular information about several genes in response to multiple abiotic stresses. Additionally, these genes may be useful for enhancement of abiotic stress tolerance in fodder crops as well a crop improvement under unfavorable environmental conditions.