4.2. Forage Yield of Napier Grass Genotypes
Forage biomass yield is one of the major targets in forage crop improvement, where fast-growing and high LSR types are preferred [34
]. The current study revealed that TFW and TDW were higher in the wet season than in the dry season, indicating that moisture availability was a determinant factor for growth and development of Napier grass. For example, top productive genotypes BAGCE 94, BAGCE 100, and BAGCE 30 had mean TDW of 5.7 t/ha, 5.5 t/ha, and 4.9 t/ha, respectively, during the wet season, while in the dry season, their respective mean TDW were 2.5 t/ha, 1.64 t/ha, and 1.46 t/ha, respectively (Table A3
; Appendix B
). These results indicate that the potential performance for high levels of forage production is associated with adequate moisture levels in the soil. With regards to TDW in the wet season, TDW in the current study was much lower than previously reported [19
] for some Napier grass genotypes from the ILRI collection grown under Ethiopian conditions. While these variations might be attributed to differences in genotype performance, the influences of the prevailing environmental conditions and agronomic management practices among these studies could not be ruled out.
In relation to LSR, the proportion of leaf to stem was higher in the dry season than the wet season, which indicated slower stem development during the dry season. In the wet season, the highest mean LSR was obtained from genotypes CNPGL 93-08-1, BAGCE 63, and BAGCE 80, with mean values of 4.9%, 4.8%, and 4.1%, respectively, while mean LSR values for top biomass producing genotypes had low to medium LSR values. Thus, top yielding genotypes can be classified as fast growing with a higher stem proportion, which might have implications in feed nutritive quality because leaves have been shown to have a high level of feed quality compared to the stem fraction [36
Significant variation for TFW, TDW, and LSR was observed among genotypes, regardless of the season (wet or dry), indicating the existence of genetic variability among the Napier grass genotypes. As Napier grass is a perennial fodder, the identification of consistently productive cultivars across the seasons and years is an important parameter. Results from the current study showed that top biomass yielding genotypes in the dry season were also top producing during the wet season. Genotypes that show consistently high TDW throughout the wet and dry seasons were BAGCE 94, BAGCE 100, and BAGCE 53. These genotypes performed better than some of the current ILRI ‘best bet’ genotypes planted in a replicated trial in adjacent plots (Table A1
and Table A2
, Appendix A
), indicating the potential of these genotypes to be used for improved biomass yield in the tested environment. Under the Brazilian environment, however, BAGCE 53 was an early flowering and low biomass producing genotype [37
], which indicates differential performance of this genotype in the respective environments.
The cumulative annual yield also reflects that these top yielding genotypes produced the highest annual forage yield and can therefore be selected for stable forage production across environments (Table 8
). Furthermore, the observation of relatively high and considerable GCV and PCV values for TFW and TDW in the current study indicates the importance of the genotypic effect in the expression of these traits.
4.3. Genotypic Performance for Feed Nutritional Quality
Nutritional quality of forage crops depends mainly on the digestibility and amount of essential nutrients [38
]. Results from the feed quality analyses revealed the presence of genotypic variability both in wet and dry seasons, but the GCV and PCV values were low for feed nutritional quality traits in this study, similar to a previous report [39
] that showed low genotypic and phenotypic coefficient of variation for quality traits. In the present study, the mean NDF value of studied genotypes was higher than the maximum expected mean NDF value for forage grasses [40
]; however, the observed mean value for IVOMD and CP contents were higher than the minimum requirement for maintaining ruminants [42
]. In livestock production, energy is one of the limiting factors in animal performance [44
]. ME is the commonly used trait for evaluating energy content of feed [45
]. Napier grass genotypes were within the range of ME content for tropical grasses [44
]. Genotypes that had the highest ME content were BAGCE 86, CNPGL 96-24-1, and CNPGL 93-08-1, both in wet and dry seasons. Overall, the observed nutritional performance of genotypes indicated that these genotypes could be an important resource for improving feed quality.
Generally, top biomass yielding genotypes had the highest fiber components, i.e., NDF and ADF, while the respective, CP, IVOMD, and ME values were low. In contrast, low forage biomass yielding genotypes had high CP, IVOMD, and ME, with low fiber components. It is also interesting to note that purple Napier grass genotypes, such as CNPGL 93-18-2, CNPGL 93-08-1, and CNPGL 92-133-3, had high CP and ME contents. These findings were consistent with the observed negative correlation between fiber components and CP, IVOMD, and ME. A previous report [46
] also indicated that an increase in fiber components reduces cellular nutrients, such as crude protein and digestibility. Furthermore, a PCA partitioned the genotypes into three cluster groups; for example, genotypes in cluster one showed high CP, IVOMD, and ME values, while genotypes in cluster two showed high fiber components and forage biomass yield in both wet and dry seasons. The observed values of feed nutritional quality traits were highly dependent on the season for all Napier grass genotypes in this study. For example, fiber components NDF and ADF were higher in the wet season, while IVOMD, CP, and ME were lower in the wet season. A decline in organic matter digestibility and metabolizable energy during the wet season is associated with increased phenological development and forage production, which stimulates stem production, resulting in a higher stem proportion [47
]. Functionally, this decline in digestibility is attributed to an increase in structural components and cell maturation [28
]. Therefore, the increased concentration of CP, IVOMD, and ME during the dry season could potentially compensate for reduced forage biomass as reflected by relatively lower TDW and higher LSR in the dry season. However, it should be noted that our findings are not in agreement with the results of Reference [49
], who reported a decline in CP, IVOMD, and ME values and an increase in NDF, ADF, and ADL in the dry season. These differences might be attributed to the phenological differences of plants at time of harvest.
In line with the scaling relationship between TDW and feed quality traits, the slopes were significantly different from 1 and tended to increase in the dry season compared to the wet season, which might show a flexible relationship between TDW and feed quality traits. The difference in CP, IVOMD, and ME between wet and dry seasons for the same TDW might indicate: (1) an increased structural development, such as stem elongation and reduced leaf area, which would negatively affect these traits; and (2) accurate comparison of genotypes for feed quality traits can be done by considering the effect of plant size [28
]. In addition, the rapid decline in the dry season for CP, IVOMD, and ME as compared to the wet season might be attributed to an abiotic stress response due to increased temperature and reduced soil moisture in the dry season that could trigger fiber deposition within the cell wall [50
]. The allometry based on TDW explained about 30% of the variation; hence, the residual variation would be an important factor to test the significant variation of feed quality traits among genotypes (Table 7
). Furthermore, the observed low R2
value would indicate that prediction based on biomass yield alone might not explain the changes in feed quality traits (Table 7
Top forage biomass yielding genotypes produced the highest cumulative annual ME yield, indicating increased annual forage biomass yield in high yielding genotypes complements the observed low ME content per kilogram of dry matter. This emphasizes that energy production, per se, coupled with forage biomass production is crucial for characterizing and selecting Napier grass genotypes for livestock/dairy production. Therefore, the efficiency of selection for improved feed quality performance is influenced by how traits are associated. For example, selection only for higher forage biomass yield could compromise CP, IVOMD, and ME. Since no genotypes were entirely high in TDW and ME, exploitation of plant breeding and marker assisted selection (MAS) could be an alternative strategy to develop improved Napier grass for both forage biomass yield and feed quality traits.
4.4. Markers Associated with Dry Weight Yields and Metabolizable Energy
Eighty-two markers (SNPs and SilicoDArTs) associated with annual dry weight yield and metabolizable energy were identified, using both the mixed model (MM) and ANOVA statistical model, in marker-trait association analysis. The MM was corrected with pairwise kinship matrix data, while the ANOVA model did not include any correction. As compared to MM, the ANOVA detected a higher number of markers associated with the traits. However, we report here only on markers detected by both models for robust selection. Many of the identified markers detected most of the high performing genotypes identified in the agronomic performance trial. Ten markers (most of them SNPs) were highly discriminant between high and low yielding genotypes. For example, the heterozygous form of one SNP marker (ID number 23617359) identified the top performing accessions for TDW and ME. However, four additional genotypes (BAGCE_93, BAGCE_1, BAGCE_97, and BAGCE_56, all high yielding) also showed the same heterozygous form of the maker. By using a combination of two SNP markers (ID numbers 23617359 and 30283369), two of the accessions (BAGCE_93 and BAGCE_1) were excluded, showing that the use of a combination of markers could improve the diagnostic ability of the markers.
The DArTseq genotyping platform [52
] produces short sequence reads, averaging about 55 nt in length, corresponding to each of the SilicoDArT and SNP markers [12
]. In this study, the short sequence reads corresponding to the associated markers were compared with previously reported Napier grass genomic [30
] and transcriptome [31
] sequences. Very few of the DArTseq sequence reads aligned with the assembled genomic sequences, as only assembled sequences longer than 200 nt were used in the comparison. However, many of the DArTseq reads aligned with the transcriptome sequences, indicating that many of the associated markers are found within the gene coding regions. DNA variation in the coding regions is likely to provide a significant contribution to adaptation and productivity [53
]. One of these sequences (corresponding with marker 23644354) was annotated as an ‘enhanced ethylene response protein’, which is one of the genes involved in source-sink communication and sucrose-mediated regulation of starch synthesis [54
A greater number of associations were detected on chromosome 3, which might indicate the position of quantitative trait locus (QTL) governing biomass yield and metabolizable energy. One of these associated markers was annotated as cytochrome P450. In a previous GWAS analysis in pearl millet [15
], cytochrome P450 was associated with plant population, grain number, panicle number, and tiller numbers. Plant cytochrome P450s are involved in a wide range of biosynthetic pathways and play critical roles in the synthesis of lignins and various fatty acid conjugates, hormones, pigments, defense compounds, and signaling molecules [55
The identified markers could be useful in the implementation of marker assisted selection in Napier grass to improve the efficiency and precision of selecting genotypes for higher dry weight and metabolizable energy. When compared to the field phenotyping and evaluation, the use of the markers is simpler and can save time, resources, and effort, as the selection can be carried out at the seedling stage. In addition, the markers can be important in the identification and mapping of QTLs controlling the traits [56
]. In most of the associated markers, the minor allele frequency was associated with higher dry weight yield and metabolizable energy, hence, increasing the frequency of the minor alleles in the population by breeding could improve the productivity of Napier grass with crucial implications for livestock productivity.