An optimisation of biofuel feedstock can be achieved using plant breeding for increasing stover yield and quality. Stover quality is associated to the composition of the cell wall and the potential for saccharification (42). Only one of the QTLs found was significantly associated with saccharification efficiency in this study, and coincides in the same bin than those previously described for glucose yield (24). With respect to other studies, we describe new regions related to saccharification efficiency in bins 1.05, 6.07 and 10.07. According to the above mentioned co-localizations we should take into account that we are referring to QTLs detected at the bin scale and QTLs detected in bi-parental populations corresponds sometimes to a different vegetal material or pre-treatment method.
Genetic markers and genes associated with these traits can allow the establishment of breeding programs based on genomic selection or marker-assisted selection for increasing stover and saccharification yields to avoid heavy and expensive field evaluations and laboratory assays.
In the next paragraphs, we support the reasons for the proposed genes involved in plant development, growth, and assimilation of nutrients as probable candidate genes for the QTL involved in stover yield.
Nitrogen supply is one of the major factors limiting growth and productivity in crops, affecting both grain and stover yields. Therefore, we propose glutamate synthase 1 (Zm00001d029732) gene as candidate gene for the QTL qStoverYield_1_1 because the enzyme glutamate synthase is essential for ammonia assimilation in plants and has been proposed as a key target enzyme to improve nitrogen assimilation efficiency. Chichkova et al. (43) found a direct relation between this gene and variability for biomass as they observed increases in shoot weight and shoot total nitrogen and carbon contents in tobacco transgenic plants overexpressing NADH-glutamate synthase.
In addition, stover yield is determined by plant development and growth, processes that are greatly limited by biotic and abiotic stresses (1,44). Authors, have reported that biomass yield increase can be achieved through enhancing mechanisms of stress tolerance (45–47). As generation of reactive oxygen species (ROS) occurs at stress conditions, plant mechanisms to protect from ROS damage could contribute to enhanced tolerance to stress because oxidative stress has a negative effect on biomass and plant fitness (48). Consequently, glutathione S-transferase (GST) genes that lie within the confidence intervals of QTLs for stover yield such as Zm00001d041772 which is located within qStoverYield_3_1 QTL could be highlighted as candidate genes because GST contribute to minimise ROS species (49). In the same way, for qStoverYield_3_2 we also spot as candidate the L-ascorbate peroxidase 2 gene (Zm00001d041939), involved in ascorbate-glutathione cycle and detoxification of hydrogen peroxide (50).
Another gene can be proposed as candidate gene for QTL qStoverYield_3_1, the Zar9 (Zm00001d041774), because it belongs to the Auxin-Regulated Genes involved in Organ Size (ARGOS) family of genes, that controls plant growth and organ size and have been assigned as key factor determining yield (51–53). Guo et al. (54) tested the effects of the maize overexpression of a Zar gene and found increased stalk, ear, and total dry biomasses, and leaf area. Also, the GRAS gene family plays a crucial role in diverse plant growth and development processes by being involved in gibberellin signalling. Within the confidence interval of qStoverYield_3_2, we highlight a gene encoding a GRAS tf as possible candidate for this QTL.
For the QTL qStoverYield_5_1, we highlight the gene (Zm00001d012925) that codifies for the α- subunit of an anthranilate synthase, the first enzyme in the tryptophan biosynthesis pathway, that has been suggested as a broader regulator in auxin production (55). Lu et al. (56) linked polymorphisms at a anthranilate synthase gene in rice to variability for yield; meanwhile higher accumulation of anthranilate synthase α-subunit in transgenic poplars could result in enhanced growth through the regulation of auxin biosynthesis (57).
Regarding saccharification efficiency, among the genes found within the confidence interval of each QTL, and based on previous knowledge, we can point out those involved in the phenylpropanoid pathway, especially in monolignol biosynthesis and polymerization, and the ones involved in cell wall deconstruction, and recalcitrance. Biomass hydrolysis is a key factor in lignocellulosic deconstruction and the complex polysaccharide matrix in the cell wall limits the accessibility to cellulose conferring recalcitrance to the whole structure and reducing saccharification efficiency (1,3). Despite its low abundance in maize stems and leaves, pectins play a role in limiting saccharification efficiency (58,59) so genes involved in pectin biosynthesis could be good candidates for saccharification efficiency QTLs. That is the case of the gene Zm00001d038947 (within qSACC_6_1 QTL), a galacturonosyl transferase involved in biosynthesis of homogalacturan (HG) which is the main pectic polysaccharide and is involved in cell adhesion and cell wall plasticity (60). Downregulation of enzymes involved in HG biosynthesis modifies pectin organization and composition; leading to a significant reduction in cell wall cross-linking, and recalcitrance to pre-treatment and deconstruction (61–63). Interestingly, we also found that two pectinesterases (Zm00001d030622 and Zm00001d030643) are within the confidence interval of another saccharification efficiency QTL (qSACC_1_2). Pectinesterases are a group of pectinases that modify pectins during cell wall development and can consequently affect the cell wall architecture (64).
On the other hand, lignin has been pointed out as the most important polymer in the determination of biomass recalcitrance (65). This role has led to the identification of the genes involved in monolignol biosynthesis and polymerization, as well as the transcription factors that regulate lignin synthesis. The maize MYBs are classified into 37 groups (66), according to their phylogeny, expression patterns, and also structural and functional characteristics. In our association study, we identified two genes encoding MYB tf probably involved in the phenylpropanoid pathway that lie within the confidence intervals of QTL for saccharification efficiency (qSACC_1_2 and qSACC_6_1): MYB 100 (Zm00001d030644), clustered in G13 “metabolism” and MYB 130 (Zm00001d038930) in G28 “phenylpropanoid pathway”. A branch of the phenylpropanoid pathway leads to the synthesis of phenolic precursors and monomers of lignin; and the other branch to flavonoids, stilbenes and coumarins. The closest Arabidopsis ortholog of MYB 130 is a gene involved in anthocyanin metabolism. As suggested by Barrière et al. (67), MYB 130 may not be directly involved in biosynthesis of lignified cell walls; still it could be indirectly involved in phenylpropanoid pathway through regulation of other compounds (66,67). Among Arabidopsis ortholog genes of maize MYB 100 classified in the same group, AtMYB61 was shown to participate in Arabidopsis lignin biosynthesis (68,69), and it produced ectopic lignification when over-expressed in Arabidopsis plants, besides being involved in establishing the xylem to phloem ratio during secondary growth (68). Another ortholog, HvMYB33, is expressed in lignifying tissue of barley (70); meanwhile AtMYB 50, AtMYB 55 and AtMYB 86 may be also involved in regulating secondary cell wall biosynthesis (71).
In the phenylpropanoid pathway that leads to the synthesis of lignin monolignols, Coumaroyl-CoA is converted into caffeoyl-CoA through the formation of quinate or shikimate esters by a hydroxycinnamoyl transferase (HCT) (72). Here, we found a gene encoding a hydroxycinnamoyl transferase (Zm00001d030542) that lies within the confidence interval for the saccharification efficiency QTL in chromosome 1 (qSACC_1_1). The downregulation of this enzyme has been shown to change lignin composition by enriching H units and decreasing the S:G ratio (73–75). Increases in H produce a greater frequency of resistant inter-unit bonds, and this strengthening of the cell wall leads to less amenability and degradability (76–78). Therefore, gene Zm00001d030542 appears as a promising candidate gene for improving saccharification efficiency.
Finally, cell wall composition and organisation is a remarkably polygenic character and is influenced by hormonal and developmental factors. Interestingly, we found a gene encoding a Gibbellerin 2-oxidase (Zm00001d038996), which irreversibly catalyzes the deactivation of bioactive gibbellerin, within the confidence interval of the saccharification efficiency QTL qSACC_6_1. Gibberellic acid (GA) has been shown to regulate lignin biosynthesis and morphogenesis, at higher amounts of bioactive GA, levels of lignification in plant tissues are increased suggesting that lignification and biomass recalcitrance could be optimized by targeting gibberellin biosynthesis (79).