Cell wall digestibility of perennial ryegrass : an association mapping approach

Subtitle:Celwandverteerbaarheid van Engels raaigras : een associatiemappingstrategie

Lolium perenne L. (perennial ryegrass) is the most prevalent grass species on dairy farms in temperate regions of the world. Its energy content is among the highest of forage grasses, but not as high as that of more expensive concentrate, which is an essential part of a cow's diet to opti-mize milk yields. Increasing the available energy content of ryegrass, measured as the total digestibility (OMD), would be economically beneficial for the farmer, as it would reduce the amount of concentrate to be added. Moreover, ryegrass often contains an excess in protein, resulting in nitrogen pollution, which would also be reduced if OMD were higher. Past breeding efforts have been successful in increasing the content of water-soluble carbohydrates at the expense of the cell wall portion (NDF), with a consequential positive impact on OMD. However, NDF is important for rumen functioning and the cow's health, and for this reason, we focus on breeding for an improved cell wall digestibility (NDFD). The cell wall is rich in energy, under the form of cellulose and hemicellu-lose. These structural carbohydrates are partially digested by rumen microorganisms. However, these microorganisms cannot break down lignin, a polymer that functions as a glue in the cell wall, and cannot access cellulose and hemicellulose easily. Therefore, decreasing the lignin content or modifying the lignin composition could improve the digestibility of the carbohydrate fraction of the cell wall, and would vastly increase the energy released per gram ryegrass forage. Reducing the amount of ferulic acids, which can cross-link lignin to hemicellulose, may have a similar effect. Here, we present a study that considers both phenotypic and marker-assisted selection (MAS) to improve the cell wall digestibility of perennial ryegrass. In order to obtain useful molecular markers for MAS, an association mapping is conducted where genetic variation in single-nucleotide polymorphisms (SNPs) is correlated with phenotypic variation in lignin content and NDFD. It is known that NDFD decreases with the maturity of the plant. There-fore, we harvested a diverse set of genotypes at a fixed maturity stage, namely at heading. A large variability in NDFD was found at this stage. However, the cell wall of blade, and to a lesser extent stem, is still harder to digest when harvest (and thus heading) occurs later. This is due to vegetative tillers being older when harvested later. To select plants with the highest NDFD independent from this confounder, a harvest-date (HD) correction was applied. Although the genetic diversity and heritability are lower than for NDF, NDFD has a relatively larger impact on OMD. As a result, the response to selection is similar for both traits. As NDF has been successfully used as criterion for improving OMD in the past, we expect the same for NDFD. We conclude that NDFD is a relevant trait for breeding. Our estimates predict a potential to increase milk yields by 2% by selecting the highest-NDFD genotypes in the studied material. A strong relationship was found between lignin content and NDFD, mak-ing it a worthy substitute for phenotypic selection if ruminal fluid (used for NDFD analysis) is not readily available. For phenotypic selection at the plant level, selecting on HD-corrected KL (Klason lignin) is equivalent to selecting on HD-corrected NDFD. For TL (total lignin), even a higher breeding efficiency is expected than for NDFD. For phenotypic selection, separation of organs is considered infeasible, however, for association mapping, organ-specific HD-corrected TL content is recommended as trait, for its high accuracy and high expected heritability. The effects of esterified ferulates (estFA) and diferulates (diFA) on plant NDFD are lim-ited after NIRS prediction, and these traits are therefore not recommend-ed for breeding purposes. Nevertheless, the effect of ferulates should be investigated further. To conduct association mapping, we selected and sequenced 127 genes from 22 gene families related to lignin or ferulic acid biosynthesis. How-ever, gene families contain many genes (paralogs) coding for structurally similar enzymes with possibly different functions. Based on a literature study, we prioritized ryegrass genes in four monolignol biosynthesis gene families, namely 4CL, COMT, CAD and CCR, each containing a large num-ber of genes in ryegrass. Considering the conservation of essential resi-dues in protein sequences, we were able to put forward hypotheses on functional redundancy of paralogs within each gene family. We later used this information for prioritizing genes for marker development. Six molecular markers were significantly associated with NDFD or (pro-tein-corrected) KL and were selected as candidates for validation. Alleles with a large beneficial effect were discovered in the following genes: LpHCALDH1, LPHCALDH11, LpWRKY5, LpCAD1, LpC4H3 and LpLAC1. Once validated, priority should go to finding these beneficial alleles in current elite material. Increasing the frequency of beneficial alleles in a popula-tion is feasible, but pyramiding and fixing several beneficial alleles in a single genotype will not be a sinecure. Nevertheless, in order to develop an elite ryegrass variety with an improved cell wall digestibility, only a few markers may suffice, in combination with phenotypic or genomic selection.

Publication year:2016

Accessibility:Open