Association study between the gibberellic acid insensitive gene and leaf length in a Lolium perenne L. synthetic variety

Abstract

Background: Association studies are of great interest to identify genes explaining trait variation since they deal with more than just a few alleles like classical QTL analyses. They are usually performed using collections representing a wide range of variability but which could present a genetic substructure. The aim of this paper is to demonstrate that association studies can be performed using synthetic varieties obtained after several panmictic generations. This demonstration is based on an example of association between the gibberellic acid insensitive gene (GAI) polymorphism and leaf length polymorphism in 'Herbie', a synthetic variety of perennial ryegrass.

Methods: Leaf growth parameters, consisted of leaf length, maximum leaf elongation rate (LERmax) and leaf elongation duration (LED), were evaluated in spring and autumn on 216 plants of Herbie with three replicates. For each plant, a sequence of 370 bp in GAI was analysed for polymorphism.

Results: Genetic effect was highly significant for all traits. Broad sense heritabilities were higher for leaf length and LERmax with about 0.7 in each period and 0.5 considering both periods than for LED with about 0.4 in each period and 0.3 considering both periods. GAI was highly polymorphic with an average of 12 bp between two consecutive SNPs and 39 haplotypes in which 9 were more frequent. Linkage disequilibrium declined rapidly with distance with r 2 values lower than 0.2 beyond 150 bp. Sequence polymorphism of GAI explained 8-14% of leaf growth parameter variation. A single SNP explained 4% of the phenotypic variance of leaf length in both periods which represents a difference of 33 mm on an average of 300 mm.

Conclusions: Synthetic varieties in which linkage disequilibrium declines rapidly with distance are suitable for association studies using the "candidate gene" approach. GAI polymorphism was found to be associated with leaf length polymorphism which was more correlated to LERmax than to LED in Herbie. It is a good candidate to explain leaf length variation in other plant material.

Figure 1

Description of the creation of the variety 'Herbie'.

Figure 2

Example of the Eq.1 function fitted between leaf length and thermal time. Leaf elongation rate obtained by derivation of the function is shown. Are indicated: the maximum leaf length (Ym), the starting time of elongation with a leaf length of 5 mm (tc), the time when the speed elongation is maximal (tm), the time when the leaf finishes growing (te) and the leaf elongation duration (LED).

Figure 3

Representation of GRAS protein structure from Bolle [38]. DELLA and VHIID protein domains and leucine (Leu) rich regions are indicated. The arrows show the amplified part of GAI studied.

Figure 4

Pattern of gametic linkage disequilibrium (LD) decays. LD was measured between SNPs pairs, with r², from haplotypic data of GAI gene inferred by PHASE software. A logarithm fitting-curve is presented in black (R² = 0.09).

Figure 5

Haplotype tree of the SNP polymorphism from the GAI gene. The haplotypes are shown in Table 6. Only haplotypes present more than 10 times were taken into account. The arrows indicate the transitions with a Psim higher than 0.05 in the tree-scanning analysis. The tree was obtained from the program PhyloDraw (contact: jhchoi@pearl.cs.pusan.ac.kr) on the basis of the treefile output from PHYLIP.