A gene encoding an abscisic acid biosynthetic enzyme (LsNCED4) collocates with the high temperature germination locus Htg6.1 in lettuce (Lactuca sp.)

Abstract

Thermoinhibition, or failure of seeds to germinate when imbibed at warm temperatures, can be a significant problem in lettuce (Lactuca sativa L.) production. The reliability of stand establishment would be improved by increasing the ability of lettuce seeds to germinate at high temperatures. Genes encoding germination- or dormancy-related proteins were mapped in a recombinant inbred line population derived from a cross between L. sativa cv. Salinas and L. serriola accession UC96US23. This revealed several candidate genes that are located in the genomic regions containing quantitative trait loci (QTLs) associated with temperature and light requirements for germination. In particular, LsNCED4, a temperature-regulated gene in the biosynthetic pathway for abscisic acid (ABA), a germination inhibitor, mapped to the center of a previously detected QTL for high temperature germination (Htg6.1) from UC96US23. Three sets of sister BC(3)S(2) near-isogenic lines (NILs) that were homozygous for the UC96US23 allele of LsNCED4 at Htg6.1 were developed by backcrossing to cv. Salinas and marker-assisted selection followed by selfing. The maximum temperature for germination of NIL seed lots with the UC96US23 allele at LsNCED4 was increased by 2-3°C when compared with sister NIL seed lots lacking the introgression. In addition, the expression of LsNCED4 was two- to threefold lower in the former NIL lines as compared to expression in the latter. Together, these data strongly implicate LsNCED4 as the candidate gene responsible for the Htg6.1 phenotype and indicate that decreased ABA biosynthesis at high imbibition temperatures is a major factor responsible for the increased germination thermotolerance of UC96US23 seeds.

Fig. 1

Pedigree of NILs developed for the introgression of the Htg6.1 QTL containing LsNCED4 using backcrossing and marker-assisted selection

Fig. 2

Candidate genes in the lettuce consensus map composed of nine linkage groups and collocations with QTL (LG6 is shown in Fig. 3). Mapped candidate genes are in bold italics. Genomic locations of QTL for seed germination traits detected with data reanalyzed for seeds grown in two environments are shown: solid bars Davis, CA, horizontal open bars Yuma, AZ. Lengths of bars denote 1-LOD confidence intervals. Phenotypic variances and additive genetic effects on germination percentage are adjacent to bars (A Salinas, B UC96US23)

Fig. 3

Genetic map of LG6 and positions, LOD thresholds and genetic effects of Htg6.1 using markers from the lettuce integrated map and SPP markers from a mapping microarray. Numbers indicate locations (in cM) of genetic markers used in CIM analysis. Solid bar Davis, CA 2002, open bar De Lier, The Netherlands 2002; diagonal lined bar Davis, CA 2006. Phenotypic variances and additive genetic effects are shown adjacent to bars. Germination/dormancy candidate genes located on LG6 are in bold italics. Solid, dotted and dashed lines in LOD graph represent Davis 2002, Netherlands 2002 and Davis 2006 populations, respectively. Solid vertical line at LOD 5 is average permutated threshold value for declaring QTL significant

Fig. 4

Germination across a temperature gradient of Salinas (filled circles), UC96US23 (filled triangles) and 15 BC₃S₂ sub-NIL seed lots derived from NILs 2 (a), 27 (b), and 86 (c) having either Salinas (A/A open circles) or UC96US23 alleles (B/B open triangles) at LsNCED4. Error bars are ±SD. Differences in germination between BC₃S₂ NIL genotypic classes were significant (p < 0.001) between 29 and 32°C

Fig. 5

Germination phenotypes at 30°C of BC₃S₂ NIL seed lots homozygous at LsNCED4 for either Salinas (A/A a, c, e) or UC96US23 alleles (B/B b, d, f) derived from NIL 2 (a, b), NIL 27 (c, d) and NIL 86 (e, f). The sub-NILs are ordered according to increasing germination percentages. The mean germination percentages averaged across sub-NILs were a, 5.1% (n = 17), b 60.7% (n = 26), c 2.7% (n = 28), d 58.3% (n = 46), e 5.4% (n = 25), and f 65.5% (n = 53). Error bars indicate ±SD

Fig. 6

Expression of LsNCED4 at 30°C in seed lots pooled according to genotype at LE0196 (Htg6.1; 0.5 cM from LsNCED4). A/A Salinas genotype, B/B UC96US23 genotype. Error bars show SE of three biological replicates