QTLs and candidate genes for desiccation and abscisic acid content in maize kernels

Abstract

Background: Kernel moisture at harvest is an important trait since a low value is required to prevent unexpected early germination and ensure seed preservation. It is also well known that early germination occurs in viviparous mutants, which are impaired in abscisic acid (ABA) biosynthesis. To provide some insight into the genetic determinism of kernel desiccation in maize, quantitative trait loci (QTLs) were detected for traits related to kernel moisture and ABA content in both embryo and endosperm during kernel desiccation. In parallel, the expression and mapping of genes involved in kernel desiccation and ABA biosynthesis, were examined to detect candidate genes.

Results: The use of an intermated recombinant inbred line population allowed for precise QTL mapping. For 29 traits examined in an unreplicated time course trial of days after pollination, a total of 78 QTLs were detected, 43 being related to kernel desiccation, 15 to kernel weight and 20 to ABA content. Multi QTL models explained 35 to 50% of the phenotypic variation for traits related to water status, indicating a large genetic control amenable to breeding. Ten of the 20 loci controlling ABA content colocated with previously detected QTLs controlling water status and ABA content in water stressed leaves. Mapping of candidate genes associated with kernel desiccation and ABA biosynthesis revealed several colocations between genes with putative functions and QTLs. Parallel investigation via RT-PCR experiments showed that the expression patterns of the ABA-responsive Rab17 and Rab28 genes as well as the late embryogenesis abundant Emb5 and aquaporin genes were related to desiccation rate and parental allele effect. Database searches led to the identification and mapping of two zeaxanthin epoxidase (ZEP) and five novel 9-cis-epoxycarotenoid dioxygenase (NCED) related genes, both gene families being involved in ABA biosynthesis. The expression of these genes appeared independent in the embryo and endosperm and not correlated with ABA content in either tissue.

Conclusions: A high resolution QTL map for kernel desiccation and ABA content in embryo and endosperm showed several precise colocations between desiccation and ABA traits. Five new members of the maize NCED gene family and another maize ZEP gene were identified and mapped. Among all the identified candidates, aquaporins and members of the Responsive to ABA gene family appeared better candidates than NCEDs and ZEPs.

Figure 1

Time course of mean water status and ABA content in kernel in parents and inbreds. The LHRF_F_3:4segregating population derived from the cross between the F2 and the F252 parental inbred lines differing in desiccation rate (four intermated cycle were performed after the second generation and before single descent). A, water content expressed as a percentage of dry weight (% DW) in the two parental inbred lines and the best (LHRF61) and worst (LHRF66) LHRF_ F_3:4families. B, ABA concentration per kernel dry matter weight (DW) or per kernel fresh matter weight (FW) in the LHRF_ F_3:4population (LHRF). C, ABA concentration per kernel water in the LHRF_ F_3:4population (LHRF). D, ABA concentration per dry matter weight (DW) in endosperm (End) and embryo (Emb) of the LHRF_ F_3:4population (LHRF). Data from LHRF are means ± SD; n = 153.

Figure 2

QTLs and candidate gene map. The LHRF_ F_3:4segregating population was used. Bins are shown on the right of chromosomes. Distances are in pcM in the LHRF_ F_3:4map (LHREF3_1201). QTLs for desiccation (blue), desiccation rate (green), growth (black) and ABA content (pink) are on the left of the chromosomes (Tables 2 and 3). The confidence intervals of the QTLs are indicated by vertical bars. Arrows highlight colocations between desiccation QTLs (blue) and growth QTLs (black) and between desiccation QTLs and ABA QTLs (pink). Gene locations in the QTL confidence interval are indicated close to their corresponding QTLs. Gene codes are detailed in Tables 3 and 4. Genes in bold were mapped by PCR in this study (Additional file 2). The others were mapped by Génoplante http://urgi.versailles.inra.fr/GnpMap/. Genes involved in kernel drying and located in the vicinity of the QTL confidence interval, are indicated in grey. ABA-emb: ABA in embryo (pg/DW); ABA-end: ABA in endosperm (pg/DW); DW: kernel dry matter weight (mg/kernel); FW: kernel fresh matter weight (mg/kernel); %DW = DW/FW× 100; Water: kernel water content (mg/kernel); Rate = (Water/FW × 100)/(thermal time interval); Slope: regression line slope of (Water/FW × 100) as a function of thermal time.

Figure 3

Transcript profiling of candidate genes related to water transfer, kernel maturation and ABA regulation. End point RT-PCR was performed on total RNA of the indicated tissues using gene-specific primers listed in Additional file 2. RNA quality and quantity were checked by total RNA loading on an agarose gel and ethidium bromide staining. The constitutively expressed 18 S rRNA gene was used as an internal control of RNA quantity. A, whole kernels without glumes at 30 to 80 DAP. B, Endosperms (End) and Embryos (Emb) at 30 to 60 DAP. The number of PCR cycles (end point) is indicated in brackets after the gene name.

Figure 4

Phylogenetic tree of NCED proteins. The five novel maize NCED protein sequences were aligned with known NCED protein sequences [39,44,45]. In addition, four other NCED protein sequences were included in the alignment: OsNCED4 [GenBank:AAW21318.1] and OsNCED5 [GenBank:AAW21317.1] from rice and PsNCED2 [GenBank:BAC10550] and PsNCED3 [GenBank:BAC10551] from Pisum sativum. An unrooted tree ofthe NCED protein sequences was obtained using the maximum Likelihood method. Branch lengths are scaled proportional to substitution rate. Bootstrap values (percent) of 500 bootstrap replicates exceeding 70% are indicated above the supported branches. The Arabidopsis and maize NCEDs are highlighted by grey and dark boxes, respectively. Maize accession numbers are indicated in Table 4. Other NCEDs include: AtNCED2 [GenBank:NP_193569], AtNCED3 [GenBank:NP_188062], AtNCED5 [GenBank:NP_174302], AtNCED6 [GenBank:NP_189064] and AtNCED9 [GenBank:NP_177960] from Arabidopsis; LeNCED [GenBank:CAB10168] from Lycopersicon esculentum; OsNCED1 [GenBank:AAW21319.1], OsNCED2 [GenBank:AAW21321.1] and OsNCED3 [GenBank:AAW21320.1] from rice; PaNCED1 [GenBank:AF224672] and PaNCED3 [GenBank:AF224671] from Phaseolus vulgaris; StNCED [GenBank:CAB76920] from Solanum tuberosum.

Figure 5

Transcript profiling of maize NCEDs and ZEPs in kernel at 40 and 60 DAP. qRT-PCR was performed on total RNA of the indicated tissues using gene-specific primers listed in Additional file 2. Gene codes are detailed in Table 4. RNA quality and quantity were checked by total RNA loading on an agarose gel and ethidium bromide staining. Transcript levels were normalized with the values obtained for the Zeastar gene, which was used as an internal reference gene, and are shown relative to NCED3 transcript levels in embryo at 40 DAP (the expression level of maize NCED3 in embryo at 40 DAP = 1). Values represent the mean of three biological replicates ± SE. When two samples show different letters above the bars, the difference between them is significant (P < 0.05). When both tissue and genotype effects were significant, a, b, λ a and λ b are indicated (see the Methods section and ANOVA in Additional file 4). End X: endosperm at x DAP; Emb X: embryo at X DAP.