Integrative QTL Identification, Fine Mapping and Candidate Gene Analysis of a Major Locus qLTG3a for Seed Low-Temperature Germinability in Rice

Abstract

Low-temperature germinability (LTG) is an important agronomic trait that can affect the planting time, planting area, and grain yield of staple crops, such as rice. However, the genetic mechanism of LTG is still unclear. In this study, a multi-parental permanent population with 208 single segment substitution lines (SSSLs) was used to conduct a genetic dissection for LTG across four cropping seasons. LTG was a typical quantitative trait with a high combined broad-sense heritability of 0.71. By comparison with the recipient parent, Huajingxian74, 24 SSSLs were identified as carrying LTG QTLs, which were further merged into integrated QTLs with shorter genetic distances by substitution mapping. Finally, 14 LTG QTLs were mapped on ten chromosomes, including seven positive-effect and seven negative-effect QTLs, with additive effect contributions ranging from 19.2 to 39.9%. qLTG3a, a main-effect and novel QTL, was confirmed by bulk segregant analysis using an F₂ segregating population, and five key recombinants were selected to develop F₃ populations for progeny testing. Marker-trait association analysis fine mapped qLTG3a to a 332.7-kb physical region between markers M6026 and M6341. Within this interval, 40 annotated genes were revealed, and three genes (Os03g0213300, Os03g0214400, and Os03g0214600) were considered as pivotal candidate genes for qLTG3a based on their sequence variations and expression patterns. Besides low temperature, qLTG3a can also enhance seed germination under standard temperature and osmotic stress. In summary, this study identified some genetic factors regulating LTG and opened a new window for breeding elite direct-seeded rice varieties. It will help reduce the climate risk in the production process of rice, which is of great significance to ensuring food security.

Keywords: Bulk segregant analysis; Candidate gene; Low temperature; Seed germination vigor; Substitution mapping.

Fig. 1

Phenotypic distribution of LTG for 208 rice SSSLs in four cropping seasons. a 2015E, b 2015L, c 2016E, d 2016L. Parameters are the number of SSSLs (N) and mean (M) and standard deviation (S) for germination of the freshly harvested seeds at 15 °C. The arrow indicates the germination percentage of the recipient parent HJX74. Broad-sense heritability (H²) of LTG was estimated by the germination rates of SSSLs in each cropping season. 2015E, 2015L, 2016E, and 2016L represent the early (E) and late (L) cropping seasons in 2015 and 2016, respectively

Fig. 2

Substitution mapping of LTG QTLs in rice. The long horizontal dark bars represent the chromosomes with the molecular markers above them. The short black horizontal bars coded from S1 to S24 represent the SSSLs detected with QTLs in at least two cropping seasons, while the gray ones coded from S25 to S28 represent the SSSLs not detected with a QTL in all cropping seasons. The intervals located LTG QTLs are shown between two vertical dotted lines with the names of QTLs under the substituted segments

Fig. 3

Chromosome location of 14 QTLs for LTG in rice. The linkage map of QTLs was constructed by using MapChart 2.2 (Voorrips 2002). SSR markers are indicated on the right of the chromosomes. Genetic distance (cM) is shown as rulers on the left margin. Black bars on each chromosome's right are the location intervals of QTLs for LTG with their names on the right. Chr chromosome

Fig. 4

Validate a major LTG QTL, qLTG3a, by an F₂ population and BSA sequencing. "aa" represents the HJX74 allele of qLTG3a, and "AA" represents the S6 allele of qLTG3a. Roles of qLTG3a in LTG during seed germination (a) and seed development (c). Dots and bars represent the means and standard deviations of LTG at each time point. Germination data were calculated based on at least four biological replicates during seed germination and nine biological replicates during seed development. b Representative germination images showing different LTG phenotypes between HJX74 and S6. Pictures were taken on the 5th, 7th, and 10th day after sowing. Bar = 1 cm. d Frequency distribution and genetic effects of qLTG3a in a 356-progeny population derived from the cross of HJX74 and S6. The donor parent of S6 is Katy. LTG was evaluated on the 7th day after sowing. The genotypes of individual plants were determined by the InDel P7 marker. The additive effects (a), dominance effects (d), and proportion of the variance explained by the QTL (R²) were estimated based on Model 1. A positive a or d value indicates that the "A" allele increased LTG. * for P < 0.0001 and ns for P ≥ 0.05. e Manhattan plot shows the distribution of SNP-index and ΔSNP-index on 12 chromosomes. The reference genome for BSA sequencing is the indica variety R498 (Shuhui498). The blue and red lines represent 95 and 99% confidence intervals, respectively. The black line shows the ΔSNP-index value of fitting results

Fig. 5

Fine mapping of qLTG3a. Five recombinants (S6_R1 to R5) derived from S6_Aa were selected to delimit the locus of qLTG3a to a 332.7-kb region flanked by M6026 and M6341. The dotted vertical lines indicate the target region of qLTG3a. The partial physical maps of chromosome 3 were constructed based on the Nipponbare reference genome (IRGSP-1.0). Black and white bars represent the chromosomal segments from S6 and HJX74, respectively. N, the number of plants in a recombinant-derived progeny population; r, marker-trait correlation coefficients for LTG. A significant r value (* for P < 0.01 and ns for P ≥ 0.05) indicates that the allele of S6 on the marked heterozygous region enhanced LTG

Fig. 6

Expression patterns of the candidate genes underlying qLTG3a in various tissues. Root, stem, and leaf were collected at the reproductive stage, and seedlings were three days old. The root sample of Os03g0213300 was set as control sample for all genes. Transcript levels were assessed by qRT-PCR using OsActin1 as an internal control. Data are means ± SD (n = three biological replicates). DAF, days after flowering

Fig. 7

Roles of qLTG3a in various environmental stress tolerance. a, b normal temperature, c, d high temperature, e, f high-salt stress, g, h osmotic stress. "aa" represents the sensitive allele of qLTG3a from HJX74 and "AA" represents the tolerant allele of qLTG3a from S6. At each time point, columns and bars represent the means and standard deviations of germination rates calculated based on three biological replicates. The significance of differences was calculated using Student's t test. *P < 0.05 and **P < 0.01. 2016E, early cropping season in 2016; 2016L, late cropping season in 2016. Bars = 1 cm