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Improving seed vigor in response to cold stress is an important breeding objective in maize that allows early sowing. Using two cold tolerant inbred lines 220 and P9-10 and two susceptible lines Y1518 and PH4CV, three connected F2:3 populations were generated for detecting quantitative trait locus (QTL) related to seed low-temperature germination ability. At 10°C, two germination traits (emergence rate and germination index) were collected from a sand bed and three seedling traits (seedling root length, shoot length, and total length) were extracted from paper rolls. Significant correlations were found among all traits in all populations. Via single-population analysis, 43 QTL were detected with explained phenotypic variance of 0.62%∼39.44%. Seventeen QTL explained more than 10% phenotypic variance; of them sixteen (94.12%) inherited favorable alleles from the tolerant lines. After constructing a consensus map, three meta-QTL (mQTL) were identified to include at least two initial QTL from different populations.
Vigorous and uniform seedlings are necessary for achieving a high yield in crop production. However, various abiotic stresses occurring after sowing impair seedling establishment (
Plant low-temperature acclimation is a complex inherited quantitative trait controlled by several minor genes, and easily influenced by environment. Quantitative trait locus (QTL) mapping is a powerful approach to study and manipulate complex traits important in agriculture. QTL for low-temperature acclimation has been conducted in rice, wheat and bean (
In this study, we employed three F2:3 populations, derived from four inbred lines, two tolerant and two susceptible to cold stress, to detect the QTL related to maize LTGA under sand bed and paper roll germination conditions. The objectives of this study were: (1) to analyze low temperature seed emergence and seedling performance of the three F2:3 populations and the four parental lines; (2) to identify QTL for LGTA from each population; (3) to integrate QTL from different populations and pinpoint mQTL for further fine-mapping or molecular breeding.
Based on a germplasm screening program targeting at seed vigor, inbred lines 220 and P9-10 were selected as cold tolerant lines, whereas PH4CV and Y1518 were susceptible. PH4CV was the paternal parent of XY335, a widely cultivated hybrid generated by the Pioneer Technology Co., Tieling, Jilin Province, China (
The density of frequency distribution of cold germination related traits in populations.
Germination experiments were performed in both sand bed and paper roll experiments in a dark chamber at 10°C. In sand bed, after sieving and washing to remove soil, nutrient and other contaminations, sand was dried at 130°C for 5 h, moistened using distilled water to 16% content, and then sprayed in a plastic box to form a 2 cm thick bed (
Phenotypic performance of the four parental lines (220, P9-10, PH4CV, and Y1518) germinated under low (10°C) and normal (25°C) temperature in two germination systems of sand bed and paper rolls.
Once a shoot broke through the sand and became visible, it was counted as emerged plant. Emergence of plants was counted from 17 to 25 days after sowing (DAS) at 2-day intervals (in total five records). Emergence rate (ER) was expressed as percentage of emerged plants at 25th DAS to the total seed used. The germination index (GI) was calculated as
where
In paper rolls, 10 sterilized seeds were sown in a moist brown germination paper (Anchor Ltd., St. Paul, MN, United States) and another sheet of humid paper was used as a cover. Then the germination paper was rolled and put erectly in a sealed plastic bag (
The data were analyzed using the
Shoots from 30 plants for each line were sampled for genomic DNA extraction following the cetyltrimethylammonium bromide (CTAB) method (
Based on the SNP markers shared by different populations, an integrated map was built according to the user manual of QTL IciMapping software (
Genes with well annotation involving in low-temperature adaption were collected from
Two groups of seedlings growing in paper rolls were cultivated under 10°C and 25°C germination conditions, respectively, shoots were collected for RNA isolation once they have similar seedling length at 15 DAS (grown under 10°C) and 3 DAS (grown under 25°C), respectively. Ten shoots in each replication were pooled and grinded in liquid nitrogen for total RNA extraction using RNAprep pure Plant Kit [Tiangen Biotech (Beijing)]. RNA samples were treated with RNase-free DNase Kit (Invitrogen) to remove DNA contamination. Followed by reverse transcription reaction of cDNA with RT MasterMix (Applied Biological Materials Inc.), qPCR analysis was performed on Applied Biosystems QuantStudio 6 (Thermo Fisher) using qPCR MasterMix solution (Applied Biological Materials Inc.). The primers used in qPCR were listed in Supplementary Table
Germination (ER and GI) and seedling (TL, RL, and SL) performance of the four parental lines was evaluated in sand bed and paper rolls, respectively (
The average values of ER among the three populations were similar, while those of the other four traits showed differences (
Coefficients of variation (CV) and broad-sense heritabilities (
Mean and heritability (
Population | Traita | Mean ± SD | CV (%) | Range | Kurtosis | Skewness | |
---|---|---|---|---|---|---|---|
220 × PH4CV | ER (%) | 60.48 | 34.50 | 4.00–98.00 | -0.38 | -0.50 | 0.84 |
GI | 2.38 | 51.08 | 0.07–6.29 | 0.00 | 0.50 | 0.83 | |
TL (cm) | 7.84 | 21.05 | 3.73–11.37 | -0.41 | -0.13 | 0.93 | |
RL (cm) | 4.66 | 25.71 | 1.58–7.78 | -0.17 | -0.05 | 0.94 | |
SL (cm) | 3.18 | 17.69 | 1.87–4.55 | -0.43 | 0.12 | 0.91 | |
220 × Y1518 | ER (%) | 59.65 | 37.70 | 4.44–97.78 | -0.86 | -0.27 | 0.85 |
GI | 2.98 | 42.70 | 0.48–6.72 | -0.42 | 0.39 | 0.82 | |
TL (cm) | 4.82 | 19.15 | 1.66–8.08 | 1.64 | 0.55 | 0.93 | |
RL (cm) | 3.09 | 23.17 | 0.53–5.78 | 1.95 | 0.53 | 0.92 | |
SL (cm) | 1.73 | 17.72 | 0.96–2.80 | 0.13 | 0.40 | 0.94 | |
P9-10 × PH4CV | ER (%) | 62.67 | 42.15 | 0–100.00 | -0.50 | -0.62 | 0.82 |
GI | 1.61 | 65.76 | 0–5.23 | 0.88 | 0.87 | 0.81 | |
TL (cm) | 6.16 | 19.03 | 2.96–10.16 | 0.45 | -0.32 | 0.94 | |
RL (cm) | 2.06 | 19.46 | 0.97–3.40 | 0.11 | 0.17 | 0.94 | |
SL (cm) | 4.06 | 22.03 | 1.53–7.36 | 0.66 | -0.23 | 0.93 | |
Within a population, the phenotypic distribution of all five traits were approximately consistent with normal distributions based on low values of skewness and kurtosis (below 1, except for kurtosis for TL and RL in 220 × Y1518;
Phenotypic correlation between emergence rate (ER), germination index (GI), total length (TL), root length (RL), and shoot length (SL) in population 220 × PH4CV, 220 × Y1518 and P9-10 × PH4CV.
Populations | Traits | ER | GI | TL | RL | SL |
---|---|---|---|---|---|---|
220 × PH4CV | ER | 1 | ||||
GI | 0.85 ∗ | 1 | ||||
TL | 0.55 ∗ | 0.49 ∗ | 1 | |||
RL | 0.46 ∗ | 0.39 ∗ | 0.97 ∗ | 1 | ||
SL | 0.64 ∗ | 0.59 ∗ | 0.86 ∗ | 0.72 ∗ | 1 | |
220 × Y1518 | ER | 1 | ||||
GI | 0.93 ∗ | 1 | ||||
TL | 0.41 ∗ | 0.47 ∗ | 1 | |||
RL | 0.32 ∗ | 0.36 ∗ | 0.96 ∗ | 1 | ||
SL | 0.51 ∗ | 0.58 ∗ | 0.77 ∗ | 0.57 ∗ | 1 | |
P9-10 × PH4CV | ER | 1 | ||||
GI | 0.87 ∗ | 1 | ||||
TL | 0.52 ∗ | 0.48 ∗ | 1 | |||
RL | 0.54 ∗ | 0.53 ∗ | 0.78 ∗ | 1 | ||
SL | 0.42 ∗ | 0.39 ∗ | 0.95 ∗ | 0.56 ∗ | 1 | |
A total of 5,179 SNP markers were scanned and resulted in 1,382, 1,500, and 1,419 markers that fit the expected 1:2:1 distribution ratio in F2:3 lines, and were polymorphic between the two parents of the population 220 × PH4CV, 220 × Y1518 and P9-10 × PH4CV, respectively (Supplementary Table
A total of 43 QTL were identified to be associated with LTGA with 19, 13, and 11 from 220 × PH4CV, 220 × Y1518 and P9-10 × PH4CV, respectively (
QTL for low temperature germination related traits represented in the consensus linkage map of the three populations of 220 × PH4CV, 220 × Y1518, and P9-10 × PH4CV. The circle, triangle, square, star and pentagon represented traits of ER, GI, TL, RL, and SL. ER and GI were collected from sand bed experiment, and TL, RL, and SL were collected from paper rolls experiment. Color red, green and blue indicated QTL detected from population 220 × PH4CV, 220 × Y1518, and P9-10 × PH4CV, respectively.
Taking advantage of the same SNP chip used for genotyping, we integrated the three maps into a consensus linkage map (cMap) based on the markers shared by populations (
After projecting the 43 initial QTL on the cMap, 12 QTL (27.9%) overlapped, resulting in 3 mQTL (
Meta-QTL (mQTL) detected from the consensus linkage map.
Name | Flanking Makers | CI (cM) | Initail QTLb | Favorable Allele | Referencec |
---|---|---|---|---|---|
M1c174799835–M1c184012303 | 164.57–223.23 | 220 or P9-10 | |||
M2c193833217–M2c199180638 | 163.75–165.86 | 220 | |||
M9c149041431–M9c151277505 | 129.89–146.48 | 220 or P9-10 | |||
The six candidate genes within the above detected QTL were homologous to published low-temperature adaption genes by BLAST analysis (Supplementary Tables
Expressions of six candidate genes in parent lines 220 and PH4CV. Expression were conducted on shoots that were collected at 15 days after sowing (DAS) and 3 DAS under low (10°C) and normal (25°C) temperature, respectively.
Improvement of maize LTGA could help farmers to sow early, which has advantages in earlier harvest and longer plant life cycle: earlier harvest could extend maize growth region to higher altitudes and longer life cycle increases biomass accumulation and yield output (
To evaluate the reliability of QTL detected in this work, we compared our identified 43 QTL to the previous QTL released from several cold related publications in maize. According to the physical position of QTL on B73 RefGen_v2, we found that 29 QTL (67.4%) overlapped with published QTL (Supplementary Table
Three mQTL were found to locate in chromosome regions that have been mentioned to harbor cold related QTL in previous reports (
Quantitative trait locus with higher effect are generally easier for gene cloning or more efficient for MAS (
The reliability of QTL analysis depends on population size, phenotypic variance, phenotyping methods and marker density, etc. In this study, we addressed some of these obstacles and detected the most promising mQTL for further gene cloning and MAS. First, we applied two different methods (sand bed and paper rolls) for germination trait evaluation at low temperature, in contrast to previous studies that only used one of the methods of sand bed, peat bed, paper rolls or field evaluation at early spring (
Second, use of three populations for phenotyping and genotyping enabled us to identify mQTL across populations. Although a number of QTL associated with cold acclimation have been identified, only few were consistent across diverse genetic backgrounds. In this work, three mQTL were identified to contain initial QTL from two populations. Furthermore, two of the identified mQTL (
Third, outstanding parental lines with contrasting cold tolerance were selected for generating populations, which contributed to efficient identification of major QTL. Of the 43 initial QTL, 3 explained more than 30% and 14 explained 10–30% phenotypic variance. Cold tolerance increasing alleles were all inherited from the tolerant lines 220 or P9-10, further supporting the efficacy of selection of suitable parental lines. In contrast, phenotypic variance of QTL identified in previous reports were generally lower than 20% (
We identified 43 QTL responsible for maize LTGA using three connected populations germinated in a sand bed and paper rolls. By constructing a consensus linkage map, three mQTL were suggested to include initial QTL that detected from different populations. In future, it is of great interest to clone genes underlying mQTL regions and uncover the molecular mechanisms of maize cold tolerance during germination.
GyW, JW, and RG designed the study; XL, GhW, GJ, LR, and LL performed the experiments; XL and JF analyzed the data; XL drafted the manuscript; TL, JW, and RG advised on data analysis and revised the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This research was supported by the National Basic Research Program of China (2014CB138200), the China Agriculture Research System (CARS-08), the special fund for Agro-Scientific Research in the Public Interest (201303002), the National Science Foundation of China (31771891), the National Key Research and Development Program of China (2016YFD0101803).
The Supplementary Material for this article can be found online at: