Low Temperature Germination Research Articles

BSA-Seq Approach Identified Candidate Region and Diagnostic Marker for Chilling Tolerance of High Oleic Acid Peanut at Germination Stage

High oleic acid peanut (HOAP) is extensively embraced in China because of its high nutritional value and enhanced oxidative stability. However, its dissemination has been severely constrained in high altitude and high latitude areas due to chilling stress during sowing, resulting in significant yield loss in these regions. Despite the lack of understanding of the molecular mechanisms underlying low temperature germination (LTG) in HOAP, discovering the quantitative trait loci (QTL) that confer this trait will undoubtedly benefit breeding efforts. In the present study, we identified putative genomic regions and single nucleotide polymorphisms (SNPs) that govern LTG tolerance of HOAP in an F2 population derived from the cross of chilling-tolerant YH65 and chilling-sensitive FL14 using bulk segregant analysis (BSA). Analysis of ΔSNP-index and Euclidean distance (ED) value association pinpointed the overlapped region to a 2.29 Mb interval on chromosome A05. The candidate interval showed that 122 genes were significantly related to response to abiotic stress and plant–pathogen interaction. Furthermore, an SNP site associated with LTG tolerance was discovered. The SNP site was employed as a Kompetitive Allele Specific PCR (KASP) marker and validated in a universal peanut panel. These findings may provide valuable insight into the molecular mechanism underpinning LTG tolerance and facilitate marker-assisted selective breeding in HOAP.

Brassinosteroid biosynthesis gene OsD2 is associated with low-temperature germinability in rice.

In rice, low-temperature germinability (LTG) is essential for stable stand establishment using the direct seeding method in temperate and high-altitude areas. Previously, we reported that the quantitative trait locus qLTG1 is associated with LTG. qLTG1 is also associated with tolerance to several abiotic stresses, such as salt and osmotic conditions. In this study, map-based cloning and sequence analysis indicated that qLTG1 is allelic to DWARF2 (OsD2), which encodes cytochrome P450 D2 (LOC_Os01g10040) involved in brassinosteroid (BR) biosynthesis. Sequence comparison of the two parental lines, Hwaseong and Oryza rufipogon (IRGC 105491), revealed five single nucleotide polymorphisms (SNPs) in the coding region. Three of these SNPs led to missense mutations in OsD2, whereas the other two SNPs were synonymous. We evaluated two T-DNA insertion mutants, viz., overexpression (OsD2-OE) and knockdown (OsD2-KD) mutants of OsD2, with the Dongjin genetic background. OsD2-KD plants showed a decrease in LTG and grain size. In contrast, OsD2-OE plants showed an increase in grain size and LTG. We also examined the expression levels of several BR signaling and biosynthetic genes using the T-DNA insertion mutants. Gene expression analysis and BR application experiments demonstrated that BR enhanced the seed germination rate under low-temperature conditions. These results suggest that OsD2 is associated with the regulation of LTG and improving grain size. Thus, OsD2 may be a suitable target for rice breeding programs to improve rice yield and LTG.

Quantitative Trait Loci Mapping Analysis for Cold Tolerance Under Cold Stress and Brassinosteroid-Combined Cold Treatment at Germination and Bud Burst Stages in Rice.

Low temperature is one of the major abiotic stresses limiting seed germination and early seedling growth in rice. Brassinosteroid (BR) application can improve cold tolerance in rice. However, the regulatory relationship between cold tolerance and BR in rice remains undefined. Here, we constructed a population of 140 backcross recombinant inbred lines (BRILs) derived from a cross between a wild rice (Dongxiang wild rice, DXWR) and a super rice (SN265). The low-temperature germination rate (LTG), survival rate (SR), plant height (PH), and first leaf length (FLL) were used as indices for assessing cold tolerance under cold stress and BR-combined cold treatment at seed germination and bud burst stages. A high-resolution SNP genetic map, covering 1,145 bin markers with a distance of 3188.33 cM onto 12 chromosomes, was constructed using the GBS technique. A total of 73 QTLs were detected, of which 49 QTLs were identified under cold stress and 24 QTLs under BR-combined cold treatment. Among these, intervals of 30 QTLs were pairwise coincident under cold stress and BR-combined cold treatment, as well as different traits including SR and FLL, and PH and FLL, respectively. A total of 14 candidate genes related to cold tolerance or the BR signaling pathway, such as CBF/DREB (LOC_Os08g43200), bHLH (LOC_Os07g08440 and LOC_Os07g08440), WRKY (LOC_Os06g30860), MYB (LOC_Os01g62410 and LOC_Os05g51160), and BRI1-associated receptor kinase 1 precursor (LOC_Os06g16300), were located. Among these, the transcript levels of 10 candidate genes were identified under cold stress and BR-combined cold treatment by qRT-PCR. These findings provided an important basis for further mining the genes related to cold tolerance or the BR signaling pathway and understanding the molecular mechanisms of cold tolerance in rice.

Exogenous Proline Optimizes Osmotic Adjustment Substances and Active Oxygen Metabolism of Maize Embryo under Low-Temperature Stress and Metabolomic Analysis

Maize (Zea mays L.) is more sensitive to low-temperature stress in the early growth period. The study was to explore the response mechanism of proline to low-temperature stress during maize seed germination. Maize varieties Xinxin 2 (low-temperature insensitive) and Damin 3307 (low-temperature sensitive) were chosen as the test materials, setting the normal temperature for germination (22 °C/10 °C, 9d), low-temperature germination (4 °C/4 °C, 5d) and normal temperature recovery (22 °C/10 °C, 4d), combined with proline (15 mmol·L−1) soaking treatment, to study its effects on the osmotic regulation system and antioxidant protection system of maize embryos. Metabolomics analysis was carried out to initially reveal the basis of the metabolic regulation mechanism. The results showed that the activities of superoxide dismutase (SOD), peroxidase (POD), ascorbic acid peroxidase (APX) and glutathione reductase (GR) were induced to some extent under low-temperature stress. The activities of SOD, POD, APX and GR were further enhanced in the soaking seeds with proline. Proline treatment improved the activities of catalase (CAT), monodehydrated ascorbic acid reductase (MDHAR) and dehydroascorbic acid (DHAR), increased the contents of ascorbic acid (AsA) and glutathione (GSH) and decreased the contents of oxidized ascorbic acid (DHA) and reduced glutathione (GSSG) under low-temperature stress. The ratio of AsA/DHA and GSH/GSSG increased. The increase in antioxidant enzyme activity and the content of antioxidants can help to maintain the stability of the AsA-GSH cycle, and effectively reduce the production rate of superoxide anion (O2•−), hydrogen peroxide (H2O2) and malondialdehyde (MDA). Based on the UPLC-MS/MS detection platform and self-built database, 589 metabolites were detected in each treated maize embryo; 262 differential metabolites were obtained, including 32 organic acids, 28 amino acids, 20 nucleotides and their derivatives, 26 sugars and alcohols, 46 lipids, 51 alkaloids, 44 phenols and 15 other metabolites. Sixty-eight metabolic pathways involving different metabolites were obtained by KEGG enrichment analysis. The results showed that proline increased the accumulation of sorbitol, planteose, erythritose 4-phosphate, arabinose and other saccharides and alcohols in response to low-temperature stress, increased the content of osmoregulation substances under low-temperature stress. Proline also restored the TCA cycle by increasing the content of α-ketoglutarate and fumaric acid. Proline increased the contents of some amino acids (ornithine, proline, glycine, etc.), alkaloids (cocamidopropyl betaine, vanillylamine, 6-hydroxynicotinic acid, etc.), phenols (phenolic ayapin, chlorogenic acid, etc.) and vitamins (ascorbic acid, etc.) in the embryo under low-temperature stress. Combined with pathway enrichment analysis, proline could enhance the low-temperature stress resistance of germinated maize embryos by enhancing starch and sucrose metabolism, arginine and proline metabolism, biosynthesis of secondary metabolites, flavonoid biosynthesis and pentose phosphate pathway.

QTL Mapping and Candidate Gene Analysis for Seed Germination Response to Low Temperature in Rice.

Low temperature is a serious threat to the seed emergence of rice, which has become one of the main limiting factors affecting rice production in the world. It is of great significance to find the candidate genes controlling low-temperature tolerance during seed germination and study their functions for breeding new rice cultivars with immense low-temperature tolerance during seed germination. In the current experiment, 120 lines of the Cheongcheong Nagdong Double Haploid (CNDH) population were used for quantitative trait locus (QTL) analysis of low-temperature germinability. The results showed a significant difference in germination under low different temperature (LDT) (15 °C, 20 °C) conditions. In total, four QTLs were detected on chromosome 3, 6, and 8. A total of 41 genes were identified from all the four QTLs, among them, 25 genes were selected by gene function annotation and further screened through quantitative real-time polymerase chain reaction (qRT-PCR). Based on gene function annotation and level of expression under low-temperature, our study suggested the OsGPq3 gene as a candidate gene controlling viviparous germination, ABA and GA signaling under low-temperature. This study will provide a theoretical basis for marker-assisted breeding and lay the basis for further mining molecular mechanisms of low-temperature germination tolerance in rice.

The qLTG1.1 candidate gene CsGAI regulates low temperature seed germination in cucumber.

The CsGAI gene, identified by map-based, was involved in regulating seed germination in low temperature via the GA and ABA signaling pathways. Low temperature reduces the percentage of seeds germinating and delays seed germinating time, thus posing a threat to cucumber production. However, the molecular mechanism regulating low temperature germination in cucumber is unknown. We here dissected a major quantitative trait locus qLTG1.1 that controls seed germination at low temperature in cucumber. First, we fine-mapped qLTG1.1 to a 46.3-kb interval, containing three candidate genes. Sequence alignment and gene expression analysis identified Csa1G408720 as the gene of interest that was highly expressed in seeds, and encoded a highly conserved, low temperature-regulated DELLA family protein CsGAI. GUS expression analysis indicated that higher promoter activity underscored higher transcriptional expression of the CsGAI gene. Consistent with the known roles of GAI in ABA and GA signaling during germination, genes involved in the GA (CsGA2ox, CsGA3ox) and ABA biosynthetic pathways (CsABA1, CsABA2, CsAAO3 and CsNCED) were found to be differently regulated in the tolerant and sensitive genotypes under low temperatures, and this was reflected in differences in their ratio of GA-to-ABA. Based on these data, we proposed a working model explaining how CsGAI integrates the GA and ABA signaling pathways, to regulate cucumber seed germination at low temperature, thus providing new insights into this mechanism.

Unraveling the Genetic Architecture for Low Temperature Germinability-Related Traits in Rice Using Genome-Wide Association Study

Rice is frequently affected by cold weather at high altitudes in temperate and subtropical regions. With the popularity of direct seeding, a better understanding of the genetic mechanisms regulating cold tolerance will enable breeders to develop varieties with strong low temperature germinability (LTG). In this study, six indices including low temperature germination percentage (LTGP), relative germination percentage (RGP), relative plumule length (RPL), plumule length after 6-day recovery (PLR), plumule length recovery rate (PLRR) and recovery ability of plumule length after cold stress (RAPL) were measured to assess LTG, and carried out a genome-wide association study (GWAS) to identify QTL and candidate genes related to LTG by using a natural population comprising 211 rice accessions. A total of 18 QTL including two for LTGP, three for RGP, five for PLR, four for PLRR, two for RPL and two for RAPL were uncovered on 12 chromosome regions located in chromosome 1, 2, 4, 5, 6, 9, 10 and 12. On chromosome 2, qLTGP2 and qRGP2 were co-localized at 3.3 Mb, and qPLR2 and qPLRR2 were co-localized at 5.5 Mb; qLTGP5, qPLR5 and qPLR5 were co-localized at 27.8 Mb on chromosome 5; qPLR6 and qPLRR6 were co-localized at 5.7 Mb on chromosome 6; and qPLR12 and qPLRR12 were co-localized at 23.5 Mb on chromosome 12. These results indicated that some LTG-related traits may share the same genetic pathway. For the 18 LTG-related QTL, seven QTL (qLTGP2, qRGP2, qPLR2, qPLRR2, qLTGP5, qPLR5 and qPLR5) were reported for the first time. According to candidate gene analysis, fourteen genes from five QTL (qLTGP2, qPLR2, qLTGP5, qRAPL10 and qPLR12) were considered as candidate genes and will be further functionally validated in subsequent experiments. QTL with superior candidate genes identified in this study will be useful in improving cold tolerance in rice cultivars. The rice varieties with strong LTG identified in this study will enrich the resources of rice cultivation project.

QTL mapping and candidate gene analysis of low temperature germination in rice (Oryza sativa L.) using a genome wide association study.

Low temperature germination (LTG) is a key agronomic trait in rice (Oryza sativa L.). However, the genetic basis of natural variation for LTG is largely unknown. Here, a genome-wide association study (GWAS) was performed using 276 accessions from the 3,000 Rice Genomes (3K-RG) project with 497 k single nucleotide polymorphisms (SNPs) to uncover potential genes for LTG in rice. In total, 37 quantitative trait loci (QTLs) from the 6th day (D6) to the 10th day (D10) were detected in the full population, overlapping with 12 previously reported QTLs for LTG. One novel QTL, namely qLTG1-2, was found stably on D7 in both 2019 and 2020. Based on two germination-specific transcriptome datasets, 13 seed-expressed genes were isolated within a 200 kb interval of qLTG1-2. Combining with haplotype analysis, a functional uncharacterized gene, LOC_Os01g23580, and a seed germination-associated gene, LOC_Os01g23620 (OsSar1a), as promising candidate genes, both of which were significantly differentially expressed between high and low LTG accessions. Collectively, the candidate genes with favorable alleles may be useful for the future characterization of the LTG mechanism and the improvement of the LTG trait in rice breeding.

Exogenous Proline Alleviated Low Temperature Stress in Maize Embryos by Optimizing Seed Germination, Inner Proline Metabolism, Respiratory Metabolism and a Hormone Regulation Mechanism

Proline (Pro) is not only an important osmotic adjustment substance, but it also plays an important role in regulating plant abiotic stress resistance. The maize varieties, Xinxin 2 (low temperature insensitive) and Damin 3307 (low temperature sensitive), were chosen as materials, setting a normal temperature for germination (22 °C/10 °C, 9d), low temperature germination (4 °C/4 °C, 5d) and normal temperature recovery (22 °C/10 °C, 4d), combined with a proline (15 mmol·L−1) soaking treatment, to reveal the seed germination and regulation mechanism in maize embryos. The results showed that proline significantly improved the germination potential, germination rate, germination index and vigor index of seeds under low temperature stress, increasing the length of the coleoptile and radicle, increasing the dry and fresh weight of young buds (coleoptile + radicle), and increasing the activity of α-amylase. Proline enhanced the activities of Δ1-pyrroline-5-carboxylic acid synthetase (P5CS) and ornithine aminotransferase (OAT) in maize embryos under low temperature stress, enhanced the proline synthesis pathways, and further enhanced proline accumulation. Proline induced the activity of proline dehydrogenase (ProDH) in the early stage of low temperature stress and stress relief. Under low temperature stress, the activities of hexokinase (HXK), phosphofructokinase (PFK), pyruvate kinase (PK), isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) and glucose-6-phosphate dehydrogenase (G-6-PDH and 6-P-GDH) in maize embryos were decreased, resulting in a decrease in the glycolysis (EMP) pathway, tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP). Proline alleviated the inhibition of key enzyme activities of the EMP pathway, TCA cycle and PPP of maize embryos under low temperature stress, increased the activities of cytochrome oxidase (COX) and alternative oxidase (AOX), increased the ATP content, alleviated the inhibition of low temperature stress on main cytochrome pathway activity (ρVcyt), while further increasing the total respiratory activity (Vt) and the actual operational activity of the alternative pathway (ρValt) during seed germination at the initial stage of low temperature stress, as well as improving the inhibition of the Vt and ρValt in the middle and late stages of low temperature stress. Under low temperature stress, the content of abscisic acid (ABA) increased significantly, while gibberellin (GA), auxin (IAA) and zeatin nucleoside (ZR) decreased significantly. Proline alleviated the decrease in IAA, ZR and GA contents in maize embryos under low temperature stress, reduced the increase in the ABA content, and reduced the inhibition of low temperature on seed germination.

Transcriptome Analysis of Near-Isogenic Lines Provides Novel Insights into Genes Associated with Seed Low-Temperature Germination Ability in Maize (Zea mays L.)

sMaize originated from tropical regions and is extremely sensitive to low temperature during germination. Our previous work identified a major QTL, qp1ER1-1, for low temperature germination ability (LTGA) of maize. Here, we introgressed qp1ER1-1 from the tolerant line L220 into the sensitive line PH4CV to generate two near isogenic lines NIL220-3 and NIL220-25. When germinated under cold temperature for 25 days (Cold-25), the NILs showed similar seedling root length and shoot length to L220, but significantly higher than PH4CV. However, when germinated under cold temperature for 15 days (Cold-15) or under normal temperature (25 °C) for 3 days (CK-3), all lines showed similar seedling performance, indicating that introgression of qp1ER1-1 from L220 into PH4CV could improve LTGA of NIL220-3 and NIL220-25. The whole seedlings, including root and shoot, of Cold-15 and CK-3 were harvested for transcriptome analysis, when both stayed at a similar developmental stage. Dry seed embryo was sequenced as a non-germination control (CK-0). Compared with PH4CV, the tolerant line (L220, NIL220-3, and NIL220-25) specifically expressed genes (different expressed genes, DEGs) were identified for CK-0, Cold-15, and CK-3. Then, DEGs identified from Cold-15, but not from CK-0 or CK-3, were defined as tolerant line specifically expressed LTGA genes. Finally, 1786, 174, and 133 DEGs were identified as L220, NIL220-3, and NIL220-25 specifically expressed LTGA genes, respectively. Of them, 27 were common LTGA genes that could be identified from all three tolerant lines, with two (Zm00001d031209 and Zm00001d031292) locating in the confidence interval of qp1ER1-1. In addition, GO analysis revealed that L220 specifically expressed LTGA genes were majorly enriched in the cell division process and plasma membrane related categories. Taken together, these results provided new insight into the molecular mechanism of maize seed LTGA and facilitated the cloning of the qp1ER1-1 gene.

QTL Mapping Low-Temperature Germination Ability in the Maize IBM Syn10 DH Population.

Chilling injury poses a serious threat to seed emergence of spring-sowing maize in China, which has become one of the main climatic limiting factors affecting maize production in China. It is of great significance to mine the key genes controlling low-temperature tolerance during seed germination and study their functions for breeding new maize varieties with strong low-temperature tolerance during germination. In this study, 176 lines of the intermated B73 × Mo17 (IBM) Syn10 doubled haploid (DH) population, which comprised 6618 bin markers, were used for QTL analysis of low-temperature germination ability. The results showed significant differences in germination related traits under optimum-temperature condition (25 °C) and low-temperature condition (10 °C) between two parental lines. In total, 13 QTLs were detected on all chromosomes, except for chromosome 5, 7, 10. Among them, seven QTLs formed five QTL clusters on chromosomes 1, 2, 3, 4, and 9 under the low-temperature condition, which suggested that there may be some genes regulating multiple germination traits at the same time. A total of 39 candidate genes were extracted from five QTL clusters based on the maize GDB under the low-temperature condition. To further screen candidate genes controlling low-temperature germination, RNA-Seq, in which RNA was extracted from the germination seeds of B73 and Mo17 at 10 °C, was conducted, and three B73 upregulated genes and five Mo17 upregulated genes were found by combined analysis of RNA-Seq and QTL located genes. Additionally, the variations of Zm00001d027976 (GLABRA2), Zm00001d007311 (bHLH transcription factor), and Zm00001d053703 (bZIP transcription factor) were found by comparison of amino sequence between B73 and Mo17. This study will provide a theoretical basis for marker-assisted breeding and lay a foundation for further revealing molecular mechanism of low-temperature germination tolerance in maize.

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

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.

QTL mapping for low temperature germination in rapeseed

Rapeseed, a major oil crop in the world, is easily affected by low-temperature stress. A low temperature delays seed germination and increases seedling mortality, adversely affecting rapeseed growth and production. In the present study, a tolerant cultivar (Huyou21) was crossed with a susceptible genotype (3429) to develop a mapping population consisting of 574 F2 progenies and elucidate the genetic mechanisms of seed germination under low temperatures. Two quantitative trait loci (QTL) for low-temperature germination (LTG) were detected, one on chromosome A09 (named qLTGA9-1) and the other on chromosome C01 (named qLTGC1-1), using the QTL-seq approach and confirmed via linkage analysis in the mapping population. Further, qLTGA9-1 was mapped to a 341.86 kb interval between the SSR markers Nys9A212 and Nys9A215. In this region, 69 genes including six specific genes with moderate or high effect function variants were identified based on the Ningyou7 genome sequence. Meanwhile, qLTGC1-1 was mapped onto a 1.31 Mb interval between SSR markers Nys1C96 and Nys1C117. In this region, 133 genes including five specific genes with moderate effect function variants were identified. These specific genes within the two QTL could be used for further studies on cold tolerance and as targets in rapeseed breeding programs.

Coordinated Effect of Ascorbate Biosynthesis and Recycling in Maize Seed Germination and Seedling Establishment under Low Temperature

The impacts of low temperature occasionally encountered at higher latitude regions on maize seed germination present significant threats to yield and cultivation. Exploring the association of antioxidant system with low temperature (LT) germination could support the breeding strategies for better responding to LT disturbance. In this study, we have examined the germination rate and growth potential of a set of elite maize inbred accessions under LT and normal temperature (NT) conditions in the field. These accessions were found to have variable germination rate and growth potential when grown at LT, whereas the difference is not significant under NT. Physiological study revealed lower hydrogen peroxide content in LT tolerant accessions when compared with sensitive ones. LT-tolerant and LT-sensitive lines maintained similar content of ascorbate (AsA) and glutathione (GSH), whereas the reduced substrate content of which were significantly higher in LT-tolerant accessions. Consistently, activities of ascorbate peroxidase and dehydroascorbate reductase, the enzyme components that responsible for the AsA-GSH recycling, were much higher in LT-tolerant lines. Transcription profile revealed the increased expression of ZmVTC2 gene in LT-tolerant inbred line, which was rate limited step in AsA biosynthesis. These data indicates that the coordinated improvement of AsA biosynthesis and AsA-GSH recycling increase the pool size of the total antioxidants, which ameliorate LT-induced oxidative stress during maize seed germination.

Breeding for Low Temperature Germinability in Temperate Japonica Rice Varieties: Analysis of Candidate Genes in Associated QTLs

In temperate areas, rice deals with low temperatures that can affect plant growth and crop yield. Rapid germination is required for adequate plant establishment in the field, therefore obtaining cultivars that maintain this phenotype under suboptimal temperature conditions is a challenge for rice breeders. Our study aimed to investigate temperature-induced expression changes in genes underlying quantitative trait loci (QTLs) associated to this trait (low temperature germinability, LTG) that were detected in a previous genome wide association study (GWAS). In the context of a breeding program for japonica rice cultivars adapted to cultivation in Spain, we obtained two biparental families of lines derived from hybridization with two cold tolerant Italian cultivars, and we have studied the effect on the LTG phenotype of introgressing these QTLs. A wide region in chromosome 3 was related to significant increases in seedling growth rate at 15 °C, although the extent of the effect depended on the analyzed family. In parallel, we studied the pattern of expression during germination at different temperatures of 10 genes located in the LTG-associated QTLs, in five japonica rice cultivars and in a biparental family of recombinant inbred lines (RILs). Cold induced changes in the expression of the 10 analyzed genes, with significant differences among genotypes. Variation in LTG phenotype was consistently associated with changes in the pattern of expression of five genes from the tagged regions in rice chromosome 3, which encoded for enzymes implicated in phytohormone metabolism (OsFBK12, Os3Bglu6), oxidative stress (SPL35, OsSRO1c) and Mn homeostasis maintenance (OsMTP8.1). Differential expression induced by cold in two regulatory genes (Os02g0824000 and Os06g06400) also contributed to explain low temperature tolerance during rice germination. In conclusion, introgression in defective cultivars of favorable alleles for these genes would contribute to the genetic improvement of LTG in japonica rice varieties.

Accelerated aging predicts the emergence of rice seedlings in the field

Selecting the most appropriate vigor test for rice to predict seedling emergence remains a challenge. This is mainly attributed to the diversity of available tests that evaluate vigor; however, there is a critical need for tests that can show a high correlation with field emergence. Thus, this study aimed to determine which vigor tests are the most efficient in predicting the emergence of rice seedlings, to facilitate correct selection for quality control analyses and pre-sowing for rice seed commercialization. Across two growing seasons, 2016/17 and 2017/18, different seed lots of ‘SCS122 Miura’ rice were subjected to vigor evaluations including germination test, electrical conductivity, cold test, low temperature germination, accelerated aging, root and shoot length, seedling dry weight, emergence speed, and emergence speed index, and greenhouse and field emergence tests. A completely randomized design with four replicates was used for the experiments. All statistical analyses were performed using R software. The germination rate for ‘SCS122 Miura’ seed lots in both growing seasons was greater than 80%. In both growing seasons, unlike the similar results obtained for field emergence, in the laboratory tests, differences were observed between these same seed lots. Electrical conductivity and emergence speed showed a negative correlation with greenhouse and field emergence. The vigor tests for accelerated aging, emergence speed, and dry weight are more efficient in predicting the emergence of rice seedlings. Among the tested methods, accelerated aging (41 °C for 120 h) is one of the most efficient for predicting the emergence of rice seedlings and the best option for laboratory analysis of quality control and pre-sowing owing to its practicality and time required for execution.

Mapping QTLs controlling low-temperature germinability in rice by using single segment substitution lines derived from 4 AA-genome species of wild rice

Rice growth is sensitive to low temperature. Chilling injury is one of the main natural disasters in rice cultivation, and is damaging to rice growth at all developmental stages. Low-temperature germinability (LTG) is an important trait for seedling establishment, especially in rice direct-sowing practice. However, the genetic mechanism of LTG remains elusive. Here, we report the mapping of QTLs controlling LTG in rice using 586 single segment substitution lines (SSSL) derived from 4 AA-genome wild rice species. 21 SSSLs with the highest germination rate at low temperature in both of the two tested seasons were selected, from them 10 QTLs controlling LTG were identified by substitution mapping to locate on 6 chromosomes: chromosome 1, 3, 5, 7, 8 and 11, with an interval length of 3.45 to 20.15 cM. Five QTLs were detected from Niv-SSSls: qLTGn3-1, qLTGn5-1, qLTGn5-3, qLTGn7-1 and qLTGn7-2, their additive effects ranged from 3.72% to 19.74% in late season 2018. qLTGb1-1 and qLTGb8-2 were detected in the SSSLs with O. barthii as the donor and they accounted for at least 9.51% of the total phenotypic variation. Two QTLs, qLTGg5-2 and qLTGg8-1, were derived from O. glumaepatula, and their average additive effects were 4.28% and 20.39% in late seasons 2018, respectively. qLTGm11-1 on chromosome 11 was the only QTL identified from O. meridionalis. All QTLs derived from wild rice species improved low-temperature germination significantly. The SSSL library with wild rice species as donor is therefore a valuable resource for rice germplasm innovation and the breeding of chilling-resistant varieties.

Chickpea seed vigor evaluated by computerized seedling analysis

Computerized seedling analysis is a promising tool for assessing seed vigor, providing reliable and less subjective results. The aim of this study was to define a method for evaluating the vigor of chickpea seeds through computerized image analysis, using the Seedling Analysis System (Sistema de Análise de Plântula - SAPL®) as a basis. Two chickpea cultivars were evaluated, consisting of five seed lots of cv. BRS Aleppo and four seed lots of cv. Cícero. Test were performed on the seeds of each cultivar for initial characterization of the physiological potential of the lots, and the results were compared with the determinations made via SAPL®. The results showed high correlations between the variables obtained from computerized analysis and from traditional vigor tests, especially for the close relationship between the cold test and low temperature germination test with the corrected vigor index (r > 0.9) evaluated on the fourth day after the beginning of the test. The total length of seedlings, length of the primary root, and growth and corrected vigor indices obtained through computerized analysis of seedlings on the 4th day after sowing allowed separation of the lots regarding vigor. Computerized analysis of seedlings using SAPL® is effective in determining the vigor of chickpea seed lots.

Tibet The Seed Germination Characteristics Under Low Temperature and Spring Sowing Study of 8 Rapeseed Varieties

To explore the low temperature germination characteristics of rapeseed, 8 main cultivars were selected for germination experiments in incubators at 20℃, 16℃, 12℃ and 8℃, respectively, to measure and analyze the germination potential, germination rate, root length, bud length, etc. The results showed that there was significant difference in the germination potential of tested rapeseeds at temperatures. And the low temperature of 8℃ was significantly reduced the germination potential. With the decrease of germination temperature, the growth of roots and buds were inhibited significantly. There were significant differences both in temperatures and varieties. The germination potential, root length and bud length could be used as the main index to evaluate seed germination of rapeseed under low temperature. In addition, to screen varieties suitable for spring sowing as green manure, the spring sowing test was carried out. The study found that the fresh grass yield of “Huyouzao No.1” was 1712.53 kg/m 2 , which was much higher than other 7 varieties. Its relative root length was second only to “Huyou17” and its relative bud length next to “Huyoufei No.1” at 8℃, respectively. The results can provide a reference for early spring sowing of rapeseed as green manure.

Comparative Study on Low Temperature Germination Ability of Overwintering Green Manure

Green manure is a biological fertilizer source with complete nutrients, which provides reference for the selection of green manure resistant to low temperature germination. Six green manure crops including Brassica napus L., Orychophragmus violaceus (L.) O. E. Schulz, Medicago sativa L., Lolium perenne L., Melilotus officinalis (Linn.) Pall. and Vicia villosaRoth were selected as experimental materials to study the effects of low temperature stress on germination and seedling growth of different green manure through low temperature germination experiments. The results showed that there were significant differences in cold tolerance of the seeds of 6 medium green manure crops during germination. The germination potential, germination rate, germination index, vitality index, seedling height, root length, seedling fresh weight, soluble protein content and chlorophyll content of Brassica napus L. and Vicia villosaRoth seeds were all better than those of other green manure crops, which provided theoretical basis for the actual production of green manure.