Ca2+ deficiency triggers panicle degeneration in rice mediated by Ca2+ /H+ exchanger OsCAX1a.

Increasing rice yield has always been one of the primary objectives of rice breeding. However, panicle degeneration often occurs in rice-growing regions and severely curbs rice yield. In this study, we obtained a new apical panicle degeneration mutant, which induces a marked degeneration rate and diminishes the final grain yield. Cellular and physiological analyses revealed that the apical panicle undergoes programmed cell death, accompanied by excessive accumulations of peroxides. Following, the panicle degeneration gene OsCAX1a was identified in the mutant, which was involved in Ca2+ transport. Hydroponics assays and Ca2+ quantification confirmed that Ca2+ transport and distribution to apical tissues were restricted and over-accumulated in the mutant sheath. Ca2+ transport between cytoplasm and vacuole was affected, and the reduced Ca2+ content in the vacuole and cell wall of the apical panicle and the decreased Ca2+ absorption appeared in the mutant. RNA-Seq data indicated that the abnormal CBL (calcineurin b-like proteins) pathway mediated by deficient Ca2+ might occur in the mutant, resulting in the burst of ROS and programmed cell death in panicles. Our results explained the key role of OsCAX1a in Ca2+ transport and distribution and laid a foundation to further explore the genetic and molecular mechanisms of panicle degeneration in rice.

Adaptation to high latitudes through a novel allele of Hd3a strongly promoting heading date in rice.

KEY MESSAGE: A novel Hd3a allele strongly promoting rice heading date was identified, and it functions through florigen activation complex (FAC) and was selected during the spread of rice cultivation to high-latitude areas. Heading date is a critical agronomic trait for rice that determines the utilization of light and temperature conditions and thereby affects grain yield. Rice is a short day (SD) plant, and its photoperiodic information is processed by complex pathways and integrated by florigens to control flowering. In this study, we identified a novel allele for the florigen gene Heading date 3a (Hd3a), characterized by a C435G substitution in its coding region, by a genome-wide association study (GWAS) approach in a panel of 199 high-latitude japonica rice varieties. The C435G substitution induces plants to flower 10 days earlier in high-latitude area (long day condition). Then, we mutated C435 to G in Hd3a by prime editing and found the point mutation plants flowered 12 days earlier. Further molecular experiments showed the novel Hd3a protein can interact with GF14b protein and increase the expression of OsMADS14, the output gene of florigen activation complex (FAC). Molecular signatures of selection indicated that the novel Hd3a allele was selected during the process of rice cultivation expansion into high-latitude areas. Collectively, these results provide new insights into heading date regulation in high-latitude areas and advance improvements to rice adaptability to enhance crop yield.

Five plants per RIL for phenotyping traits of high or moderate heritability ensure the power of QTL mapping in a rice MAGIC population.

Currently, the power of QTL mapping is mainly dependent on the quality of phenotypic data in a given population, regardless of the statistical method, as the quality of genotypic data is easily guaranteed in the laboratory. Increasing the sample size per line used for phenotyping is a good way to improve the quality of phenotypic data. However, accommodating a large-scale mapping population takes a large area of rice field, which frequently results in high costs and extra environmental noises. To acquire a reasonable small sample size without a penalty in mapping power, we conducted three experiments with a 4-way MAGIC population and measured phenotypes of 5, 10, and 20 plants per RIL. Three traits including heading date, plant height, and tillers per plant were focused. With SNP- and bin-based QTL mapping, 3 major and 3 minor QTLs for heading date with high heritability and 2 major QTLs for plant height with moderate heritability were commonly detected across the three experiments, but no QTL for tillers per plant with low heritability were commonly identified. In addition, bin-based QTL mapping was more powerful than SNP-based mapping and able to rank the genetic effects of parental alleles. Thus, 5 plants per RIL for phenotyping ensure the power of QTL mapping for traits of high or moderate heritability, and bin-based QTL mapping is recommended for multiparent populations.

An ethyl methanesulfonate-induced neutral mutant-bridging method efficiently identifies spontaneously mutated genes in rice.

Spontaneous mutants are mainly obtained from tissue culture or natural occurrences in plants. The traditional strategy for identifying spontaneously mutated genes is to continuously backcross these mutants to another variety and develop a near-isogenic F2 population for map-based cloning or bulked segregant analysis. However, this strategy is time-consuming. Here, we have developed a new method to efficiently accelerate the identification process. The chemical mutagen ethyl methanesulfonate was first used to treat the wild type of the spontaneous mutants to induce thousands of neutral mutations. An induced individual without any statistically significant phenotypic changes which was compared with the wild type was chosen as the neutral mutant. The spontaneous mutant was then crossed with the neutral mutant to develop a pseudo-near-isogenic F2 population in which only the induced neutral mutations and the causal mutation were segregated in the genome. This population ensures that the variation of the mutated trait is controlled only by the spontaneously mutated gene. Finally, after sequencing the neutral mutant and the mutant-type DNA pool of the F2 population the spontaneous mutation will be identified quickly by bioinformatics analysis. Using this method, two spontaneously mutated genes were identified successfully. Therefore, the neutral mutant-bridging method efficiently identifies spontaneously mutated genes in rice, and its value in other plants is discussed.

Genome-wide association studies reveal that members of bHLH subfamily 16 share a conserved function in regulating flag leaf angle in rice (Oryza sativa).

As a major component of ideal plant architecture, leaf angle especially flag leaf angle (FLA) makes a large contribution to grain yield in rice. We utilized a worldwide germplasm collection to elucidate the genetic basis of FLA that would be helpful for molecular design breeding in rice. Genome-wide association studies (GWAS) identified a total of 40 and 32 QTLs for FLA in Wuhan and Hainan, respectively. Eight QTLs were commonly detected in both conditions. Of these, 2 and 3 QTLs were identified in the indica and japonica subpopulations, respectively. In addition, the candidates of 5 FLA QTLs were verified by haplotype-level association analysis. These results indicate diverse genetic bases for FLA between the indica and japonica subpopulations. Three candidates, OsbHLH153, OsbHLH173 and OsbHLH174, quickly responded to BR and IAA involved in plant architecture except for OsbHLH173, whose expression level was too low to be detected; their overexpression in plants increased rice leaf angle. Together with previous studies, it was concluded that all 6 members in bHLH subfamily 16 had the conserved function in regulating FLA in rice. A comparison with our previous GWAS for tiller angle (TA) showed only one QTL had pleiotropic effects on FLA and TA, which explained low similarity of the genetic basis between FLA and TA. An ideal plant architecture is expected to be efficiently developed by combining favorable alleles for FLA from indica with favorable alleles for TA from japonica by inter-subspecies hybridization.

Dominant complementary interaction between OsC1 and two tightly linked genes, Rb1 and Rb2, controls the purple leaf sheath in rice.

KEY MESSAGE: Two tightly linked genes for rice purple leaf sheath were identified via map-based cloning. Further analysis indicated that these two genes together with OsC1 co-regulating the purple leaf sheath. The purple color of the leaf sheath in rice is dependent on the accumulation of anthocyanins such as cyanidin 3-O-glucoside (C3G) and peonidin 3-O-glucoside (P3G). Although many genes related to leaf sheath color have been mapped, the genetic basis for leaf sheath color is not yet clear. Here, PSH1 (purple leaf sheath 1) was mapped to chromosome 1 using an F2 and a RIL population. Map-based cloning and transformation assays further divided PSH1 as two tightly linked bHLH genes, Rb1 and Rb2. Ectopic expression of these two genes resulted in substantial accumulation of C3G and P3G in the leaf blade, leaf sheath and pericarp. Single gene mutants displayed a faded purple leaf sheath or green leaf sheath in the top half of the leaf sheath, but double mutants displayed a green leaf sheath, indicating that both genes have dosage effects on anthocyanin synthesis. However, overexpression of Rb1 and Rb2 sharply decreased grain filling. A segregation ratio of green to purple was 15:1 observed in the F2 population from parents Minghui 63 and Xizang 2, which both had green leaf sheaths; these results demonstrate that dominant complementary interaction between OsC1 and Rb (Rb1 and Rb2) controls the purple leaf sheath. These findings systematically uncovered the genetic basis of leaf sheath color and provided alternative genes for breeding anthocyanin-rich rice.

Bin-based genome-wide association analyses improve power and resolution in QTL mapping and identify favorable alleles from multiple parents in a four-way MAGIC rice population.

KEY MESSAGE: A whole genome bin map was developed for a MAGIC population. Association studies for heading date at bin level exhibited powerful QTL mapping and identified favorable alleles. The presumed advantages of multiparent advanced generation intercross (MAGIC) population in quantitative trait locus (QTL) mapping were not fully utilized in the previous studies in which genome-wide association studies (GWAS) were conducted at only single nucleotide polymorphism level. In this study, we genotyped a rice four-way MAGIC population of 247 F7 lines and their parents by sequencing. A total of 5934 bins with an average length of 65 kb were constructed and covered 97% of the genome. The MAGIC population showed low population structure and balanced parental contributions. A bin-based GWAS for heading date identified 4 QTLs in three environments. Three major QTLs were mapped exactly to the bins where the major heading date genes DTH3, Ghd7.1 and Ghd8 were located. Multiple comparisons showed that different parental alleles had varied genetic effects. Like DTH3, the alleles of the Guichao 2/YJSM, IR34 and Cypress had larger, intermediate and no effects, respectively. Based on comparative sequencing of 8 known heading date genes undetected in this MAGIC population, only Ghd7 exhibited diverse function among parents. The failure in Ghd7 mapping was well explained by its interaction with Hd1 because Ghd7 had no effects on heading date when combined with the nonfunctional hd1 carried by all four parents. Overall, bin-based GWAS have more mapping power and higher resolution with a MAGIC population and provide favorable alleles to breeders. The use of more diversified parents is encouraged to develop a MAGIC population for detecting more QTLs for important agronomical traits.

A Novel Tiller Angle Gene, TAC3, together with TAC1 and D2 Largely Determine the Natural Variation of Tiller Angle in Rice Cultivars.

Tiller angle is one of the most important components of the ideal plant architecture that can greatly enhance rice grain yield. Understanding the genetic basis of tiller angle and mining favorable alleles will be helpful for breeding new plant-type varieties. Here, we performed genome-wide association studies (GWAS) to identify genes controlling tiller angle using 529 diverse accessions of Oryza sativa including 295 indica and 156 japonica accessions in two environments. We identified 7 common quantitative trait loci (QTLs), including the previously reported major gene Tiller Angle Control 1 (TAC1), in the two environments, 10 and 13 unique QTLs in Hainan and Wuhan, respectively. More QTLs were identified in indica than in japonica, and three major QTLs (qTA3, qTA1b/DWARF2 (D2) and qTA9c/TAC1) were fixed in japonica but segregating in indica, which explained the wider variation observed in indica compared with that in japonica. No common QTLs were identified between the indica and japonica subpopulations. Mutant analysis for the candidate gene of qTA3 on chromosome 3 indicated a novel gene, Tiller Angle Control 3 (TAC3), encoding a conserved hypothetical protein controlling tiller angle. TAC3 is preferentially expressed in the tiller base. The ebisu dwarf (d2) mutant exhibited a decreased tiller angle, in addition to its previously described abnormal phenotype. A nucleotide diversity analysis revealed that TAC3, D2 and TAC1 have been subjected to selection during japonica domestication. A haplotype analysis identified favorable alleles of TAC3, D2 and TAC1, which may be used for breeding plants with an ideal architecture. In conclusion, there is a diverse genetic basis for tiller angle between the two subpopulations, and it is the novel gene TAC3 together with TAC1, D2, and other newly identified genes in this study that controls tiller angle in rice cultivars.

QTLs for heading date and plant height under multiple environments in rice.

Both heading date and plant height are important traits related to grain yield in rice. In this study, a recombinant inbred lines (RILs) population was used to map quantitative trait loci (QTLs) for both traits under 3 long-day (LD) environments and 1 short-day (SD) environment. A total of eight QTLs for heading date and three QTLs for plant height were detected by composite interval mapping under LD conditions. Additional one QTL for heading date and three QTLs for plant height were identified by Two-QTL model under LD conditions. Among them, major QTLs qHd7.1, qHd7.2 and qHd8 for heading date, and qPh1 and qPh7.1 for plant height were commonly detected. qHd7.1 and qHd7.2 were mapped to small regions of less than 1 cM. Genome position comparison of previously cloned genes with QTLs detected in this study revealed that qHd5 and qPh3.1 were two novel QTLs. The alleles of these QTLs increasing trait values were dispersed in both parents, which well explained the transgressive segregation observed in this population. In addition, the interaction between qHd7.1 and qHd8 was detected under all LD conditions. Multiple-QTL model analysis revealed that all QTLs and their interactions explained over 80% of heading date variation and 50% of plant height variation. Two heading date QTLs were detected under SD condition. Of them, qHd10 were commonly identified under LD condition. The difference in QTL detection between LD and SD conditions indicated most heading date QTLs are sensitive to photoperiod. These findings will benefit breeding design for heading date and plant height in rice.

Duplication of OsHAP family genes and their association with heading date in rice.

Heterotrimeric Heme Activator Protein (HAP) family genes are involved in the regulation of flowering in plants. It is not clear how many HAP genes regulate heading date in rice. In this study, we identified 35 HAP genes, including seven newly identified genes, and performed gene duplication and candidate gene-based association analyses. Analyses showed that segmental duplication and tandem duplication are the main mechanisms of HAP gene duplication. Expression profiling and functional identification indicated that duplication probably diversifies the functions of HAP genes. A nucleotide diversity analysis revealed that 13 HAP genes underwent selection. A candidate gene-based association analysis detected four HAP genes related to heading date. An investigation of transgenic plants or mutants of 23 HAP genes confirmed that overexpression of at least four genes delayed heading date under long-day conditions, including the previously cloned Ghd8/OsHAP3H. Our results indicate that the large number of HAP genes in rice was mainly produced by gene duplication, and a few HAP genes function to regulate heading date. Selection of HAP genes is probably caused by their diverse functions rather than regulation of heading.

Combinations of the Ghd7, Ghd8 and Hd1 genes largely define the ecogeographical adaptation and yield potential of cultivated rice.

Rice cultivars have been adapted to favorable ecological regions and cropping seasons. Although several heading date genes have separately made contributions to this adaptation, the roles of gene combinations are still unclear. We employed a map-based cloning approach to isolate a heading date gene, which coordinated the interaction between Ghd7 and Ghd8 to greatly delay rice heading. We resequenced these three genes in a germplasm collection to analyze natural variation. Map-based cloning demonstrated that the gene largely affecting the interaction between Ghd7 and Ghd8 was Hd1. Natural variation analysis showed that a combination of loss-of-function alleles of Ghd7, Ghd8 and Hd1 contributes to the expansion of rice cultivars to higher latitudes; by contrast, a combination of pre-existing strong alleles of Ghd7, Ghd8 and functional Hd1 (referred as SSF) is exclusively found where ancestral Asian cultivars originated. Other combinations have comparatively larger favorable ecological scopes and acceptable grain yield. Our results indicate that the combinations of Ghd7, Ghd8 and Hd1 largely define the ecogeographical adaptation and yield potential in rice cultivars. Breeding varieties with the SSF combination are recommended for tropical regions to fully utilize available energy and light resources and thus produce greater yields.

A transposon insertion in the promoter of OsUBC12 enhances cold tolerance during japonica rice germination.

Low-temperature germination (LTG) is an important agronomic trait for rice (Oryza sativa). Japonica rice generally has greater capacity for germination at low temperatures than the indica subpopulation. However, the genetic basis and molecular mechanisms underlying this complex trait are poorly understood. Here, we report that OsUBC12, encoding an E2 ubiquitin-conjugating enzyme, increases low-temperature germinability in japonica, owing to a transposon insertion in its promoter enhancing its expression. Natural variation analysis reveals that transposon insertion in the OsUBC12 promoter mainly occurs in the japonica lineage. The variation detected in eight representative two-line male sterile lines suggests the existence of this allele introgression by indica-japonica hybridization breeding, and varieties carrying the japonica OsUBC12 locus (transposon insertion) have higher low-temperature germinability than varieties without the locus. Further molecular analysis shows that OsUBC12 negatively regulate ABA signaling. OsUBC12-regulated seed germination and ABA signaling mainly depend on a conserved active site required for ubiquitin-conjugating enzyme activity. Furthermore, OsUBC12 directly associates with rice SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 1.1 (OsSnRK1.1), promoting its degradation. OsSnRK1.1 inhibits LTG by enhancing ABA signaling and acts downstream of OsUBC12. These findings shed light on the underlying mechanisms of UBC12 regulating LTG and provide genetic reference points for improving LTG in indica rice.

Combination of maleic hydrazide and coumarin inhibits rice seed germination involving reactive oxygen species accumulation, ABA metabolism and starch degradation.

Pre-harvest sprouting (PHS) in cereal crops is a prevalent phenomenon that impacts grain yield and quality. Several PHS inhibitory compounds were screened and identified in previous studies, such as eugenol (EUG), maleic hydrazide (MH), coumarin (COU), etc. However, few studies have focused on the combination of PHS inhibitors, and the inhibitory mechanism remains unclear. Here, through combination tests of EUG, MH, and COU, the optimal combination of PHS inhibitors was selected as MH 20 mg L-1 + COU 100 mg L-1, which presented the lowest germination percentages. The optimal combination treatment significantly decreased the germination rate, α-amylase activity, content of soluble sugar and soluble protein, enhanced ABA content and the activity of superoxide dismutase (SOD) and peroxidase (POD), inhibited the production of superoxide anion (O2-) and hydrogen peroxide, and reduced the content of malondialdehyde (MDA); conversely, this trend is precisely the opposite in normal germination. Furthermore, gene expression analysis revealed that the optimal combination of MH and COU significantly decreased the expression level of OsAmy1A and OsAmy3D at 12 and 48 h after imbibition (HAI); and promoted the expression of OsRbohs (OsRbohA, OsRbohC, OsRbohD, OsRbohE, OsRbohH) and ABA biosynthetic genes OsNCED1, OsNCED2, and OsNCED5, especially OsNCED2 at 12 HAI, but down-regulated expression of OsRbohs and ABA catabolic genes OsABA8ox1-3 at 48 HAI. These results demonstrated that the delay in seed germination induced by MH and COU involved in ROS, ABA, and sugars; the optimal combination of MH and COU inhibited the germination process by promoting ABA biosynthesis and reducing ABA catabolism, and restraining the α-amylase activity to lower soluble sugar content. Intriguingly, although the expression of OsRbohs, which play a crucial role in generating ROS, increased in early imbibition (12h), the activity of the antioxidant enzymes SOD and POD also increased with the optimal combination treatment of MH and COU, which lead to the delay in ROS accumulation and inhibition of germination. These results have deepened our understanding of the regulatory mechanism of PHS inhibitors and provided theoretical support for the application of MH and COU in preventing sprouting before crop harvesting.

Toxicity effects of nanoplastics on soybean (Glycine max L.): Mechanisms and transcriptomic analysis.

Microplastic (MP) pollution has become a major concern in recent years. In agricultural production, MPs can not only affect the growth of crops but also affect yield. Compared with micron-sized MPs, nanoplastics (NPs) may be more harmful to plants. However, the effects of NPs on plant growth and development have attracted relatively little attention. As such, research has currently plateaued at the level of morphology and physiology, and the molecular mechanisms are still unclear. In this study, soybeans (Glycine max L.) were treated with polystyrene nanoplastics (PS-NPs) to observe phenotypic changes and measure the effects of PS-NPs on diverse aspects of soybeans. Compared to the control group, the soybean stem and root lengths were inhibited by 11.78% and 12.58%, respectively. The reactive oxygen species content and the antioxidant enzyme activities changed significantly (p < 0.05). The accumulation of manganese (Mn) and magnesium (Mg) in the roots revealed that root transmembrane transport was affected by PS-NPs stress. The content of salicylic acid 2-O-ß-glucoside was inhibited whereas the accumulation of l-tryptophan, the precursor of auxin synthesis, was significantly increased (p < 0.05) in leaves. Transcriptomic analysis showed that PS-NPs could affect soybean DNA repair, membrane protein transport, and hormone synthesis and response. This study revealed the toxicity of NPs to soybeans and that NPs affected a variety of biological processes through transcriptome and hormone metabolome analysis, which provides a theoretical basis to further study the molecular mechanism of the effects on plants.

[Discovery of QTLs increasing yield related traits in common wild rice].

Common wild rice (Oryza rufipogon) is an important genetic resource. Discovery of desirable alleles in wild rice will make important contributions to rice genetic improvement. In this study, Zhenshan 97 as the recurrent parent and wild rice as the donor parent were used to develop a BC2F1 population. One plant BC2F1-15 in the population showed distinct phenotype from Zhenshan 97 was selected to produce a population of BC2F5 by continuous self-crossing. The genotype assay of the plant BC2F1-15 with 126 polymorphic SSR markers evenly distributed on 12 chromosomes showed that it was heterozygous at 30% of the control marker loci. Four, 3, 4, 2, and 1 QTLs were detected for heading date, plant height, spikelets per panicle, grain weight, and single plant yield in the BC2F5 population, respectively. One QTL region flanked by the marker interval of RM481-RM2 on chromosome 7 had pleiotropic effects on heading date, spikelets per panicle, and grain yield per plant, and the alleles of wild rice increased phenotypic values. At the other 3 QTLs for spikelets per panicle, common wild rice had positive effects. These results clearly showed that common wild rice carried desirable alleles for yield related traits. The favorable alleles from common wild rice are new valuable genes for rice breeding.

Bin-based genome-wide association studies reveal superior alleles for improvement of appearance quality using a 4-way MAGIC population in rice.

INTRODUCTION: The multiparental population provides us the chance to identify superior alleles controlling a trait for genetic improvement. Genome wide association studies at bin level (bin-GWAS) are expected to be more power in QTL mapping than GWAS at SNP level (SNP-GWAS). OBJECTIVES: This study is to estimate genetic effects of QTL conferring grain appearance quality in rice by SNP-GWAS and bin-GWAS, compare their power in QTL mapping and identify the superior alleles of all detected QTL from 4 parents for genetic improvement. METHODS: A 4-way MAGIC population and its four founders were cultivated in two environments to dissect the genetic basis of rice grain appearance quality. Both SNP-GWAS and bin-GWAS were conducted for QTL mapping. Multiple comparison among 4 parental bin/alleles was used to identify the superior alleles. RESULTS: A total of 16 and 20 QTL associated with grain appearance quality were identified by SNP- and bin-GWAS, respectively. A minor chalkiness QTL qPGWC8.2/qDEC8 was assigned to a 30-kb genomic region, in which OsMH_08T0121900 is the potential candidate gene because its encoded protein, glucan endo-1,3-beta-glucosidase precursor is involved in the starch and sucrose metabolism pathway. The superior parental alleles for GS3, GL3.1, GW5, GW7, and Chalk5 and two QTLs were almost carried by the high-quality parents Cypress and Yuejingsimiao (YJSM), while the poor-quality parent Guichao-2 (GC2) always carried the inferior alleles. The top five recombinant inbred lines with the highest quality of grain shape and chalkiness traits all carried gene combinations of superior alleles. CONCLUSIONS: Both SNP- and bin-GWAS methods are encouraged for joint QTL mapping with MAGIC population. qPGWC8.2/qDEC8 is a novel candidate gene strongly associated with chalkiness. The superior alleles of GS3, GW5, GL3.1, GW7, Chalk5 and qPGWC8.2 were identified, and the pyramiding of these superior alleles is helpful to improve rice appearance quality.

A mini review of MAR-binding proteins.

Genomic DNA encompasses several levels of organization, the nuclear matrix mediates the formation of DNA loop domains that are anchored to matrix attachment regions (MARs). By means of specific interaction with MAR binding proteins (MARBPs), MAR plays an important regulation role in enhancing transgene expression, decreasing expression variation among individuals of different transformants and serving as the replication origin. Through these years, some MARBPs have been identified and characterized from humans, plants, animals and algae so far and the list is growing. Most of MARBPs exist in a co-repressor/co-activator complex and involve in chromosome folding, regulation of gene expression, influencing cell development and inducing cell apoptosis. This review covers recent advances that have contributed to our understanding of MARBPs.

Development of Whole-Genome Agarose-Resolvable LInDel Markers in Rice.

The level of difficulty involved in separating marker genotypes greatly determines the utilization of such marker-aided selection (MAS) by breeders. Genotyping by use of agarose gel electrophoresis is easily accepted by breeders due to its simple requirements and easy operation in the lab. Here, we extracted 19,937 large fragment insertions/deletions (LInDels) that were 30-55 bp based on two indica rice and one japonica rice reference genome sequences. Thousands of primer pairs were designed by the Primer 3 program to amplify the corresponding LInDels, and 6582 LInDel markers with unique genome loci were reserved after being tested by e-PCR; 346 of these markers were validated in a panel of 22 cultivars by running on a 1.5% agarose gel. Most LInDel markers had a considerable number of polymorphisms. The LInDel markers have an equivalent efficiency to that of the SSR and SNP markers in identifying hybrids, estimating genetic distance and developing genetic linkage maps. The hybrid genotypes of the LInDel markers exhibited three bands, which were the result of heteroduplex formation between the insertion allele and the deletion allele. Fifty-five breeding markers, including 9 intragenic markers and 46 closely linked LInDel markers, were developed for 55 known genes that are related to yield, biotic and abiotic stress tolerance. These agarose-resolvable LInDel markers will be welcomed by breeders and will play an important role in MAS.

Downregulation of DBN1 is related to vincristine resistance in colon cancer cells.

OBJECTIVE: This study was aimed to investigate the relationship between the expression of drebrin (DBN1) gene and resistance in colon cancer to reveal the mechanism of tumor drug resistance and provide a basis for the reversal of this drug resistance in tumor cells. MATERIALS AND METHODS: The human colon carcinoma cell line HCT-8 was used, and vincristine (VCR)-resistant colon cancer cell line HCT-8/V was established by gradually increasing the concentration of VCR. Polymerase chain reaction (PCR) primers were designed for DBN1 gene. The DBN1 differential expression in colon cancer sensitive and resistant cell lines was detected by fluorescence quantitative PCR. Western blot analysis was used to study DBN1 expression in the resistant cells further. RESULTS: VCR resistance of HCT-8/V cell line was established. Quantitative PCR and Western blot results showed that DBN1 expression in the resistant cell line was significantly lower, the difference being statistically significant (P < 0.05). CONCLUSION: Low DBN1 gene expression may be associated with colon cancer cell resistance to VCR.

Minor Effects of 11 Dof Family Genes Contribute to the Missing Heritability of Heading Date in Rice (Oryza sativa L.).

DNA binding with one finger (Dof) proteins are plant-specific transcription factors with important and diverse functions in seed germination, flowering time, and biotic and abiotic stresses. In this study, haplotype-based association analysis was conducted between heading date and 30 Dof family genes in a worldwide germplasm collection. Of these, 22 Dof genes were associated with heading date. Multiple comparisons among haplotypes revealed their diverse functions in promoting and suppressing heading date under short-day (SD) and long-day (LD) conditions. They cumulatively made a considerable contribution to the missing heritability of heading date. A set of knockout mutants of 30 Dof genes generated by CRISPR/Cas9-mediated genome editing technology showed that 11 and 9 Dof genes regulated heading date under LD and SD, respectively. Phenotype measurement of mutants showed that these 11 and 9 Dof genes slightly regulated heading with effects of 2-5 days under LD and SD, respectively. Both mutant and natural variation assays indicated functional redundancy in regulating heading date among Dof family genes. Nucleotide diversity analysis suggested that most Dof genes have been subjected to selection during domestication and improvement. Beyond heading date, this set of mutants is also a good resource for evaluating the function of Dof genes in regulating stress tolerance and seed germination.