Prospect of the QTL-qSB-9^(Tq) utilized in molecular breeding program of japonica rice against sheath blight

The major QTL-qSB-9^Tq conferring partial resistance to rice （Oryza sativa L.） sheath blight （Rhizoctonia solani Kvhn） has been verified on chromosome 9 of the indica rice cultivar, Teqing. In this study, the prospect of this QTL utilized in molecular breeding program of japonica rice for sheath blight resistance was investigated. Most of the japonica rice cultivars showed lower level of sheath blight resistance than the indica rice cultivars. At the corresponding site of qSB-9^Tq, nine typical japonica rice cultivars from different ecological regions or countries proved to possess the susceptible allele（s）. Introgression of qSB-9^Tq into these cultivars enhanced their resistance level by decreasing sheath blight score of 1.0 （0.5-1.3）, which indicated that qSB-9^Tq had a large potential in strengthening the resistance of japonica rice to sheath blight. The use of the three molecular markers, which were polymorphic between Teqing and many japonica rice cultivars, promotes the application of qSB-9^Tq in a concrete molecular breeding program.

Genetic mapping of quantitative trait loci in crops

Dissecting the genetic architecture of complex traits is an ongoing challenge for geneticists.Two complementary approaches for genetic mapping,linkage mapping and association mapping have led to successful dissection of complex traits in many crop species.Both of these methods detect quantitative trait loci(QTL) by identifying marker–trait associations,and the only fundamental difference between them is that between mapping populations,which directly determine mapping resolution and power.Based on this difference,we first summarize in this review the advances and limitations of family-based mapping and natural population-based mapping instead of linkage mapping and association mapping.We then describe statistical methods used for improving detection power and computational speed and outline emerging areas such as large-scale meta-analysis for genetic mapping in crops.In the era of next-generation sequencing,there has arisen an urgent need for proper population design,advanced statistical strategies,and precision phenotyping to fully exploit high-throughput genotyping.

Integration of QTL detection and marker assisted selection for improving resistance to Fusarium head blight and important agronomic traits in wheat

Fusarium head blight(FHB), caused by Fusarium graminearum, is one of the most destructive wheat(Triticum aestivum L.) diseases worldwide. Identification of quantitative trait loci(QTL) conferring FHB resistance followed by marker assisted selection(MAS) is an efficient approach to breed FHB-resistant varieties. In this study, 38 additive QTL and 18 pairs of epistatic QTL for FHB resistance were detected in four environments using a population of recombinant inbred lines(RILs) derived from varieties Neixiang 188 and Yanzhan 1. Six QTL clusters were located on chromosomes 2D, 4B, 4D, 5A, 5D and 7B, suggesting possible polytrophic functions. Six elite lines with good FHB resistance and agronomic traits were selected from the same population using the associated markers. Our results suggest that MAS of multiple QTL will be effective and efficient in wheat breeding.

Identification and cell wall analysis of interspecific hybrids between Oryza sativa and Oryza ridleyi

Oryza ridleyi is an allotetraploid wild species with the HHJJ genome, and Oryza sativa is a diploid cultivated rice that has the AA genome. Although the wide hybrid between the two species is difficult to obtain, we overcome this difficulty by young embryo rescue. An obvious heterosis was primarily found for the plant height, tillering ability, vegetative vigor, etc. However, the hybrid panicle and culm traits were found to resemble that of the wild rice parent, O. ridleyi, for the long awns, exoteric purple stigma, grain shattering, dispersed panicles, and culm mechanical strength. Genomic in situ hybridization （GISH） analysis was subsequently performed on the mitotic metaphase chromosome of the root tips, and we determined that the hybrid is an allotriploid with 36 chromosomes and its genomic constitution is AHJ. Chemical analyses conducted on the culm of O. sativa, O. ridleyi, and their interspecific hybrids showed that major changes occurred in the xylose, glucose, and arabinose concentrations, which are correlated with the specific hemicellulose polymer and cellulose components that are important in the primary cell walls of green plants. Meanwhile, the culm anatomical analyses indicated that additional large vascular bundles and an extra sclerenchyma cell layer were found in O. ridleyi. Additionally, further thickening of the secondary cell walls of the cortical fiber sclerenchyma cells and the phloem companion cells was discovered in O. ridleyi and in the interspecific hybrids. These results imply that there may be a potential link between culm mechanical strength and culm anatomical structure.

Genetic analysis of the seed dehydration process in maize based on a logistic model

Seed moisture at harvest is a critical trait affecting maize quality and mechanized production,and is directly determined by the dehydration process after physiological maturity.However,the dynamic nature of seed dehydration leads to inaccurate evaluation of the dehydration process by conventional determination methods.Seed dry weight and fresh weight were recorded at 14 time points after pollination in a recombinant inbred line(RIL)population derived from two inbred lines with contrasting seed dehydration dynamics.The dehydration curves of RILs were determined by fitting trajectories of dry weight accumulation and dry weight/fresh weight ratio change based on a logistic model,allowing the estimation of eight characteristic parameters that can be used to describe dehydration features.Quantitative trait locus(QTL)mapping,taking these parameters as traits,was performed using multiple methods.Single-trait QTL mapping revealed 76 QTL associated with dehydration characteristic parameters,of which the phenotypic variation explained(PVE)was 1.03%to 15.24%.Multipleenvironment QTL analysis revealed 21 related QTL with PVE ranging from 4.23%to 11.83%.Multiple-trait QTL analysis revealed 58 QTL,including 51 pleiotropic QTL.Combining these mapping results revealed 12 co-located QTL and the dehydration process of RILs was divided into three patterns with clear differences in dehydration features.These results not only deepen general understanding of the genetic characteristics of seed dehydration but also suggest that this approach can efficiently identify associated genetic loci in maize.

Efficiencies of Generating Selectable Marker-Free Transgenic Rice with Different Transformation Methods

To study the efficiency of generating selectable marker-free （SMF） transgenic rice, two transformation methods were employed for four rice varieties （Wuxiangjing 9, Longtefu, Xieqingzao and Zhenshan 97）. One method is by using a single twin T-DNA binary vector pYH592 in one Agrobacterium strain, which is composed of two separate T-DNA regions （one carrying an antisense Wx gene and the other carrying a HPTgene）. The other one, named as two-strain/two-vector system, is by using two separate binary vectors in two separate Agrobacterium cultures. The results indicated that the average co-transformation frequencies of the antisense Wx gene and the HPT gene were 10.1% and 45.0%, respectively, for the four rice varieties. And the SMF transgenic plants selected from the offsprings of co-transformants were 55.6% and 60.0% in the two-strain/two-vector and twin T-DNA vector binary systems, respectively.

Molecular evolution and functional divergence of HAK potassium transporter gene family in rice(Oryza sativa L.)

The high-affinity K＋ （HAK） transporter gene family is the largest family in plant that functions as potassium transporter and is important for various aspects of plant life. In the present study, we identified 27 members of this family in rice genome. The phylogenetic tree divided the land plant HAK transporter proteins into 6 distinct groups. Although the main characteristic of this family was established before the origin of seed plants, they also showed some differences between the members of non-seed and seed plants. The HAK genes in rice were found to have expanded in lineage-specific manner after the split of monocots and dicots, and both segmental duplication events and tandem duplication events contributed to the expansion of this family. Functional divergence analysis for this family provided statistical evidence for shifted evolutionary rate after gene duplication. Further analysis indicated that both point mutant with positive selection and gene conversion events contributed to the evolution of this family in rice.

A combination of leaf rust resistance gene Lr34 and lesion mimic gene lm significantly enhances adult plant resistance to Puccinia triticina in wheat

Leaf rust caused by Puccinia triticina is an economically-important disease in wheat worldwide.A combination of different types of resistance genes may significantly enhance rust resistance under rust-favorable conditions.To investigate the interactions between the rust resistance gene Lr34 and the lesion mimic gene lm on 1BL in Ning 7840,a segregating F8-10 population of 180 recombinant inbred lines was developed from Ning 7840/Chokwang and evaluated for both lesion mimic expression and leaf rust response at the adult plant stage in a greenhouse.A major quantitative trait locus(QTL),derived from Sumai 3,was co-localized with Lr34 on chromosome 7D and explained 41.5% of phenotypic variations for rust severity and 22.1% for leaf tip necrosis(LTN).The presence of Lr34 was confirmed by Lr34-specific markers cssfr1 and cssfr2 in Ning 7840 and Sumai 3.Unlike Lr34,lm conditioned a spontaneous lesion mimic phenotype and had a significant effect on reducing uredinial size,and a smaller effect on severity.Additive effects were observed between lm and Lr34 for severity and LTN,and an epistatic effect was observed for infection type.Single marker analysis also identified several other QTL with minor effects on severity,infection type,or LTN.

Generating of rice OsCENH3-GFP transgenic plants and their genetic applications

In order to investigate rice functional centromeres, OsCENH3-GFP chimeric gene was constructed and transformed into the indica rice variety, Zhongxian 3037, mediated by Agrobacturium. The integration of the exogenous genes in the transgenic plants was confirmed by PCR and Southern blotting. The transgenic plants grow normally during their whole life time, just like Zhongxian 3037. No significant defects were detected in either mitosis or meiosis of the transgenic plants. The overlapping of GFP signals and anti-CENH3 foci in both mitotic and meiotic cells from T0 and T1 generation plants indicated that GFP had been successfully fused with CENH3, so the GFP signals can well represent the CENH3 locations on each chromosome. To evaluate the applicability of the transgenic plants to other genetic studies, fluorescence in situ hybridization (FISH) using rice centromeric tandem repetitive sequence CentO as the probe was conducted on the zygotene chromosomes of pollen mother cells (PMCs). It has been revealed that the GFP signals are overlapping with CentO FISH signals, showing that CentO is one of the key elements constituting rice functional centromeres. Immunofluorescent staining using anti-α-tublin antibody and anti-PAIR2 antibody on the chromosomes during mitosis and meiosis stages of the transgenic plants further reveals that OsCENH3-GFP transgenic plants can be widely used for studying rice molecular biology, especially for tagging functional centromeres in both living cells and tissues.

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.)

Gibberellin （GA） 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs （GA3 or GA4） can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs （GA12 and/or GA53） as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.

Framework for dissection of complex cytonuclear epistasis by a two-dimensional genome scan

Epistasis between cytoplasmic and nuclear genes is the primary genetic component of complex quantitative traits.Genetic dissection of cytonuclear epistasis is fundamentally important to understand the genetic architecture of complex traits.In this study,a two-dimensional genome scan strategy was employed to evaluate the contribution of cytoplasm,quantitative trait loci (QTL),QTL×QTL interactions and QTL×QTL×cytoplasm interactions to the phenotypic variation.The p-value and parameter value for each genetic effect were calculated by multiple regression analysis.A stepwise approach was suggested to build confidence in candidate QTL on the basis of q-value estimation,false discovery rate calculation and Bonferroni adjustment.A fine-scale grid scan strategy was proposed for further analysis of peaks of interest.Plant height in maize was used as an example to illustrate the efficiency of the two-dimensional genome scan strategy.

Genetic analysis and gene cloning of a triangular hull 1 (tri1) mutant in rice (Oryza sativa L.)

Grain shape and size are two key factors that determine rice yield and quality. In the present study, a rice triangular hull mutant (tri1) was obtained from the progeny of japonica rice variety Taipei 309 treated with 60Co γ-rays. Compared to the wild type, the tri1 mutant presents a triangular hull, and exhibits an increase in grain thickness and protein content, but with a slight decrease in plant height and grain weight. Genetic analysis indicated that the mutant phenotype was controlled by a recessive nuclear gene which is stably inherited. Using a map-based cloning strategy, we fine-mapped tri1 to a 47-kb region between the molecular markers CHR0122 and CHR0127 on the long arm of chromosome 1, and showed that it co-segregates with the molecular marker CHR0119. According to the rice genome sequence annotation there are six predicated genes within the mapped region. Sequencing analysis of the mutant and the wild type indicated that there was a deletion of an A nucleotide in exon 3 of the OsMADS32 gene, which could result in a downstream frameshift mutation and premature termination of the predicted polypeptide. Both semi-quantitative and real-time RT-PCR analyses showed that this gene expressed highly in young inflorescences, while expressed at very low levels in other tissues. These results implied that the OsMADS32 gene could be a candidate of TRI1. Taken together, the results of this study lay the foundation for further investigation into the molecular mechanisms regulating rice caryopsis development.

Genomic selection methods for crop improvement:Current status and prospects

With marker and phenotype information from observed populations, genomic selection(GS) can be used to establish associations between markers and phenotypes. It aims to use genome-wide markers to estimate the effects of all loci and thereby predict the genetic values of untested populations, so as to achieve more comprehensive and reliable selection and to accelerate genetic progress in crop breeding. GS models usually face the problem that the number of markers is much higher than the number of phenotypic observations.To overcome this issue and improve prediction accuracy, many models and algorithms,including GBLUP, Bayes, and machine learning have been employed for GS. As hot issues in GS research, the estimation of non-additive genetic effects and the combined analysis of multiple traits or multiple environments are also important for improving the accuracy of prediction. In recent years, crop breeding has taken advantage of the development of GS.The principles and characteristics of current popular GS methods and research progress in these methods for crop improvement are reviewed in this paper.

BZR1 Interacts with HY5 to Mediate Brassinosteroid- and Light-Regulated Cotyledon Opening in Arabidopsis in Darkness

Light and brassinosteroid （BR） are two central stimuli that regulate plant photomorphogenesis. Although previous phenotypic and physiological studies have implied possible interactions between BR and light in regulating photomorphogenesis, the underlying molecular mechanism（s） remain largely unknown. In the present study, we identified a physical connection between the BR and light signaling pathways, which was mediated by the BR-regulated transcription factor BZR1 and light-regulated transcription factor HY5 in Arabidopsis thaliana. Genetic evidence showed that the gain-of-function bzrl-lD mutant in the BR signaling pathway and loss-of-function hy5-215 mutant in the light signaling pathway exhibited closed cotyledons under BR-deficient and dark-grown conditions and both bzrl-lD and hy5-215 mutants were able to suppress the cotyledon opening phenotype of the BR-insensitive mutants bril-5 and bin2-1. Biochemical studies demonstrated that BZR1 interacts with HY5 both in vitro and in vivo and ectopic expression of HY5 considerably reduces the accumulation of BZR1 protein. In addition, HY5 specifically interacts with the dephosphorylated form of BZR1 and attenuates BZRI＇s transcriptional activity in regulating its target genes related to cotyledon opening. Our study provides a molecular framework for coordination of BR and light signals in regulating cotyledon opening, an important process in photomor- phogenesis in plants.

基于育种值预测的基因组选择方法的比较(英文)

利用高密度分子标记,在基因组水平上预测育种值已经在动植物遗传育种研究中得到应用,但是高密度标记也带来许多统计和计算上的问题.为了解决这些基因组选择中的问题,产生了很多不同的方法,包括RR-BLUP,GBLUP,Bayes A,Bayes B,Bayes Cπ和Bayesian LASSO等.本文将这些方法用于一组小麦数据集的分析,同时模拟了不同数目QTL和不同遗传率情况下各种方法分析结果的差异.研究结果表明:在确定基因组选择方法时,要充分考虑所研究性状的遗传结构.如果确认某种性状由较少的大效应QTL控制时,各种方法预测能力的差异较大,应选择Bayes Cπ.如果QTL数目中等,各种方法预测能力的绝对差异较小,但是仍然发现Bayes A优于其他方法.如果性状由大量的微效基因决定,各种方法之间几乎找不到显著的差异,不过此时无论是在模拟分析还是在小麦实际产量的预测中,RR-BLUP都略优于其他方法,说明在这种情况下RR-BLUP是有效的方法.

CAPS/dCAPS Designer: a web-based high-throughput dCAPS marker design tool

Dear Editor,An increasing number of single-nucleotide polymorphisms(SNP)have been identified and extensively used in genetics studies with advances in next-generation sequencing(NGS)and SNP arrays.However,due to frequent calling errors,the accuracy of SNPs of interest must usually be validated.In recent years,the single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction(Ni PTag,the commercial technology named KASP),a relatively new and powerful SNP

Rice OsGL 1-1 Is Involved in Leaf Cuticular Wax and Cuticle Membrane

Cuticular wax forms a hydrophobic barrier on aerial plant organs; it plays an important role in protecting a plant from damage caused by many forms of environmental stress. In the present study, we characterized a rice leaf wax-deficient mutant osgll-1 derived from a spontaneous mutation, which exhibited a wax-deficient and highly hydro- philic leaf phenotype. We cloned the OsGLI-1 gene by the map-based cloning method and performed a complementation test to confirm the function of the candidate gene. Molecular studies revealed that OsGLI-1 was a member of the OsGL1 family, and contained regions that were homologous to some regions in sterol desaturases and short-chain dehydro- genases/reductases. Compared to the wild-type, the osgll-1 mutant showed decreased cuticular wax deposition, thinner cuticular membrane, decreased chlorophyll leaching, increased rate of water loss, and enhanced sensitivity to drought. OsGL 1-1 is expressed ubiquitously in rice. The transient expression of OsGLl-l-green fluorescent protein fusion protein indicated that OsGLI-1 is localized in the cytoplasm, plasma membrane, and nucleus.

Centromere inactivation in a dicentric rice chromosome during sexual reproduction

T0135 is a variant selected from the progeny of a rice line telotrisomic for the short arm of chromosome 11(2n+11S·).Fluorescent in situ hybridization(FISH)results indicated that T0135 contained two telocentric chromosomes,which have two centromere-specific molecular markers(5S rDNA)for chromosome 11;thus T0135 is a newly-described rice chromosome variant with two dicentric chromosomes,named 22+11L·+11L·+11S·11S·+11S·11S·(22 represents the 22 chromosomes excluding chromosome 11 in the rice genome,"·"represents the centromere).To investigate the genetic stability of the rice dicentric chromosomes during sexual reproduction,we observed the chromosome types in the progeny.Ninety-four percent of the progeny had the same chromosome type as the parental line.This result indicates that the dicentric chromosomes are mostly stable during mitosis and meiosis.Immunofluorescence analysis for centromere specific histone H3(CENH3)revealed that only one centromere is active and the other centromere is inactivated in the rice dicentric chromosomes.