Effects of Nitrogen Application Time on Caryopsis Development and Grain Quality of Rice Variety Yangdao 6

A pot experiment was conducted to study the effects of different nitrogen application time （during the tillering or the booting stages） with the same nitrogen rates on the caryopsis development and grain quality of rice variety Yangdao 6. The increased nitrogen fertilizer （urea）, especially applied during the booting stage, could evidently increase the milled rice rate, head rice rate and protein content in rice grains compared with the control （no nitrogen application）, and decrease chalky grain rate and amylose content. Moreover, the increased nitrogen fertilizer significantly affected the caryopsis development and enhanced the grain weight when nitrogen applied during the tillering and the booting stages, especially during the booting stage. During caryopsis development the increased nitrogen fertilizer applied during the tillering and booting stages could obviously decrease the total starch and amylose contents, but not obviously for the amylopectin content in rice grain. Increased topdressing of nitrogen fertilizer, especially applied during the booting stage, had significant effect on the development and structures of amyloplasts and proteinoplasts. That is, it could change the distribution, number and shape of amyloplasts and proteinoplasts in the endosperm cells especially in grain abdomen, Compared with the control the arrangements of amyloplasts and proteinoplasts were closer, with more numbers, higher density and less interspaces each ohter. Furthermore, most amyloplasts showed polyhedron under the increased nitrogen fertilizer level.

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 hese methods for crop improvement are reviewed in this paper.

InDel and SNP Markers and Their Applications in Map-based Cloning of Rice Genes

High-density markers are necessary for map-based cloning of rice genes, but the currently available markers are not satisfactory enough. InDel (insertion-deletion length polymorphism) and SNP (single nucleotide polymorphism) are the new generation of molecular markers and can basically meet the need of fine mapping. InDel and SNP markers can be developed through bioinformatics. These markers are valuable markers with the characters of low cost, high specificity and stability. This article introduced the methods for designing InDel and SNP markers, taking the mapping of a rice rolled leaf gene as an example. In addition, some key factors in improving the design efficiency were also discussed.

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.

Effects of High Temperature on Antioxidant Enzymes and Lipid Peroxidation in Flag Leaves of Wheat During Grain Filling Period

On the basis of the phytotron, the effects of high temperature （daily average temperature 25, 30, 35 and 40℃, respectively） on antioxidant enzymes and lipid peroxidation in flag leaves of wheat at 50% relative air moisture during grain fastest filling stage [19-21 days after anthesis （DAA）] were studied. The wheat cultivars tested were Yangmai 9 with weak-gluten and Yangmai 12 with medium-gluten. Compared with 25℃, the higher the temperature was, the higher was the MDA content in flag leaves, while lower were the SOD, POD, and CAT activities. SOD and CAT activities in Yangmai 12 appeared to be more sensitive to high temperature than that in Yangmai 9. But POD activity in Yangmai 12 was less sensitive to high temperature. MDA content in Yangmai 12 was higher than that in Yangmai 9. The 1000-grain weight declined with increase in temperature.

Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

Somatic hybridization is performed to obtain significant cytoplasmic male sterility （CMS） lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-IA, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-IB, and the abortive process of SaNa-IA were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-IA was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-IA aborted in the pollen mother cell （PMC） stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-IA is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

Co-location of QTL for Sclerotinia stem rot resistance and flowering time in Brassica napus

Sclerotinia stem rot(SSR) caused by Sclerotinia sclerotiorum(Lib.) de Bary is one of the most devastating diseases of Brassica napus worldwide. Both SSR resistance and flowering time(FT) adaptation are major breeding goals in B. napus. However, early maturing rapeseed varieties, which are important for rice-rapeseed rotation in China, are often highly susceptible to SSR. Here, we found that SSR resistance was significantly negatively correlated with FT in a natural population containing 521 rapeseed inbred lines and a double haploid(DH) population with 150 individual lines, both of which had great variation in FT. Four chromosomal regions on A2, A6, C2, and C8 affecting both SSR resistance and FT were identified using quantitative trait loci(QTL) mapping after constructing a high-density genetic map based on single nucleotide polymorphism markers in the DH population.Furthermore, we aligned QTL for the two traits identified in the present and previous studies to the B. napus reference genome, and identified four colocalized QTL hotspots of SSR resistance and FT on A2(0–7.7 Mb), A3(0.8–7.5 Mb), C2(0–15.2 Mb), and C6(20.2–36.6 Mb). Our results revealed a genetic link between SSR resistance and FT in B.napus, which should facilitate the development of effective strategies in both early maturing and SSR resistance breeding and in map-based cloning of SSR resistance QTL.

Systematic Identification of Rice ABC1 Gene Family and Its Response to Abiotic Stress

Members of the activity of bc1 complex (ABC1) family are protein kinases that are widely found in prokaryotes and eukaryotes. Previous studies showed that several plant ABC1 genes participated in the abiotic stress response. Here, we present the systematic identification of rice and Arabidopsis ABC1 genes and the expression analysis of rice ABC1 genes. A total of 15 and 17 ABC1 genes from the rice and Arabidopsis genomes, respectively, were identified using a bioinformatics approach. Phylogenetic analyses of these proteins suggested that the divergence of this family had occurred and their main characteristics were established before the monocot-dicot split. Indeed, species-specific expansion contributed to the evolution of this family in rice and Arabidopsis after the monocot-dicot split. Intron/exon structure analysis indicated that most of the orthologous genes had similar exon sizes, but diverse intron sizes, and the rice genes contained larger introns, moreover, intron gain was an important event accompanying the recent evolution of the rice ABC1 family. Multiple sequence alignment revealed one conserved amino acid segment and four conserved amino acids in the ABC1 domain. Online subcellular localization predicted that nine rice ABC1 proteins were localized in chloroplasts. Real-time RT-PCR established that the rice ABC1 genes were primarily expressed in leaves and the expression could be modulated by a broad range of abiotic factors such as H2O2, abscisic acid, low temperature, drought, darkness and high salinity. These results reveal that the rice ABC1 gene family plays roles in the environmental stress response and specific biological processes of rice.

Effects of 1,2,4-Trichlorobenzene and Mercury Ion Stress on Ca^2+ Fluxion and Protein Phosphorylation in Rice

The effects of 5 mg/L 1,2,4-trichlorobenzene （TCB） and 0.1 mmol/L mercury ion （Hg^2＋） stresses on Ca^2＋ fluxion and protein phosphorylation in rice seedlings were investigated by isotope exchange kinetics and in vitro phosphorylation assay. The Ca^2＋ absorption in rice leaves and Ca^2＋ transportation from roots to leaves were promoted significantly in response to Hg^2＋ and TCB treatments for 4-48 h. The Ca^2＋ absorption peaks presented in the leaves when the rice seedlings were exposed to Hg^2＋ for 8-12 h or to TCB for 12-24 h. Several Ca^2＋ absorption peaks presented in the roots during rice seedlings being exposed to Hg^2＋ and TCB, and the first Ca^2＋ absorption peak was at 8 h after being exposed to Hg^2＋ and TCB The result of isotope exchange kinetic analysis confirmed that short-term （8 h） Hg^2＋ and TCB stresses caused Ca^2＋ channels or pumps located on plasmalemma to open transiently. The phosphorylation assay showed that short-term TCB stress enhanced protein phosphorylation in rice roots （TCB treatment for 4-8 h） and leaves （TCB treatment for 4-24 h）, and short-term （4-8 h） Hg^2＋ stress also enhanced protein phosphorylation in rice leaves. The enhancement of protein phosphorylation in both roots and leaves corresponded with the first Ca^2＋ absorption peak, which confirmed that the enhancement of protein phosphorylation caused by TCB or Hg^2＋ stress might be partly triggered by the increases of cytosolic calcium. TCB treatment over 12 h inhibited protein phosphorylation in rice roots, which might be partly due to that TCB stress suppressed the protein kinase activity. Whereas, Hg^2＋ treatment inhibited protein phosphorylation in rice roots, and Hg^2＋ treatment over 12 h inhibited protein phosphorylation in rice leaves. This might be attributed to that not only the protein kinase activity, but also the expressions of phosphorylation proteins were restrained by Hg^2＋ stress.

Meta-analysis of QTL for Fusarium head blight resistance in Chinese wheat landraces

Epidemics of Fusarium head blight(FHB), incited by Fusarium graminearum Schwabe, in wheat cause significant reductions in grain yield and quality. Numerous quantitative trait loci(QTL) for FHB resistance have been reported from Chinese sources. However, the relationships among QTL from different landraces have not been characterized. We earlier mapped QTL for FHB resistance using low-density maps developed from five recombinant inbred line(RIL) populations involving Chinese landraces ‘Haiyanzhong’(HYZ),‘Wangshuibai’(WSB), ‘Baishanyuehuang’(BSYH), ‘Huangfangzhu’(HFZ), and‘Huangcandou’(HCD) as FHB resistant parents. In this study, we used maps of single nucleotide polymorphisms(SNP) developed from the five populations and identified 31 QTL on 16 chromosomes;10 QTL were new. We constructed a consensus map and identified six meta-QTL(MQTL) and SNP within the MQTL regions using meta-analysis. Two of the MQTL were on chromosome 3 BS(3 BSd and 3 BSc), and one on each of chromosomes 3A, 2D, 3D,and 4D. Twenty-two SNP closely linked to MQTL were converted into breeder friendly Kompetitive Allele Specific Polymerase Chain Reaction(KASP) assays, which should be useful for marker-assisted selection in breeding programs.

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.

Transcriptional proffling between yellow-and black-seeded Brassica napus reveals molecular modulations on flavonoid and fatty acid content

Brassica napus is an important cash crop broadly grown for the vegetable and oil values.Yellow-seeded B.napus is preferred by breeders due to its improved oil and protein quality,less pigments and lignin compared with the blackseeded counterpart.This study compared the differences in flavonoid and fatty acid contents between yellow rapeseed from the progenies of B.napus-Sinapis alba somatic hybrids and the black-seeded counterpart using RNA-seq analysis.Through HPLC-PDA-ESI(-)/MSanalysis,it was found that phenylpropanoids and flavonoids(i.e.,isorhamnetin,epicatechin,kaempferol,and other derivatives)in yellow seed were significantly lower than those in black seed.The fatty acid(FA)content in yellow rapeseed was higher than that in black rapeseed due to the variation of C16:0,C18:0,C18:1,C18:2,and C18:3 contents.RNA-seq analysis of seeds at four and five weeks after flowering(WAF)indicated that differentially expressed genes(DEGs)between black and yellow rapeseeds were enriched in flavonoid and FA biosynthesis,including BnTT3,BnTT4,BnTT18,and BnFAD2.Also,genes related to FA biosynthesis,desaturation and elongation(FAD3,LEC1,FUS3,and LPAT2)in yellow seed were up-regulated compared to those in black seed,while genes involved in beta-oxidation cycle(AIM1 and KAT2)of yellow seed were down-regulated compared to those in black seed.The DEGs related to the variation of flavonoids,phenylpropanoids,and FAs would help improve the knowledge of yellow seed character in B.napus and promote rapeseed improvement.

Molecular Cytogenetic Analysis of Spontaneous Interspecific Hybrid Between Oryza sativa and Oryza minuta

Genomic in situ hybridization (GISH) is a powerful tool to characterize parental chromosomes in interspecific hybrids, including the behaviour of autosynapsis and chromosome pairing. It was used to distinguish the chromosomes of Oryza sativa from wild species in a spontaneous interspecific hybrid and to investigate the chromosome pairing at metaphase I in meiosis of the hybrid in this study. The hybrid was a triploid with 36 chromosomes according to the chromosome number investigated in mitosis of root tips. During metaphase I of meiosis in the hybrid, less chromosome pairing was observed and most of the chromosomes existed as univalent. Based on GISH and FISH (Fluorescent in situ hybridization) analyses, the chromosomes of the hybrid were composed of genomes A, B and C. Thus, it was believed that the hybrid was the result of natural hybridization between cultivated rice and wild species O. minuta which was planted in experimental fields.

QTLs for Sugar Content of Stalk in Sweet Sorghum(Sorghum bicolor L.Moench)

High sugar content of sorghum stalk is an important factor in the sorghum silage production. To identify the genomic regions controlling sugar content and to develop molecular markers linked to sugar content in sweet sorghum, we used an F2：3 segregating population consisting of 207 individuals derived from a cross between a high sugar content inbred line, Early Folger, and a normal inbred line, N32B, for genetic linkage mapping and quantitative trait locus （QTL） analysis. We constructed a genetic linkage map spanning 983.5 cM based on a total of 327 markers comprising 31 restriction fragment length polymorphism （RFLP） markers, 254 amplified fragment length polymorphism （AFLP） markers, and 42 simple sequence repeat （SSR） markers. In the 20 linkage groups detected, 98.2% of markers aligned to the 10 linkage groups of sorghum. Variations in sugar content at different growth stages and among internodes suggested that the sugar content of middle internodes is stable and suitable for measuring at early dough stage. The broad sense heritability （hB0 of sugar content was 0.64 and 0.62 estimated from the data of F3 families and each parent in 2003 and 2004. We identified one and two QTLs accounting for 22.2 to 25.0% of phenotypic variance using simple interval mapping method in 2003 and 2004, respectively. These two QTLs showed a negative additive effect, and over-dominance effect. A QTL on LG-D was detected in both two years. Above results will be help us to understand the genetic mechanism of sugar content in sorghum and the QTL detected in this study might be useful in the improvement of sugar content by marker-assisted selection.

Analysis of cytosine methylation in early generations of resynthesized Brassica napus

DNA methylation, an important epigenetic modification, serves as a key function in the polyploidization of numerous crops. In this study, early generations of resynthesized Brassica napus （F1,S1-S3）, ancestral parents B. rapa and B. oleracea were analyzed to characterize their DNA methylation status during polyploidization, applying DNA methylation-sensitive amplifica- tion polymorphism （MSAP） and high-performance liquid chromatography methods. In F, 53.4% fragments were inherited from both A- and C-genomes. Besides, 5.04 and 8.87% fragments in F were inherited from A- and C- genome, respectively. 5.85 and 0.8% fragments were newly appeared and disappeared in resynthesized B. napus, respectively. 13.1% of these gene sites were identified with methylation changes in F, namely, hypermethylation （7.86%） and hypomethylation （5.24%）. The lowest methylation status was detected in F （38.7%） compared with in S1-S3. In S3, 40.32% genes were methylated according to MSAP analysis. Sequencing of methylated fragments indicated that genes involved in multiple biological processes were modified, including transcription factors, protein modification, and transporters. Expression ananlysis of DNA methyltransferase I and DNA methyltransferase chromomethylase 3 in different materials was consistent to the DNA methylation status. These results can generally facilitate dissection of how DNA methylation contributes to genetic stability and improvement of B. napus during polypLoidization.

A Preliminary Study of Mapping Genes Underlying Complex Traits Based on Chromosome Segment Substitution Lines

Complex traits are the features whose properties are determined by multiple factors, which can be genetic or environmental. Most of economically important characteristics of plants and animals belong to this special catego-

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.

A Rank-Sum Testing Method for Multi-Trait Comprehensive Ranking and Its Application

The rank-sum test is a nonparametric method used in variety evaluation. However, the hypothesis testing of the method hasn＇t been established for multi-trait comprehensive ranking. In this paper, under null hypothesis H0： the variety＇s ranking on each trait is random, the theoretical distribution of sum of ranks （SR） was firstly derived and further used to obtain the critical values for multi-trait comprehensive evaluation in rank-sum testing. A new C＋＋ class and its basic arithmetic were defined to deal with the miscount caused by the precision limitation of built-in data type in common statistical software under large number of varieties and traits. Finally, an application of the theoretical results was demonstrated using five starch viscosity traits of 12 glutinous maize varieties. The proposed method is so simple and convenient that it can be easily used to rank different varieties by multiple traits.

Comparison of Supervised Clustering Methods for the Analysis of DNA Microarray Expression Data

Several typical supervised clustering methods such as Gaussian mixture model-based supervised clustering （GMM）, k- nearest-neighbor （KNN）, binary support vector machines （SVMs） and multiclass support vector machines （MC-SVMs） were employed to classify the computer simulation data and two real microarray expression datasets. False positive, false negative, true positive, true negative, clustering accuracy and Matthews＇ correlation coefficient （MCC） were compared among these methods. The results are as follows： （1） In classifying thousands of gene expression data, the performances of two GMM methods have the maximal clustering accuracy and the least overall FP＋FN error numbers on the basis of the assumption that the whole set of microarray data are a finite mixture of multivariate Gaussian distributions. Furthermore, when the number of training sample is very small, the clustering accuracy of GMM-Ⅱ method has superiority over GMM- Ⅰ method. （2） In general, the superior classification performance of the MC-SVMs are more robust and more practical, which are less sensitive to the curse of dimensionality, and not only next to GMM method in clustering accuracy to thousands of gene expression data, but also more robust to a small number of high-dimensional gene expression samples than other techniques. （3） Of the MC-SVMs, OVO and DAGSVM perform better on the large sample sizes, whereas five MC-SVMs methods have very similar performance on moderate sample sizes. In other cases, OVR, WW and CS yield better results when sample sizes are small. So, it is recommended that at least two candidate methods, choosing on the basis of the real data features and experimental conditions, should be performed and compared to obtain better clustering result.

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.