Genome-wide association with transcriptomics reveals a shade-tolerance gene network in soybean

Shade tolerance is essential for soybeans in inter/relay cropping systems.A genome-wide association study(GWAS)integrated with transcriptome sequencing was performed to identify genes and construct a genetic network governing the trait in a set of recombinant inbred lines derived from two soybean parents with contrasting shade tolerance.An improved GWAS procedure,restricted two-stage multi-locus genome-wide association study based on gene/allele sequence markers(GASM-RTM-GWAS),identified 140 genes and their alleles associated with shade-tolerance index(STI),146 with relative pith cell length(RCL),and nine with both.Annotation of these genes by biological categories allowed the construction of a protein–protein interaction network by 187 genes,of which half were differentially expressed under shading and non-shading conditions as well as at different growth stages.From the identified genes,three ones jointly identified for both traits by both GWAS and transcriptome and two genes with maximum links were chosen as beginners for entrance into the network.Altogether,both STI and RCL gene systems worked for shade-tolerance with genes interacted each other,this confirmed that shadetolerance is regulated by more than single group of interacted genes,involving multiple biological functions as a gene network.

Improving Rice Blast Resistance by Mining Broad-Spectrum Resistance Genes at Pik Locus

Magnaporthe oryzae is known for its genetic diversity and pathogenic variability,leading to rapid breakdown of resistance in rice.Incorporating multiple broad-spectrum blast resistance genes into rice cultivars would extend disease resistance longevity.Effective resistance breeding in rice therefore requires continual enrichment of the reservoir of resistance genes and alleles.We conducted a large-scale screen of rice blast resistance in about 2000 rice accessions.Among them,247 accessions showed at least medium resistance to the natural infection of rice blast and 7 novel Pik alleles were identified from them.Variations in gene sequences were then correlated with the phenotypic trait to enable the identification of favorable alleles.Among the seven novel Pik alleles,the resistant rate of Pik-R0/ME/7017 donors was greater than 80%,and the disease score was less than 3.Through molecular marker-assisted backcross breeding,we successfully transferred the three Pik alleles,Pik-R0/ME/7017,into an elite cultivated line Kongyu 131 to obtain BC_(3)F_(2)lines,which showed enhanced resistance to rice blast compared with the recurrent parent.Assessment of these near-isogenic lines in the greenhouse using 31 isolates of M.oryzae from Heilongjiang Province of China revealed that the resistant levels of the BC_(3)F_(2)lines with Pik-R0/ME/7017 were significantly higher than those of the established cloned resistance genes Pik-m and Pi1.Exploring such alleles will enrich our gene library for resistance to rice blast.

Association of HLA Alleles(A, B, DRB1) and HIV-1 Infection in the Han Population of Hubei, China

The HIV susceptibility and resistance alleles in the HLA genes were determined by investigating the distribution characteristics of the HLA alleles（A, B, and DRB1） in HIV-infected individuals of the Han population in Hubei, and by comparing these alleles with HIV-negative individuals from the same area. A cohort of 424 HIV-1 infected individuals were chosen as study subjects, and 836 HIV-negative healthy subjects from the same area served as the control population. HLA-A, B, and DRB1 allele typing was performed using polymerase chain reaction-sequence-specific oligonucleotide probes（PCR-SSOP） and polymerase chain reaction-sequencing based typing（PCR-SBT） techniques. Arlequin ver3.0 was used to analyze the allele and haplotype frequencies of HLA-A, B, and DRB 1, whereas Epi Info 7 and SPSS18.0 was used to analyze the differences in the HLA alleles between the HIV-1 positive and HIV-1 negative groups. A＊02：03, DRB1＊01：01, and DRB1＊15：01 alleles and their haplotypes as well as the HLA_Bw4-Bw6 hybrid showed a protective effect on HIV-1 infection. After adjusting for confounding factors such as age and sex, multivariate logistic regression analysis revealed that B＊15：02G, DRB1＊01：01, and DRB1＊15：01 subtypes were the resistance genes of HIV-1 infection, while B＊13：01 might increase susceptibility to HIV-1 infection. The correlation between A＊02：06 and B＊15：01G subtypes and HIV-1 susceptibility was independent of the age and sex of the host. This study demonstrated the influence of genetic factors in humans such as HLA polymorphism on individuals to resist HIV-1 infection. Association studies of HLA polymorphism, susceptibility/resistance to HIV-1 infection, and hosts＇ genetic background are of significant importance for research on HIV-1 pathogenesis and vaccine design.

Nonfunctional alleles of long-day suppressor genes independently regulate flowering time

Due to the remarkable adaptability to various environments, rice varieties with diverse flowering times have abeen domesticated or improved from Oryza rufipogon.Detailed knowledge of the genetic factors controlling flowering time will facilitate understanding the adaptation mechanism in cultivated rice and enable breeders to design appropriate genotypes for distinct preferences. In this study,four genes（Hd1, DTH8, Ghd7 and OsPRR37） in a rice long-day suppression pathway were collected and sequenced in 154, 74,69 and 62 varieties of cultivated rice（Oryza sativa）respectively. Under long-day conditions, varieties with nonfunctional alleles flowered significantly earlier than those with functional alleles. However, the four genes have different genetic effects in the regulation of flowering time： Hd1 and Os PRR37 are major genes that generally regulate rice flowering time for all varieties, while DTH8 and Ghd7 only regulate regional rice varieties. Geographic analysis and network studies suggested that the nonfunctional alleles of these suppression loci with regional adaptability were derived recently and independently. Alleles with regional adaptability should be taken into consideration for genetic improvement. The rich genetic variations in these four genes,which adapt rice to different environments, provide the flexibility needed for breeding rice varieties with diverse flowering times.

Authors' response to “Maternal age as a potential explanation of the role of the L allele of the serotonin transporter gene in anxiety and depression in Asians”

In the letter to the editor, Dr. Comings et al. proposed a potential explanation of our findings that the L allele rather than S allele of 5-HTTLPR was associated with higher anxiety levels and reduced amygdala-prefrontal cortex （PFC） connectivity in Han Chinese[1], which demonstrated an ＇allele reversal＇ in the genetics of the 5-HTTLPR gene in Asians versus Caucasians. The authors alleged that this ＇allele reversal＇ might simply result from maternal age and suggested that we test this on our datasets. Unfortunately,

β-fibrinogen gene -455A/G polymorphism and plasma fibrinogen level in Chinese stroke patients

Abstract Objectives To investigate the relationship between the β-fibrinogen gene -455A/G polymorphism and plasma fibrinogen level and to determine the influence of the mutation on ischemic stroke. Methods Ninety-one patients (63.5±10.1 years) with ischemic stroke and 74 elderly control subjects (60.6±10.8 years) without any thromboembolic events and 98 healthy blood donators as young control (37.5±13.3 years) were enrolled in this trial. The β-fibrinogen gene -455A/G polymorphism was analyzed for all subjects by PCR-RFLP with the restrictive enzyme Hae Ⅲ, while plasma fibrinogen levels were obtained from the prothrombin time (PT) assay. For statistical analysis, the parameters were compared between any two different groups by the unpaired Student’s t test and the Chi-square test. Before analysis, log transformations for concentrations of fibrinogen were carried out.Results H2 allele frequency was higher in male ischemic stroke patients than in the elderly control (22.7% vs 7.1%, χ 2=5.56, P【0.02). There was no significant difference between the female groups. In those patients without any thromboembolic events (both elderly and young control groups), the frequency of H2 decreased with age (≤40, 21.3%; 41-59, 15.4%; and ≥60, 10.2%). In the male elderly and young control groups, the level of plasma fibrinogen was lower in the H1H1 genotype (287±96*!mg/dl and 234±58*!mg/dl) than in H1H2 and H2H2 (331±44*!mg/dl and 307±55*!mg/dl; t=2.53 and 9.67, P【0.05). In the female elderly groups, this tendency was not found.Conclusion Plasma fibrinogen expression is affected by the β-fibrinogen gene -455A/G polymorphism, and the H2 allele may be a risk factor for ischemic stroke in Chinese males.

Efficient allelic replacement in rice by gene editing： A case study of the NRT1.1B gene

Summary Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. A single nucleotide polymorphism in the NRT1.1B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Herein, we precisely replaced the japonica NRT1.1B allele with the indica allele, in just one generation, using CRISPR/Cas9 gene-editing technology. No additional selective pressure was needed to enrich the precise replacement events.

Precise gene replacement in plants through CRISPR/Cas genome editing technology:current status and future perspectives

CRISPR/Cas,as a simple,versatile,robust and cost-effective system for genome manipulation,has dominated the genome editing field over the past few years.The application of CRISPR/Cas in crop improvement is particularly important in the context of global climate change,as well as diverse agricultural,environmental and ecological challenges.Various CRISPR/Cas toolboxes have been developed and allow for targeted mutagenesis at specific genome loci,transcriptome regulation and epigenome editing,base editing,and precise targeted gene/allele replacement or tagging in plants.In particular,precise replacement of an existing allele with an elite allele in a commercial variety through homology-directed repair(HDR)is a holy grail in genome editing for crop improvement as it has been very difficult,laborious and time-consuming to introgress the elite alleles into commercial varieties without any linkage drag from parental lines within a few generations in crop breeding practice.However,it still remains very challenging in crop plants.This review intends to provide an informative summary of the latest development and breakthroughs in gene replacement using CRISPR/Cas technology,with a focus on achievements,potential mechanisms and future perspectives in plant biological science as well as crop improvement.

Single-cell transcriptome analysis reveals widespread monoallelic gene expression in individual rice mesophyll cells

Monoallelic gene expression refers to the phenomenon that all transcripts of a gene in a cell are expressed from only one of the two alleles in a diploid organism. Although monoallelic gene expression has been occasionally reported with bulk transcriptome analysis in plants, how prevalent it is in individual plant cells remains unknown. Here, we developed a single-cell RNA-seq protocol in rice and investigated allelic expression patterns in mesophyll cells of indica （93-11 ） and japonica （Nipponbare） inbred lines, as well as their F1 reciprocal hybrids. We observed pervasive monoallelic gene expression in individual mesophyll cells, which could be largely explained by stochastic and independent transcription of two alleles. By con- trast, two mechanisms that were proposed previously based on bulk transcriptome analyses, parent-of- origin effects and allelic repression, were not well supported by our data. Furthermore, monoallelically expressed genes exhibited a number of characteristics, such as lower expression levels, narrower H3K4me3/H3K9acJH3K27me3 peaks, and larger expression divergences between 93-11 and Nipponbare. Taken together, the development of a single-cell RNA-seq protocol in this study offers us an excellent opportunity to investigate the origins and prevalence of monoallelic gene expression in plant cells.