Pleiotropism of the Photoperiod-Insensitive Allele of Hd1 on Heading Date,Plant Height and Yield Traits in Rice

Five populations segregated in isogenic backgrounds and three sets of near isogenic lines (NILs) overlapping in a 362.3-kb region covering heading date gene Hd1 were developed from the indica rice cross Zhenshan97 (ZS97)/Milyang 46 (MY46). They were used to analyze the effects of Hd1 on heading date, plant height and yield traits. In a background of the parental mixtures, the photoperiod-sensitive allele derived from ZS97 functioned in promoting and delaying flowering in the natural short-day and long-day conditions, respectively. In the background of ZS97, no response to the photoperiod was observed, whereas the photoperiod-insensitive allele derived from MY46 functioned in delaying flowering, increasing plant height, and enhancing grain productivity. The additive effects estimated in two NIL sets were 6.14 and 6.14 d for heading date, 4.46 and 5.55 cm for plant height, 10.82 and 11.54 for the number of spikelets per panicle, 6.82 and 8.00 for the number of grains per panicle, and 2.16 and 2.23 g for grain yield per plant, which explained 94.1% and 96.3%, 70.5% and 84.8%, 52.4% and 55.2%, 28.9% and 39.2%, and 36.5% and 26.9% of the phenotypic variances, respectively. Since the photoperiod-insensitive allele of Hd1 confers a long vegetative phase, it is a good candidate for breeding rice varieties with high yielding potential for low latitudes.     

Gene diagnosis and targeted breeding for blast-resistant Kongyu 131 without changing regional adaptability

The fungus Magnaporthe oryzae threatens the rice production of Kongyu 131 (KY131), a leading japonica variety in Northeast China. In this study, two rice lines, KP1 and KP2-Hd1, were obtained by introgressing the blast resistance genes Pi1 and Pi2 into KY131, respectively. However, both lines headed later than KY131. RICE60K SNP array analysis showed that Hd1 closely linked to Pi2 was introgressed into KP2-Hd1, and the linkage drag of Hd1 was broken by recombination. On the other hand, no known flowering genes were introgressed into KP1. Gene diagnosis by resequencing six flowering genes showed that KP1 carried functional Hd16 and Ghd8 alleles. Due to its suppression role in heading under long-day conditions, Ghd8 was chosen as the target for gene editing to disrupt its function. Four sgRNAs targeting different sites within Ghd8 were utilized to induce large-deletion mutations, which were easy to detect via agarose gel electrophoresis. All the ghd8-mutated KP1 lines were resistant to rice blast disease and headed earlier than the control KP1, even than KY131, under natural long-day conditions, which ensures its growth in Northeast China. This study confirmed that a combination of gene diagnosis and targeted gene editing is a highly efficient way to quickly eliminate undesired traits in a breeding line.     

Phylogeography and population expansion of Western Himalayan highly-erratically migratory painted lady butterfly (Lepidoptera: Nymphalidae: Vanessa cardui)

Understanding population genetics, genetic diversity and the migration pattern of ecologically important species is crucial for effective conservation plans. We studied mitochondrial cytochrome c oxidase subunit-I (COI) gene variation in painted lady butterfly (Vanessa cardui) from different altitudinal gradients of Uttarakhand (Western Himalayas), India. We sequenced COI) gene of 91 samples and 22 COI sequences retrieved from GenBank as representatives of different localities for comparative analysis. We observed a total of 16 haplotypes, of which seven haplotypes were observed in Uttarakhand with intra-species sequence divergence ranges from 0.001 to 0.009. Haplotype (Hd) and nucleotide diversity (π) were comparatively low and averaging 0.635 and 0.002, respectively. The neutrality test, mismatch distribution graph and observed pattern of diversity indices (Hd and π) revealed population expansion. It was further confirmed by the Median Joining Network, which exhibited a star-like topology from the core haplotype, while the Bayesian phylogenetic tree displayed a mixing of haplotypes from different localities to an identical clade. Haplotype organization in the Bayesian tree and Median Joining Network was not clustered as per their topographical affinity, indicating incomplete lineage sorting and late species population development or colonization in the novel area.     

Hd1, Ghd7, and DTH8 synergistically determine the rice heading date and yield-related agronomic traits

Heading date determines the seasonal and regional adaptation of rice(Oryza sativa L.) varieties and is mainly controlled by photoperiod sensitivity(PS). The core heading date genes Hd1, Ghd7, DTH8, and PRR37 act synergistically in regulating the PS. In this study, we systematically analyze the heading date,PS, and agronomic traits of eight homozygous lines with various combinations of Hd1, Ghd7, and DTH8 alleles in the prr37 background under long-day(LD) and short-day(SD) conditions, respectivel...     

The Effect of the Crosstalk between Photoperiod and Temperature on the Heading-Date in Rice

Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions.     

Characterization and detection of epistatic interactions of 3 QTLs, Hd1, Hd2, and Hd3, controlling heading date in rice using nearly isogenic lines

To characterize quantitative trait loci (QTLs), we used marker-assisted selection (MAS) to develop three nearly isogenic lines (NILs) differing only for the presence of a single, specific QTL (QTL-NILs) –Hd1, Hd2, and Hd3 – for heading date in rice. The three lines contained the chromosomal region of the target QTL from donor variety Kasalath(indica) in the genetic background of var. Nipponbare (japonica). To analyze epistatic interactions in pairs of these QTLs, we also used MAS to develop four combined QTL-NILs with 2 of the 3 QTLs or with all 3. Different daylength treatment testing of the QTL-NILs revealed that the three QTLs control photoperiod sensitivity. Genetic analysis of F₂ populations derived from crosses between the three QTL-NILs with a single QTL using molecular markers revealed the existence of epistatic interactions between Hd1 and Hd2, and Hd2 and Hd3. These interactions were also confirmed by the analysis of combined QTL-NILs under different daylength conditions. The existence of an epistatic interaction between Hd1 and Hd3 was also clarified. Based on these results, we suggest that the Kasalath allele of Hd3 does not affect photoperiod sensitivity by itself but that it is involved in enhancement of the expression of the Nipponbare alleles of Hd1 and Hd2. Received: 22 October 1999 / Accepted: 21 March 2000     

Genetic dissection of a genomic region for a quantitative trait locus, <Emphasis Type="Italic">Hd3</Emphasis>, into two loci, <Emphasis Type="Italic">Hd3a</Emphasis> and <Emphasis Type="Italic">Hd3b</Emphasis>, controlling heading date in rice

The rice photoperiod sensitivity gene Hd3 was originally detected as a heading date-related quantitative trait locus localized on chromosome 6 of rice. High-resolution linkage mapping of Hd3 was performed using a large segregating population derived from advanced backcross progeny between a japonica variety, Nipponbare, and an indica variety, Kasalath. To determine the genotype of Hd3, we employed progeny testing under natural field and short-day conditions. As a result, two tightly linked loci, Hd3a and Hd3b, were identified in the Hd3 region. Nearly-isogenic lines for Hd3a and Hd3b were selected from progeny using marker-assisted selection. The inheritance mode of both Hd3a and Hd3b was found to be additive. Analysis of daylength response in nearly-isogenic lines of Hd3a and Hd3b showed that the Kasalath allele at Hd3a promotes heading under short-day conditions while that at Hd3b causes late heading under long-day and natural field conditions.     

t-plasminogen activator inhibitor-1 polymorphism in idiopathic pulmonary arterial hypertension

AIM:

The aim of the present study was to identify the possible genotypic association of 3’UTR Hind III polymorphism of Plasminogen activator Inhibitor-1 (PAI-1) gene with idiopathic pulmonary arterial hypertension (IPAH).

BACKGROUND:

IPAH is a disorder with abnormally raised mean pulmonary arterial pressure and increase in the resistance to blood flow in pulmonary artery. One of the pathological features seen is development of intraluminal thrombin deposition leading to thrombosis. Plasminogen activator inhibitor-1 is an important inhibitor of the fibrinolytic system; its up-regulation may suppress fibrinolysis and result in an increased risk of thrombosis.

METHOD:

Blood samples from 54 IPAH patients and 100 healthy voluntary donors were analyzed by PCR-RFLP method for 3’UTR Hind III polymorphism.

RESULTS AND DISSCUSSION:

A significant association of Hd2 allele with the disease was observed. Raised mean level of right ventricular systolic pressure was observed in the Hd2/Hd2 genotypic patients, strengthening the role of Hd2 allele in the disease progression. Our data suggests an association of Hd2/Hd2 genotype, which may lead to the up-regulation of PAI-1 gene leading to increased levels of PAI-1, which is seen in IPAH. PAI-1 competes with plasminogen activators and hinders the normal mechanism of plasminogen activation system and leads to thrombosis and formation of plexiform lesions in the lung tissue, further strengthening its role in tissue remodeling and disease progression.     

Development of a candidate gene marker for Rf 1 based on a PPR gene in cytoplasmic male sterile CMS-D2 Upland cotton

The cytoplasmic male sterility (CMS) system is convenient and efficient for hybrid seed production in Upland cotton (Gossypium hirsutum L.). However, it has not been widely used because of limited restorer lines carrying the restorer gene Rf ₁ in the CMS-D2 system. In this study, the fertility segregation in a backcross (BC8F1) population of 409 individuals and an F2 population of 695 plants confirmed that the fertility restoration was determined by one dominant restorer gene (Rf ₁ ). A sequence alignment showed that 13 Rf ₁ -linked simple sequence repeat marker sequences were distributed on nine scaffolds of chromosome 9 in the sequenced D5 genome of G. raimondii Ulbrich. Ten pentotricopeptide repeat (PPR)-like genes were identified on two scaffolds, including Scaffold 333 where nine PPR-like genes were clustered in a region of about 160 kb. Among them, PPR-like gene Cotton_D_gene_10013437 was identified as the candidate for the Rf ₁ gene through a comparative sequence analysis of the homologous gene among sterile (A), maintainer (B) and restorer (R) lines, and co-segregation analysis. Compared with the non-restoring lines, the restorer had a 9-nucleotide (nt) insertion and a single nucleotide polymorphism (SNP) 8 nt upstream of the insertion at the 3′ untranslated regions (3′ UTRs) in this gene. A cleaved amplified polymorphic sequence (CAPS) marker named CAPS-R was developed from the SNP site using the restriction enzyme DraI, and was further used to track the restorer gene and its homozygous or heterozygous status in molecular breeding for restorer lines. A marker-assisted selection system using the Rf ₁ -specific CAPS-R marker and a CMS-D2 cytoplasm-specific SCAR marker was established to distinguish the three-line hybrids from other genotypes.     

Identification of perennial ryegrass (Lolium perenne (L.)) and meadow fescue (Festuca pratensis (Huds.)) candidate orthologous sequences to the rice Hd1(Se1) and barley HvCO1 CONSTANS-like genes through comparative mapping and microsynteny

Microsynteny with rice and comparative genetic mapping were used to identify candidate orthologous sequences to the rice Hd1(Se1) gene in Lolium perenne and Festuca pratensis. A F. pratensis bacterial artificial chromosome (BAC) library was screened with a marker (S2539) physically close to Hd1 in rice to identify the equivalent genomic region in F. pratensis. The BAC sequence was used to identify and map the same region in L. perenne. Predicted protein sequences for L. perenne and F. pratensis Hd1 candidates (LpHd1 and FpHd1) indicated they were CONSTANS-like zinc finger proteins with 61-62% sequence identity with rice Hd1 and 72% identity with barley HvCO1. LpHd1 and FpHd1 were physically linked in their respective genomes (< 4 kb) to marker S2539, which was mapped to L. perenne chromosome 7. The identified candidate orthologues of rice Hd1 and barley HvCO1 in L. perenne and F. pratensis map to chromosome 7, a region of the L. perenne genome which has a degree of conserved genetic synteny both with rice chromosome 6, which contains Hd1, and barley chromosome 7H, which contains HvCO1.     

Roles of the Hd5 gene controlling heading date for adaptation to the northern limits of rice cultivation

During the diversification of cultivated rice after domestication, rice was grown in diverse geographic regions using genetic variations attributed to the combination of alleles in loci for adaptability to various environmental conditions. To elucidate the key gene for adaptation in rice cultivars to the northern limit of rice cultivation, we conducted genetic analyses of heading date using extremely early-heading cultivars. The Hd5 gene controlling heading date (flowering time) generated variations in heading date among cultivars adapted to Hokkaido, where is the northernmost region of Japan and one of the northern limits of rice cultivation in the world. The association of the Hd5 genotype with heading date and genetical analysis clearly showed that the loss-of-function Hd5 has an important role in exhibiting earlier heading among a local population in Hokkaido. Distinct distribution of the loss-of-function Hd5 revealed that this mutation event of the 19-bp deletion occurred in a local landrace Bouzu and that this mutation may have been selected as an early-heading variety in rice breeding programs in Hokkaido in the early 1900s. The loss-of-function Hd5 was then introduced into the rice variety Fanny from France and contributed to its extremely early heading under the presence of functional Ghd7. These results demonstrated that Hd5 plays roles not only in generating early heading in variations of heading date among a local population in Hokkaido, but also in extremely early heading for adaptation to northern limits of rice cultivation.     

Chromosome-scale haplotype-phased genome assemblies of the male and female lines of wild asparagus (Asparagus kiusianus), a dioecious plant species

Asparagus kiusianus is a disease-resistant dioecious plant species and a wild relative of garden asparagus (Asparagus officinalis). To enhance A. kiusianus genomic resources, advance plant science, and facilitate asparagus breeding, we determined the genome sequences of the male and female lines of A. kiusianus. Genome sequence reads obtained with a linked-read technology were assembled into four haplotype-phased contig sequences (∼1.6 Gb each) for the male and female lines. The contig sequences were aligned onto the chromosome sequences of garden asparagus to construct pseudomolecule sequences. Approximately 55,000 potential protein-encoding genes were predicted in each genome assembly, and ∼70% of the genome sequence was annotated as repetitive. Comparative analysis of the genomes of the two species revealed structural and sequence variants between the two species as well as between the male and female lines of each species. Genes with high sequence similarity with the male-specific sex determinant gene in A. officinalis, MSE1/AoMYB35/AspTDF1, were presented in the genomes of the male line but absent from the female genome assemblies. Overall, the genome sequence assemblies, gene sequences, and structural and sequence variants determined in this study will reveal the genetic mechanisms underlying sexual differentiation in plants, and will accelerate disease-resistance breeding in garden asparagus.     

Assessment of genetic diversity in salt-tolerant rice and its wild relatives for ten SSR loci and one allele mining primer of salT gene located on 1st chromosome

A heterogeneous collection of rice genotypes which included seven salt-tolerant rice lines, one salt-sensitive improved line, one wild rice (Oryza rufipogon) and one salt-tolerant wild rice relative (Porteresia coarctata) was screened with ten salt-tolerance-linked simple sequence repeat markers, of which nine were from the Saltol QTL mapped on rice 1st chromosome and the rest one from 8th chromosome, having high phenotypic variance for salt tolerance. Variation in molecular weight (in the form of base pairs) of the different amplified products using RM primers was used to find out the genetic relationship among the studied rice genotypes. Genomic DNA of the studied genotypes was also amplified with a reported allele mining primer for a salt-inducible gene (salT). The amplified products were sequenced and aligned to find out the closeness among the rice lines for the studied gene. Dendrogram derived from marker profiles showed partial similarity with salT gene-derived tree. Commonly, all the salt-tolerant lines were grouped into a single cluster, including IR36 (a salt-sensitive line) to which O. rufipogon (the wild rice) and P. coarctata (the wild rice relative) joined separately. The taxonomic identity and evolutionary relationship among the three groups (rice, wild rice and wild rice relative) were bioinformatically analysed using the nucleotide sequence of the studied salT gene.     

RNA interference-mediated resistance to maize dwarf mosaic virus

Maize dwarf mosaic virus (MDMV) is a widespread pathogen that causes serious yield loss to maize crops. A hairpin RNA expression vector was constructed herein to overcome the low efficiency of cultural protection against MDMV and to improve the MDMV resistance mediated by a shorter transgenic inverted-repeat sequence. This expression vector contained a 451 bp inverted-repeat sequence, homologous to the protease gene (P1) of MDMV. It was used for the Agrobacterium tumefaciens-mediated transformation of maize calli induced from a susceptible inbred line. A total of 17 T₂ transgenic lines were identified by both specific PCR amplification and Southern blot hybridization. Of these lines, 15 were evaluated for MDMV resistance in inoculation field trials under two environments. The relative replication levels of the P1 gene were analyzed by quantitative real-time (qRT)-PCR. Results demonstrated that all of the 15 T₂ lines showed an enhanced resistance to MDMV in comparison with that of the non-transformed parent line. Six lines were deemed to be ‘resistant’ with an average disease index below 25 %, which was not significantly different from that of the resistant control. The relative replication levels of the virus gene were significantly reduced in these resistant T₂ transgenic lines. The efficiency of virus gene silencing was directly related to the transgene copy numbers presented in these transgenic lines.     

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.     

Natural Variation of the RICE FLOWERING LOCUS T 1 Contributes to Flowering Time Divergence in Rice

In rice (Oryza sativa L.), there is a diversity in flowering time that is strictly genetically regulated. Some indica cultivars show extremely late flowering under long-day conditions, but little is known about the gene(s) involved. Here, we demonstrate that functional defects in the florigen gene RFT1 are the main cause of late flowering in an indica cultivar, Nona Bokra. Mapping and complementation studies revealed that sequence polymorphisms in the RFT1 regulatory and coding regions are likely to cause late flowering under long-day conditions. We detected polymorphisms in the promoter region that lead to reduced expression levels of RFT1. We also identified an amino acid substitution (E105K) that leads to a functional defect in Nona Bokra RFT1. Sequencing of the RFT1 region in rice accessions from a global collection showed that the E105K mutation is found only in indica, and indicated a strong association between the RFT1 haplotype and extremely late flowering in a functional Hd1 background. Furthermore, SNPs in the regulatory region of RFT1 and the E105K substitution in 1,397 accessions show strong linkage disequilibrium with a flowering time–associated SNP. Although the defective E105K allele of RFT1 (but not of another florigen gene, Hd3a) is found in many cultivars, relative rate tests revealed no evidence for differential rate of evolution of these genes. The ratios of nonsynonymous to synonymous substitutions suggest that the E105K mutation resulting in the defect in RFT1 occurred relatively recently. These findings indicate that natural mutations in RFT1 provide flowering time divergence under long-day conditions.