qGL3.4 controls kernel size and plant architecture in rice.

Kernel size and plant architecture play important roles in kernel yield in rice. Cloning and functional study of genes related to kernel size and plant architecture are of great significance for breeding high-yield rice. Using the single-segment substitution lines which developed with Oryza barthii as a donor parent and an elite indica cultivar Huajingxian74 (HJX74) as a recipient parent, we identified a novel QTL (quantitative trait locus), named qGL3.4, which controls kernel size and plant architecture. Compared with HJX74, the kernel length, kernel width, 1000-kernel weight, panicle length, kernels per plant, primary branches, yield per plant, and plant height of near isogenic line-qGL3.4 (NIL-qGL3.4) are increased, whereas the panicles per plant and secondary branches per panicle of NIL-qGL3.4 are comparable to those of HJX74. qGL3.4 was narrowed to a 239.18 kb interval on chromosome 3. Cell analysis showed that NIL-qGL3.4 controlled kernel size by regulating cell growth. qGL3.4 controls kernel size at least in part through regulating the transcription levels of EXPANSINS, GS3, GL3.1, PGL1, GL7, OsSPL13 and GS5. These results indicate that qGL3.4 might be beneficial for improving kernel yield and plant architecture in rice breeding.

Rice NIN-LIKE PROTEIN 4 plays a pivotal role in nitrogen use efficiency.

Nitrogen (N) is one of the key essential macronutrients that affects rice growth and yield. Inorganic N fertilizers are excessively used to boost yield and generate serious collateral environmental pollution. Therefore, improving crop N use efficiency (NUE) is highly desirable and has been a major endeavour in crop improvement. However, only a few regulators have been identified that can be used to improve NUE in rice to date. Here we show that the rice NIN-like protein 4 (OsNLP4) significantly improves the rice NUE and yield. Field trials consistently showed that loss-of-OsNLP4 dramatically reduced yield and NUE compared with wild type under different N regimes. In contrast, the OsNLP4 overexpression lines remarkably increased yield by 30% and NUE by 47% under moderate N level compared with wild type. Transcriptomic analyses revealed that OsNLP4 orchestrates the expression of a majority of known N uptake, assimilation and signalling genes by directly binding to the nitrate-responsive cis-element in their promoters to regulate their expression. Moreover, overexpression of OsNLP4 can recover the phenotype of Arabidopsis nlp7 mutant and enhance its biomass. Our results demonstrate that OsNLP4 plays a pivotal role in rice NUE and sheds light on crop NUE improvement.

[The platform of breeding by design based on the SSSL library in rice].

Breeding by design is a new concept proposed in the beginning of the century. It refers to the breeding of varieties by crop design utilizing favorable alleles dispersed in different genetic resources in a genome. In the past 20 years, we have proposed a "three-step" strategy to carry out the research on breeding by design in rice. Firstly, we constructed a library of chromosomal single-segment substitution lines (SSSLs) by using of Huajingxian74 (HJX74), an elite xian (indica) variety from South China as the recipient and 43 accessions of seven species of rice AA genome as donors. The genes in the substituted segments of SSSLs were then detected. Breeding by design was conducted by selecting the favorable genes from the SSSL library. Our practice indicates that the SSSL library is a powerful platform for breeding by design and various "traits", "lines" and "varieties" of rice can be designed and bred by utilizing abundant genes in the SSSL library. Here, we introduce the platform of the HJX74-SSSL library and our work of breeding by design on the platform. It will provide a case study for crop design.

Root-derived long-distance signals trigger ABA synthesis and enhance drought resistance in Arabidopsis.

Vascular plants have evolved intricate long-distance signaling mechanisms to cope with environmental stress, with reactive oxygen species (ROS) emerging as pivotal systemic signals in plant stress responses. However, the exact role of ROS as root-to-shoot signals in the drought response has not been determined. In this study, we reveal that compared with wild-type plants, ferric reductase defective 3 (frd3) mutants exhibit enhanced drought resistance concomitant with elevated NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) transcript levels and abscisic acid (ABA) contents in leaves as well as increased hydrogen peroxide (H2O2) levels in roots and leaves. Grafting experiments distinctly illustrate that drought resistance can be conferred by the frd3 rootstock regardless of the scion genotype, indicating that long-distance signals originating from frd3 roots promote an increase in ABA levels in leaves. Intriguingly, the drought resistance conferred by the frd3 mutant rootstock is weakened by the CAT2-overexpressing scion, suggesting that H2O2 may be involved in long-distance signaling. Moreover, the results of comparative transcriptome and proteome analyses support the drought resistance phenotype of the frd3 mutant. Taken together, our findings substantiate the notion that frd3 root-derived long-distance signals trigger ABA synthesis in leaves and enhance drought resistance, providing new evidence for root-to-shoot long-distance signaling in the drought response of plants.

[Single nucleotide polymorphism (SNP) and its application in rice].

Single nucleotide polymorphisms (SNPs) distribute numerously and high-density throughout rice (Oryza sativa L.) genome. A total of 80,127 SNP sites were identified in rice genome, and one SNP every 154 bp was found between two rice subspecies indica and japonica. The SNP rate is 0.65%. SNPs also are very considerable among within-subspecies cultivars, even it can be found between closely related cultivars, in which it has been difficult to find polymorphic sites by conventional methods. The frequency of SNPs in rice genome varied between chromosomes, moreover it showed uneven distribution of polymorphism-rich and -poor regions along each chromosome. Several routes have been used for identification of SNP in rice, such as sequencing PCR products of DNA samples, screening SNPs in SSR fragments, and searching for SNPs through the rice genome sequences and EST database. A number of genotyping systems have been developed to identify SNPs in rice genome. High automation in SNPs identification has become a very convenient operation by the automatized systems. SNPs can be converted to CAPS (cleaved amplified polymorphic sequence) or dCAPS (derived-CAPS), and allele-specific PCR markers. SNP has shown huge potential in establishing rice genetic maps, genes cloning and functional genomics, MAS (marker assisted selection) in rice breeding, and studying on classification and evolution of germplasm.

[Developing single segment substitution lines (SSSLs) in rice (Oryza sativa L.) using advanced backcrosses and MAS].

A novel population consisted of 86 single segment substitution lines (SSSLs) were developed from advanced backcrosses between the recipient, Huajingxian 74 and six donors by microsatellite marker-assisted selection (MAS). Fifty-two SSSLs were selected in BC3F2, and 34 others were selected in BC3F3. Every SSSL contains a single chromosome segment introgressed from one donor on the genetic background of Huajingxian 74. The substituted segments in SSSLs were distributed on 12 rice chromosomes. The estimated length of the substituted segments in SSSLs ranged from 1.5 cM to 56.3 cM with an average of 23.0 cM. Total of the substituted segments covered 57.1% of the rice genome.

[Chromosome mapping of the S-b locus for F1 pollen sterility in cultivated rice (Oryza sativa L.) with RAPD markers].

S-b is one locus for F1 pollen sterility in cultivated rice (Oryza sativa L.), and the genotype of Taichung 65 (abridged as T65) is Sj/Sj, while its isogenic line, TISL2 is Si/Si at this locus. The results of pollen fertility analysis showed that the pollen of T65 and TISL2 were fertile, but the F1 plant from T65 x TISL2 produced only 40.6% fertile pollens, and the type of sterile pollen was stainable abortive. In F2 population from the cross T65 x TISL2 and BC1F1 population from the cross T65/TISL2/T65, the individuals could be classified into plants with normal pollens and plants with semi-sterile pollens, and the ratio of number of these two types of plants agreed well with the Mendel segregation ratio in 1:1. A total of 53 fertile F2 plants were testcrossed with T65, and all of them showed sterility F1 pollen in. These results demonstrate that the F1 pollen sterility is controlled by a single gene locus S-b, and the allelic interaction of S-bi and S-bj causes the pollen carrying S-bj allele abortive. A total of 187 RFLP markers and 500 RAPD primers were used to screen the polymorphism between T65 and TISL2; only H08-1300 and Y09-1500, two bands amplified by RAPD primer H08 and Y09 were found to be polymorphic. Purified H08-1300 and Y09-1500 were used as probe to hybridize with DNAs from T65 and TISL2, and the results indicated that H08-1300 and Y09-1500 appeared to be single copy in the T65 and TISL2 genome, then the RAPD marker were successfully converted into RFLP marker. The two markers were then used to perform segregation analysis, the results from co-segregation analysis of the genotypes of these two markers and the phenotypes of pollen fertility with F2 population indicated that the S-b was linked to H08-1300 and Y09-1500, and the genetic distances between each marker and the locus were 1.3 cM and 6.6 cM, respectively. To determine the chromosomal position of the S-b locus, H08-1300 was cloned and its two ends were partially sequenced. The homologous comparative analysis of these sequences with published rice sequences with BLAST was performed, and 540 bp of left end sequence of H08-1300 showed 86% homologous with the sequence of rice PAC clone P0033D06 (Accession No. AC079357), and 94% homologies of 101 bp at right end were also observed. Clone P0033D06 had been anchored by RFLP markers R3166 that was located on 18.8 cM position of rice chromosome 5 by Japan Rice Genome Program, which suggested that the S-b locus was mapped on chromosome 5 and tightly linked with R3166. The gene mapping result from this study suggests that using the rice genomic sequences published to determine the chromosome position of RAPD marker, as well as linked genes, would be a useful approach in tagging new genes.

[Molecular mapping of the fertility restorer gene Rf-4 for WA cytoplasmic male sterility in rice].

The cytoplasmic male sterility for wild-abortive (CMS-WA) has been wildly used for hybrid rice breeding in China. The fertility restoration of CMS-WA is controlled mainly by two independent and dominant nuclear fertility restoring genes, Rf-3 and Rf-4. To map the Rf-4 gene with molecular markers, rice YAC clones of RGP, Japan were used to create new molecular marker. YAC contigs located between RFLP markers R1877 and G2155 on chromosome 10 were confirmed by hybridization with 12 RFLP probes. Six YAC clones, Y4630, Y2670, Y4892, Y2111, Y3821 and Y5528 were identified. Chromosome DNAs of the YAC clones were prepared and separated by CHEF. A total of 119 probes were created by sub-cloning of the YAC DNAs. RFLPs were screened between Zhenshan 97A and its near-isogenic lines with Rf-4Rf-4 genotype. Two probes, Y3-8 from Y4892 and Y1-10 from Y4630, were found to be polymorphic. Using F2 populations from crosses between Zhenshan 97A and its near-isogenic lines ZSR11, Y3-8 and Y1-10 were mapped to Rf-4 locus with genetic distances of 0.9 cM and 3.2 cM, respectively.

[Identification of QTLs on substituted segments in single segment substitution lines of rice].

The quantitative trait loci (QTLs) for 21 traits of agronomy importance on ten substituted chromosomal segments were identified using single segment substitution lines (SSSLs) in rice, which were developed by the use of Taichung 65 as a recipient and zhai-ye-qing and dee-geo-woo-gen as donors respectively. Total length of the substituted segments in the SSSLs was 230.00 cM, which was 12.62% of rice genome. T-test was used to detect QTLs in the condition that the difference in phenotypes between a SSSL and the recipient parent (Taichung 65) was significant at the 0.1% level (P< or = 0.001). A total of 57 QTLs for 17 traits had been detected on the ten substituted segments, which were on chromosomes 1, 3, 5, 7, 8, 10 and 12 respectively. The additive effect percentages of the QTLs ranged from 1.10% to 89.73%. Among of 57 QTLs, fifteen QTLs had the additive effect percentage over 10%, thirty QTLs varied from 3% to 10% and other twelve QTLs were below 3%.

[Genetic analysis of a heading-delayed mutant by T-DNA insertion in rice (Oryza sativa L.)].

Transposon tagging was used to isolate genes in higher plant. In this study, a delayed heading mutant caused by T-DNA insertion in rice was identified. Genetic analysis of the mutant showed that the three types of phenotype, normal heading, delayed heading and overly delayed heading in the segregating populations derived from the T-DNA heterozygotes fit the ratio of 1:2:1. Test for Basta resistance showed the delayed heading plants were all resistant while the normal heading plants were susceptible, and the ratio of resistant and susceptible plants was 3:1, which indicated that the delayed heading mutant was co-segregated with Basta resistance. The delayed heading mutant caused by T-DNA insertion was confirmed by T-DNA detection using PCR method. This delayed heading mutant will be used for isolation of the tagged gene in rice.

Cytological mechanism of pollen abortion resulting from allelic interaction of F1 pollen sterility locus in rice (Oryza sativa L.).

Pollen abortion is one of the major reasons causing the inter-subspecific F(1) hybrid sterility in rice and is due to allelic interaction of F(1) pollen sterility genes. The microsporogenesis and microgametogenesis of Taichung 65 and its three F(1) hybrids were comparatively studied by using techniques of differential interference contrast microscopy, semi-thin section light microscopy, epifluorescence microscopy and TEM. The results showed that there were differences among the cytological mechanisms of pollen abortion due to allelic interaction at the three F(1) pollen sterility loci. The allelic interaction at S-a locus resulted in microspores unable to extend the protoplasm membrane with the enlargement of the microspore at the middle microspore stage and finally producing empty abortive pollen. The allelic interaction at S-b locus caused asynchronous development of microspores at the middle microspore stage producing stainable abortive pollen. The allelic interaction at S-c locus mainly led to the non-dissolution of the generative cell wall and finally caused the hybrid F(1) mainly producing stainable abortive pollen. Genotypic identification indicated that the abortive pollen were those with S ( j ) allele.

Development of a wide population of chromosome single-segment substitution lines in the genetic background of an elite cultivar of rice (Oryza sativa L.).

Naturally occurring allelic variations underlying complex traits are useful resources for the functional analysis of plant genes. To facilitate the genetic analysis of complex traits and the use of marker-assisted breeding in rice, we developed a wide population consisting of 217 chromosome single-segment substitution lines (SSSLs) using Oryza sativa L. 'Hua-Jing-Xian74' (HJX74), an elite Indica cultivar, as recipient, and 6 other accessions, including 2 Indica and 4 Japonica, as donors. Each SSSL contains a single substituted chromosome segment derived from 1 of the 6 donors in the genetic background of HJX74. The total size of the substituted segments in the SSSL population was 4695.0 cM, which was 3.1 times that of rice genome. To evaluate the potential application of these SSSLs for quantitative trait loci detection, phenotypic variations of the quantitative traits of days to heading and grain length in the population consisting of 210 SSSLs were observed under natural environmental conditions. The results demonstrated that there was a wide range of phenotypic variation in the traits in the SSSL population. These genetic materials will be powerful tools to dissect complex traits into a set of monogenic loci and to assign phenotypic values to different alleles at the locus of interest.