The MYB transcription factor Baymax1 plays a critical role in rice male fertility.

Key message Rice male fertility gene Baymax1, isolated through map-based cloning, encodes a MYB transcription factor and is essential for rice tapetum and microspore development.Abstract The mining and characterization of male fertility gene will provide theoretical and material basis for future rice production. In Arabidopsis, the development of male organ (namely anther), usually involves the coordination between MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic helix-loop-helix) members. However, the role of MYB proteins in rice anther development remains poorly understood. In this study, we isolated and characterized a male sterile mutant (with normal vegetative growth) of Baymax1 (BM1), which encodes a MYB protein. The bm1 mutant exhibited slightly lagging meiosis, aborted transition of the tapetum to a secretory type, premature tapetal degeneration, and abnormal pollen exine formation, leading to ultimately lacks of visible pollens in the mature white anthers. Map-based cloning, complementation and targeted mutagenesis using CRISPR/Cas9 technology demonstrated that the mutated LOC_Os04g39470 is the causal gene in bm1. BM1 is preferentially expressed in rice anthers from stage 5 to stage 10. Phylogenetic analysis indicated that rice BM1 and its homologs in millet, maize, rape, cabbage, and pigeonpea are evolutionarily conserved. BM1 can physically interacts with bHLH protein TIP2, EAT1, and PHD (plant homeodomain)-finger member TIP3, respectively. Moreover, BM1 affects the expression of several known genes related to tapetum and microspore development. Collectively, our results suggest that BM1 is one of key regulators for rice male fertility and may serve as a potential target for rice male-sterile line breeding and hybrid seed production.

UMP Kinase Regulates Chloroplast Development and Cold Response in Rice.

Pyrimidine nucleotides are important metabolites that are building blocks of nucleic acids, which participate in various aspects of plant development. Only a few genes involved in pyrimidine metabolism have been identified in rice and the majority of their functions remain unclear. In this study, we used a map-based cloning strategy to isolate a UMPK gene in rice, encoding the UMP kinase that phosphorylates UMP to form UDP, from a recessive mutant with pale-green leaves. In the mutant, UDP content always decreased, while UTP content fluctuated with the development of leaves. Mutation of UMPK reduced chlorophyll contents and decreased photosynthetic capacity. In the mutant, transcription of plastid-encoded RNA polymerase-dependent genes, including psaA, psbB, psbC and petB, was significantly reduced, whereas transcription of nuclear-encoded RNA polymerase-dependent genes, including rpoA, rpoB, rpoC1, and rpl23, was elevated. The expression of UMPK was significantly induced by various stresses, including cold, heat, and drought. Increased sensitivity to cold stress was observed in the mutant, based on the survival rate and malondialdehyde content. High accumulation of hydrogen peroxide was found in the mutant, which was enhanced by cold treatment. Our results indicate that the UMP kinase gene plays important roles in regulating chloroplast development and stress response in rice.

Fine mapping and candidate gene analysis of qHD5, a novel major QTL with pleiotropism for yield-related traits in rice (Oryza sativa L.).

KEY MESSAGE: A major QTL for heading date, qHD5, was fine-mapped to a 52.59-kb region on the short arm of rice chromosome 5. Heading date (HD) is one of the most important traits that enables rice to adapt to seasonal differences and specific growth conditions in diverse growing regions. In this study, a major-effect quantitative trait locus (QTL), qHD5, was resolved as a single Medelian factor that causes NIL(BG1) and NIL(XLJ) (two near-isogenic lines (NILs) used in our study) to have at a minimum of 10-day difference in HD under both long-day and short-day conditions in rice. qHD5 was initially mapped to a 309.52-kb genomic region in our previous study. Here, using an advanced BC4F3 population and map-based cloning, we further narrowed the location of qHD5 to a 52.59-kb region between the H71 and RD502 markers. Sequence analysis revealed that Os05g03040, which putatively encodes an AP2 (APETALA2) transcription factor, has six single nucleotide polymorphisms (SNPs) between NIL(BG1) and NIL(XLJ). On this basis, this gene was concluded to be the most probable candidate gene for qHD5. Our results also showed that Hd3a, RFT1, Hd1, Ehd1, and Ghd7 were differentially expressed in the two NILs. Moreover, qHD5 was found to affect yield-related traits such as flag leaf width, flag leaf length, branch number, and 1000-grain weight.

Dissecting yield-associated loci in super hybrid rice by resequencing recombinant inbred lines and improving parental genome sequences.

The growing world population and shrinkage of arable land demand yield improvement of rice, one of the most important staple crops. To elucidate the genetic basis of yield and uncover its associated loci in rice, we resequenced the core recombinant inbred lines of Liang-You-Pei-Jiu, the widely cultivated super hybrid rice, and constructed a high-resolution linkage map. We detected 43 yield-associated quantitative trait loci, of which 20 are unique. Based on the high-density physical map, the genome sequences of paternal variety 93-11 and maternal cultivar PA64s of Liang-You-Pei-Jiu were significantly improved. The large recombinant inbred line population combined with plentiful high-quality single nucleotide polymorphisms and insertions/deletions between parental genomes allowed us to fine-map two quantitative trait loci, qSN8 and qSPB1, and to identify days to heading8 and lax panicle1 as candidate genes, respectively. The quantitative trait locus qSN8 was further confirmed to be days to heading8 by a complementation test. Our study provided an ideal platform for molecular breeding by targeting and dissecting yield-associated loci in rice.

qHD5 encodes an AP2 factor that suppresses rice heading by down-regulating Ehd2 expression.

Heading date is crucial for rice reproduction and the geographical expansion of cultivation. We fine-mapped qHD5 and identified LOC_Os05g03040, a gene that encodes an AP2 transcription factor, as the candidate gene of qHD5 in our previous study. In this article, using two near-isogenic lines NIL(BG1) and NIL(XLJ), which were derived from the progeny of the cross between BigGrain1 (BG1) and Xiaolijing (XLJ), we verified that LOC_Os05g03040 represses heading date in rice through genetic complementation and CRISPR/Cas9 gene-editing experiments. Complementary results showed that qHD5 is a semi-dominant gene and that the qHD5XLJ and qHD5BG1 alleles are both functional. The homozygous mutant line generated from knocking out qHD5XLJ in NIL(XLJ) headed earlier than NIL(XLJ) under both short-day and long-day conditions. In addition, the homozygous mutant line of qHD5BG1 in NIL(BG1) also headed slightly earlier than NIL(BG1). All of these results show that qHD5 represses the heading date in rice. Transient expression showed that the qHD5 protein localizes to the nucleus. Transactivation activity assays showed that the C-terminus is the critical site that affects self-activation in qHD5XLJ. qRT-PCR analysis revealed that qHD5 represses flowering by down-regulating Ehd2. qHD5 may have been selected during indica rice domestication.

Microsatellite analysis of homozygosity progression of heterozygous genotypes segregating in the rice subspecies cross Pei'ai64s/Nipponbare.

Progression to homozygosity of heterozygous genotypes was studied in a cross of the rice subspecies Pei'ai64s and Nipponbare, using a set of 157 polymorphic microsatellite (SSR) markers. The segregation of heterozygous genotypes ranged from 49.13% in the F(2) population to 4.52% in the F(6) population (progression value 11.15%). The heterozygous genotypes were widely distributed in 180 F(2) plants, 330 F(6) lines, and 157 SSR markers. Homozygosity progression showed a wide distribution in plants and SSR markers but not in chromosomes. The segregation of heterozygous genotypes was not significant between populations but varied greatly in F(2) plants, F(6) lines, and SSR markers. The correlation between the progression to homozygosity and the heterozygosity of SSR markers was significant at the chromosome level. The segregation of heterozygous genotypes in plants, SSR markers, and chromosomes was not completely in accordance with Mendel's law. This information will help rice geneticists and breeders to understand heterozygous genotype segregation at the DNA level and to screen special markers for breeding.

[Progress on genetics of rice grain shape trait and its related gene mapping and cloning].

Increase of crop production is the primary goal of crop breeding. Rice grain shape is a quantitative trait that is directly related to yield traits and has a close relationship with quality traits. The evaluation of grain shape is mainly grain length, grain width, grain thickness, length/width, and length/thickness. In recent years, the quantitative genetic research on rice grain shape has made a significant progress and a number of important genes associated with rice grain shape have been cloned. This paper reviews the classic genetic analysis on rice grain traits, QTL mapping, grain shape gene cloning and functional analysis, and their application in rice breeding for super high yield.

Dissection of Closely Linked Quantitative Trait Locis Controlling Grain Size in Rice.

Grain size is a key constituent of grain weight and appearance in rice. However, insufficient attention has been paid to the small-effect quantitative trait loci (QTLs) on the grain size. In the present study, residual heterozygous populations were developed for mapping two genetically linked small-effect QTLs for grain size. After the genotyping and the phenotyping of five successive generations, qGS7.1 was dissected into three QTLs and two were selected for further analysis. The qTGW7.2a was finally mapped into a 21.10 kb interval containing four annotated candidate genes. Transcript levels assay showed that the expression of the candidates LOC_Os07g39490 and the LOC_Os07g39500 were significantly reduced in the NIL-qTGW7.2aBG1 . The cytological observation indicated that qTGW7.2a regulated the grain width through controlling the cell expansion. Using the same strategy, qTGW7.2b was fine-mapped into a 52.71 kb interval containing eight annotated candidate genes, showing a significant effect on the grain length and width with opposite allelic directions, but little on the grain weight. Our study provides new genetic resources for yield improvement and for fine-tuning of grain size in rice.

Mutation detection of KRAS by high-resolution melting analysis in Chinese with gastric cancer.

KRAS proteins play an important role in regulating cell functions. A series of studies has revealed that mutations of KRAS are involved in gastric carcinogenesis. However, mutation status of KRAS remains unclear in gastric cancer from Chinese Mainland. It has been proved that KRAS mutation associates with resistance to epidermal growth factor receptor (EGFR) inhibitors. In this study, KRAS mutations were detected in 52 gastric adenocarcinomas from Northern China. High-resolution melting analysis (HRMA) was used and positive samples were confirmed by direct sequencing. Of the 52 cancers, KRAS mutations were found in 5 (9.6%). All cancers with KRAS mutation were from male patients. Frequencies of KRAS mutation were 14.3% (3/21) and 6.5% (2/31) in differentiated and undifferentiated cancers; 25% (1/4) and 8.3% (4/48) in early and advanced wall penetration cancers; and were 13.3% (2/15) and 8.1% (3/37) in without and with lymph node metastasis cancers, respectively. There was no significant correlation between KRAS mutation and clinicopathological features. There were 3 mutation types in the 5 mutations, including 2 G12D, 1 G12V and 2 G13D mutations. All codon 12 mutations were found in patients with lymph node metastasis and at advanced stage, whereas all codon 13 mutations were found in patients without lymph node metastasis and at early stage. These results support KRAS mutation may only be involved in carcinogenesis of partial gastric cancers and the different mutation types of KRAS may take part in development of gastric cancer at different stages. The resistance of partial gastric cancer patients to EGFR inhibitors may be induced by KRAS mutation.

Progress in research and development on hybrid rice: a super-domesticate in China.

BACKGROUND: China has been successful in breeding hybrid rice strains, but is now facing challenges to develop new hybrids with high-yielding potential, better grain quality, and tolerance to biotic and abiotic stresses. This paper reviews the most significant advances in hybrid rice breeding in China, and presents a recent study on fine-mapping quantitative trait loci (QTLs) for yield traits. SCOPE: By exploiting new types of male sterility, hybrid rice production in China has become more diversified. The use of inter-subspecies crosses has made an additional contribution to broadening the genetic diversity of hybrid rice and played an important role in the breeding of super rice hybrids in China. With the development and application of indica-inclined and japonica-inclined parental lines, new rice hybrids with super high-yielding potential have been developed and are being grown on a large scale. DNA markers for subspecies differentiation have been identified and applied, and marker-assisted selection performed for the development of restorer lines carrying disease resistance genes. The genetic basis of heterosis in highly heterotic hybrids has been studied, but data from these studies are insufficient to draw sound conclusions. In a QTL study using stepwise residual heterozygous lines, two linked intervals harbouring QTLs for yield traits were resolved, one of which was delimited to a 125-kb region. CONCLUSIONS: Advances in rice genomic research have shed new light on the genetic study and germplasm utilization in rice. Molecular marker-assisted selection is a powerful tool to increase breeding efficiency, but much work remains to be done before this technique can be extended from major genes to QTLs.

Effects of Hd2 in the presence of the photoperiod-insensitive functional allele of Hd1 in rice.

The role of photoperiod sensitivity (PS) of flowering genes have become well recognized in rice, whereas little attention has been drawn to the non-PS component of these genes, especially to their influence on gene-by-gene interactions. Rice populations in which the photoperiod-sensitive allele at Hd1 has become insensitive to photoperiod but continued to affect heading date (HD) were used in this study to fine-map a quantitative trait locus (QTL) for HD and analyze its genetic relationship to Hd1 The QTL was delimitated to a 96.3-kb region on the distal end of the long arm of chromosome 7. Sequence comparison revealed that this QTL is identical to Hd2 In the near-isogenic line (NIL) populations analyzed, Hd1 and Hd2 were shown to be photoperiod insensitive and have pleiotropic effects for HD, plant height and yield traits. The two genes were found to largely act additively in regulating HD and yield traits. The results indicate that non-PS components of flowering genes involved in photoperiod response play an important role in controlling flowering time and grain yield in rice, which should allow breeders to better manipulate pleiotropic genes for balancing adaptability and high-yielding accumulation.

[QTL mapping and interaction analysis of genotype x environment (Fe2+ -concentrations ) for mesocotyl length in rice (Oryza sativa L.)].

A recombinant inbred lines (RILs) population derived from a cross between Zhenshan97B and Miyang46 was used for detecting QTLs with additive effects and additive-by-additive epistasis for rice mesocotyl length. A linkage map consisting of 207 DNA markers,distributing on the 12 chromosomes of rice,was employed for QTL mapping by using software QTL Mapper 1.6 of mixed linear model. Rice mesocotyl length under germination conditions with 4 different FeSO4 concentrations (0, 1.79, 7.16, 14.32 mmol/L) was measured 7 days after planting. A total of 6 QTLs with significant additive effects on chromosome 1, 5 and 9, with variance explained of 3.5%-11.4%, eleven QTLs with significant additive x additive epistatic effects on chromosome 1, 2, 3, 4, 5, 8 were detected, with variance explained of 4.5%-8.1%. In addition, one QTL for environmental interaction (Fe2+ -concentrations) was detected.

[Analysis of additive and AE interaction effects of QTLs controlling plant height, heading date and panicle number in rice (Oryza sativa L.)].

Plant height (PH), heading date (HD) and productive panicle number (PN) are important agronomic trait in rice. Appropriate plant height, heading date and panicle number are prerequisites for the desired high and stable yield level in rice breeding programs. A recombinant inbred line (RIL) population consisting of 304 individuals was derived from a cross between indica varieties Zhong156 and Gumei2, from which a linkage map consisting of 168 RFLP, SSLP, RAPD and RGA markers that distribute on all the 12 rice chromosomes was constructed, and covers 1447.9 cM of the rice genome. The parents and 304 F9 lines were grown in the paddy field in China National Rice Research Institute (CNRRI), Hangzhou, China in 2001. The experiments were carried out in two seasons followed a randomized complete block design. QTLMapper 1.01 was applied to detect QTLs and QTL x environment (QE) interaction for HD (heading data), PH (plant height) and PN (panicle number), and conditional mapping for PH and PN was performed as well. A total of 15 QTLs with significant additive effects were detected, among which 4 QTLs had significant QE interaction. Ten QTLs with additive x additive epistatic effects for PH, HD and PN were detected, among which none showed significant epistatisis x environment interaction. These QTLs explained 12.12%, 1.38% and 5.00% of the total phenotypic variance for PH, HD and PN, respectively, and contributions were generally lower due to the strong epistatic effects. In conditional QTL analysis, the numbers of QTLs showing significant additive and epistatic effects were 7 and 6 for PH, and 3 and 3 for PN, respectively. Among the QTLs having significant additive effects for PH, qPH7-2 showed both additive effects and QE interaction, qPH7-1 and qPH10 showed QE interaction only, and the remaining 4 QTLs showed additive effects only. Each of the 3 QTLs having significant additive effects for PN did not display significant QE interaction. No epistatic QE interactions was detected. In addition, conditional QTL analysis indicated that the expression of QTLs for PH and PN may vary depending on the QTLs for HD.

Locating QTLs controlling several adult root traits in an elite Chinese hybrid rice.

This study aimed to elucidate the genetics of the adult root system in elite Chinese hybrid rice. Several adult root traits in a recombinant inbred line (RIL) population of Xieyou 9308 and two backcross F1 (BCF1) populations derived from the RILs were phenotyped under hydroponic culture at heading stage for quantitative trait locus (QTL) mapping and other statistical analysis. There a total of eight QTLs detected for the root traits. Among of them, a pleiotropic QTL was repeatedly flanked by RM180 and RM5436 on the short arm of chromosome 7 for multiple traits across RILs and its BCF1 populations, accounting for 6.88% to 25.26% of the phenotypic variances. Only additive/dominant QTLs were detected for the root traits. These results can serve as a foundation for facilitating future cloning and molecular breeding.

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.

How willing are the public to pay for anti-hypertensive drugs for primary prevention of cardiovascular disease: a survey in a Chinese city.

BACKGROUND: Current recommendations on drug treatment of hypertension for primary prevention of cardiovascular disease are primarily determined by the evidence of effectiveness, disregard the resources available and values of people, and recommend a universally fixed risk cutoff for initiating drug treatment. The guidelines may have over-estimated the willingness of the public to accept and pay for these drugs and a fixed cutoff may not fit all populations. Moreover, the public may have been misinformed and are unable to make the right decision even if they are consulted. We conducted this study to address these issues and to describe the gap between current policy and what the public truly want. METHODS: A cross-sectional survey with face-to-face interviews of rural and urban residents in northern China. Before providing any information, we asked the residents whether they would accept drug treatment if they had hypertension and also asked them to estimate the 5-year cardiovascular risk in untreated hypertension and the benefits from anti-hypertensive drugs. We then informed the participants of necessary information and asked them above what benefit they would be willing to pay the current cost, and how much they would be willing to pay for the actual benefit, for anti-hypertensive drugs out of pocket. RESULTS: Eight hundred and eighty-seven rural residents and 921 urban residents were interviewed with a response rate of 97%. Ninety-five percent [95% confidence interval (CI) 94-96%] of the residents said they would take anti-hypertensive drugs if they had hypertension, although 91% (95% CI 89-92%) said they did not have sufficient knowledge to make a decision. Seventy-eight percent (95% CI 76-80%) believed that anti-hypertensive drugs were primarily to lower blood pressure or relieve symptoms. They over-estimated the cardiovascular risk of untreated hypertension by approximately 12 times and the absolute benefit of drug treatment by 20 times. Given the actual absolute benefit of the drugs, only 23% (95% CI 21-25%) were willing to pay the current annual cost of $500 Ren Min Bi (US$73.3, euro 54.8 as of 8 May 2009) for these drugs. Given the current cost, they were, on average, willing to pay for the drugs only when the 5-year cardiovascular disease risk was as high as 35% (95% CI 31-38%) or even higher. CONCLUSION: The public in China are significantly misinformed and considerably over estimate the risk of hypertension and the benefit of treatment. The public's willingness to pay for anti-hypertensive drugs is much lower than the current guidelines implicitly assume. The willingness to pay should be considered, along with other factors, when prescribing anti-hypertensive drugs to an individual patient or making hypertension guidelines for a population.