Phosphorylation of OsTGA5 by casein kinase II compromises its suppression of defense-related gene transcription in rice.

Plants manage the high cost of immunity activation by suppressing the expression of defense genes during normal growth and rapidly switching them on upon pathogen invasion. TGAs are key transcription factors controlling the expression of defense genes. However, how TGAs function, especially in monocot plants like rice with continuously high levels of endogenous salicylic acid (SA) remains elusive. In this study, we characterized the role of OsTGA5 as a negative regulator of rice resistance against blast fungus by transcriptionally repressing the expression of various defense-related genes. Moreover, OsTGA5 repressed PTI responses and the accumulation of endogenous SA. Importantly, we showed that the nucleus-localized casein kinase II (CK2) complex interacts with and phosphorylates OsTGA5 on Ser-32, which reduces the affinity of OsTGA5 for the JIOsPR10 promoter, thereby alleviating the repression of JIOsPR10 transcription and increasing rice resistance. Furthermore, the in vivo phosphorylation of OsTGA5 Ser-32 was enhanced by blast fungus infection. The CK2 α subunit, depending on its kinase activity, positively regulated rice defense against blast fungus. Taken together, our results provide a mechanism for the role of OsTGA5 in negatively regulating the transcription of defense-related genes in rice and the repressive switch imposed by nuclear CK2-mediated phosphorylation during blast fungus invasion.

Integration of transcriptomic and proteomic analyses reveals several levels of metabolic regulation in the excess starch and early senescent leaf mutant lses1 in rice.

BACKGROUND: The normal metabolism of transitory starch in leaves plays an important role in ensuring photosynthesis, delaying senescence and maintaining high yield in crops. OsCKI1 (casein kinase I1) plays crucial regulatory roles in multiple important physiological processes, including root development, hormonal signaling and low temperature-treatment adaptive growth in rice; however, its potential role in regulating temporary starch metabolism or premature leaf senescence remains unclear. To reveal the molecular regulatory mechanism of OsCKI1 in rice leaves, physiological, transcriptomic and proteomic analyses of leaves of osckI1 allele mutant lses1 (leaf starch excess and senescence 1) and its wild-type varieties (WT) were performed. RESULTS: Phenotypic identification and physiological measurements showed that the lses1 mutant exhibited starch excess in the leaves and an obvious leaf tip withering phenotype as well as high ROS and MDA contents, low chlorophyll content and protective enzyme activities compared to WT. The correlation analyses between protein and mRNA abundance are weak or limited. However, the changes of several important genes related to carbohydrate metabolism and apoptosis at the mRNA and protein levels were consistent. The protein-protein interaction (PPI) network might play accessory roles in promoting premature senescence of lses1 leaves. Comprehensive transcriptomic and proteomic analysis indicated that multiple key genes/proteins related to starch and sugar metabolism, apoptosis and ABA signaling exhibited significant differential expression. Abnormal increase in temporary starch was highly correlated with the expression of starch biosynthesis-related genes, which might be the main factor that causes premature leaf senescence and changes in multiple metabolic levels in leaves of lses1. In addition, four proteins associated with ABA accumulation and signaling, and three CKI potential target proteins related to starch biosynthesis were up-regulated in the lses1 mutant, suggesting that LSES1 may affect temporary starch accumulation and premature leaf senescence through phosphorylation crosstalk ABA signaling and starch anabolic pathways. CONCLUSION: The current study established the high correlation between the changes in physiological characteristics and mRNA and protein expression profiles in lses1 leaves, and emphasized the positive effect of excessive starch on accelerating premature leaf senescence. The expression patterns of genes/proteins related to starch biosynthesis and ABA signaling were analyzed via transcriptomes and proteomes, which provided a novel direction and research basis for the subsequent exploration of the regulation mechanism of temporary starch and apoptosis via LSES1/OsCKI1 in rice.

Membrane phospholipids remodeling upon imbibition in Brassica napus L. seeds.

Successful seed germination depends on the rapid repair of cell membrane damaged by dry storage. However, little is known about the reorganization of lipids during this process. In this study, the changes of intracellular redox environment, cell membrane integrity, lipid composition, and expression of genes related to phospholipid metabolism were assessed during imbibition of Brassica napus seeds. A total number of 443 lipids belonging to 7 categories were detected by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). In the 24 h-imbibed seeds, the relative content of triacylglycerol was lower than in dry seeds, while the relative content of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), especially PC (36:2, number of carbons in the acyl chains: number of double bonds), PC (36:3), and PE (36:3) were higher than those in dry seeds. Meanwhile, the content and unsaturation levels of phospholipids increased, indicating membrane lipids remodeling during seed imbibition. The plasma membrane integrity, which was measured by the relative electrolyte leakage (REL) of the membrane and FM4-64 fluorescent dye, was improved upon imbibition, confirming that cell membrane was repaired after 24 h-imbibition. The reduction of H2O2 content, redox potential, and malondialdehyde (MDA) content indicated that the degree of membrane lipid peroxidation was significantly decreased upon imbibition. Gene expression analysis showed that the differential expression of genes for key enzymes occurred in the plateau phase of the imbibition curve, i.e. after 8 h-to 24 h-imbibition. Moreover, the differential expression of genes such as those encoding phospholipase C (PLC), phospholipase D (PLD), triacylglycerol lipase (TAG lipase), choline/ethanolamine phosphotransferase (CEPT), and phosphatidylserine synthase (PTDSS2) during imbibition indicated that membrane lipid remodeling was related to complex metabolic pathways, among which the degradation of triacylglycerol and the synthesis of phospholipids using diacylglycerol might play an important role during membrane remodeling.

Oxidative damage and antioxidative indicators in 48 h germinated rice embryos during the vitrification-cryopreservation procedure.

KEY MESSAGES: Cu/Zn SOD and other genes may be critical indicators of a stress response to reactive oxygen species (ROS) accumulation in 48 h germinated rice embryos subjected to vitrification cryopreservation. In the current study, reactive oxygen species (ROS) accumulation was investigated in 48 h germinated rice embryos during the vitrification-cryopreservation process. We found that vitrification-cryopreservation significantly affected ROS levels, especially superoxide anion levels, in 48 h germinated rice embryos. Malonaldehyde content in the apical meristems of germinated embryos was significantly positively correlated with the rate of superoxide anion generation and the highest levels of malonaldehyde content were reached after vitrification treatment. Cell viability in 48 h germinated embryos was significantly negatively correlated with the rate of superoxide anion generation, malonaldehyde content, and electrolyte leakage. Spatial and temporal patterns in ROS accumulation in these embryos existed during the vitrification procedure. Among the vitrification-cryopreservation treatments we assessed, the preculture treatment was found to stimulate superoxide anion generation and to activate the response system in the apical meristems of germinated embryos. Loading treatments motivated the catalase and ascorbate peroxidase activities. During the vitrification-dehydration treatment, oxidative stress reached the highest levels causing an antioxidative response. This response involved antioxidant enzymes promoting detoxification of ROS. Based on a comprehensive correlation analysis involving ROS accumulation, cell viability, the activities of antioxidant enzymes, and gene expression profiles, Cu/Zn SOD, CAT1, APX7, GR2, GR3, MDHAR1, and DHAR1 may be critical indicators of oxidative stress affected by the vitrification-cryopreservation treatments. The investigation of these antioxidative responses in 48 h germinated rice embryos may, therefore, provide useful information with respect to plant vitrification-cryopreservation.

Dwarf and deformed flower 1, encoding an F-box protein, is critical for vegetative and floral development in rice (Oryza sativa L.).

Recent studies have shown that F-box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1-1 (ddf1-1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1-1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume-like organs and pistil-like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F-box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B-class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F-box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.

[Genetic analysis and gene mapping for a salt tolerant mutant at seedling stage in rice].

A salt tolerant mutant at seedling stage was obtained from an M2 population of radiation mutagenesis of an indica rice cultivar R401. The mutant seedlings could survive under the treatment of sodium chloride solution at the concentration of 150 mmol/L, while the wild-type control seedlings withered and died. An F2 population was developed from a cross between a japonica cultivar Nipponbare and the salt tolerant mutant. By investigating the performance of the F2 population under the stress of 150 mmol/L NaCl solution, we found that the mutant phenotype was caused by the recessive mutation of a single gene, temporarily designated SST(t). Bulked segregant analysis (BSA) based on the F2 mapping population revealed that SST(t) is located on chromosome 6. By analyzing 137 typical salt-tolerant F2 plants using molecular markers, SST(t) was mapped in a 2.3 cM (or 406 kb) interval between InDel markers ID26847 and ID27253, with genetic distances of 1.2 cM and 1.1 cM to the two markers, respectively.

Dynamic Changes in Membrane Lipid Metabolism and Antioxidant Defense During Soybean (Glycine max L. Merr.) Seed Aging.

Seed viability depends upon the maintenance of functional lipids; however, how membrane lipid components dynamically change during the seed aging process remains obscure. Seed storage is accompanied by the oxidation of membrane lipids and loss of seed viability. Understanding membrane lipid changes and their effect on the cell membrane during seed aging can contribute to revealing the mechanism of seed longevity. In this study, the potential relationship between oxidative stress and membrane lipid metabolism was evaluated by using a non-targeted lipidomics approach during artificial aging of Glycine max L. Merr. Zhongdou No. 27 seeds. We determined changes in reactive oxygen species, malondialdehyde content, and membrane permeability and assessed antioxidant system activity. We found that decreased non-enzymatic antioxidant contents and catalase activity might lead to reactive oxygen species accumulation, resulting in higher electrolyte leakage and lipid peroxidation. The significantly decreased phospholipids and increased glycerolipids and lysophospholipids suggested that hydrolysis of phospholipids to form glycerolipids and lysophospholipids could be the primary pathway of membrane metabolism during seed aging. Moreover, the ratio of phosphatidylcholine to phosphatidylethanolamine, double bond index, and acyl chain length of phospholipids were found to jointly regulate membrane function. In addition, the observed changes in lipid metabolism suggest novel potential hallmarks of soybean seed aging, such as diacylglycerol 36:4; phosphatidylcholine 34:2, 36:2, and 36:4; and phosphatidylethanolamine 34:2. This knowledge can be of great significance for elucidating the molecular mechanism underlying seed aging and germplasm conservation.

Proteome analysis of maize seeds: the effect of artificial ageing.

Previous understanding of the mechanism of seed ageing is largely based on observations on imbibed seeds rather than dry seeds. The present research was conducted to investigate whether seed ageing has effects on the dry seeds through proteome analysis. Maize (Zea mays cv. Dabaitou) seeds were artificially aged at 50°C (13.58% moisture content) for 5 or 13 days, and the total protein was extracted from embryos of the dry seeds. Two-dimensional electrophoresis was performed and the differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. A total of 40 proteins were identified, in which 16 proteins were upregulated, indicating that artificial ageing affected the proteome of the dry seeds. Proteomic studies revealed that the signal transduction and transcription were disturbed by artificial ageing, which might lead to reduced protection against ageing. Artificial ageing also increased proteases and broke down stored proteins, impaired metabolism and energy supply, and ultimately resulted in seed deterioration. Proteins involved in metabolism and energy were the largest downregulated protein group, with regard to glycolysis, tricarboxylic acid cycle, the electron transport chain and oxidative phosphorylation. The downregulation of these proteins, together with reduction in the specific activity of glucose-6-phosphate dehydrogenase, and the content of glucose 6-phosphate, pyruvic acid and ATP in aged seeds, suggested the important roles of the mobilization of stored carbohydrates and energy supply in seed ageing and seed vigor. The present work provides new information about the proteomic changes during seed ageing and provides a possible mechanism for seed deterioration.

[Genetic analysis and gene mapping of DDF1, a pleiotropic gene involving in both vegetable and reproductive growth in rice].

There are many pleiotropic genes playing key roles in regulating both vegetative growth and reproductive development in plants. A dwarf mutant of rice with deformed flowers, named as ddf1, was identified from indica rice breeding lines. Genetic analysis indicated that ddf1 was resulted from the recessive mutation of a single gene, temporarily named as DDF1. This result suggested that DDF1 is a pleiotropic gene, which controls both vegetative growth and reproductive development in rice. To map this gene, an F2 population was developed by crossing the ddf1 heterozygote with the tropical japonica rice variety DZ60. By means of bulked segregant analysis and small population-based linkage analysis using the published RM-series rice SSR markers, DDF1 was preliminarily mapped in a region between markers RM588 and RM587 on chromosome 6 with the genetic distances of 3.8 cM and 2.4 cM to the two markers, respectively. By developing new SSR markers in this interval according to the published rice genome sequence, we further mapped DDF1 in a 165 kb interval. The results will facilitate cloning of DDF1.

Timing for antioxidant-priming against rice seed ageing: optimal only in non-resistant stage.

Seed deterioration due to ageing strongly affects both germplasm preservation and agricultural production. Decelerating seed deterioration and boosting seed viability become increasingly urgent. The loss of seed viability is inevitable even under cold storage. For species with short-lived seed or for regions with poor preservation infrastructure where cold storage is not readily available, seed enhancement is more reliable to increase seed viability and longevity. Antioxidant priming as a way of seed enhancement usually improves seed germination. As for post-priming survival, however, significant uncertainty exists. The controversy lies particularly on seeds of high germination percentage (GP > 95%) whose viability is hardly improvable and the benefits of priming depend on prolonging seed longevity. Therefore, this study timed antioxidant priming to prolong the longevity of high-viability seeds under artificially accelerated ageing (AAA). Rice (Nipponbare) seeds (GP > 97%) under room-temperature-storage (RTS) for 6 months. were resistant to AAA first with little viability loss for a certain period, the resistant stage. This resistance gradually vanished without GP change, during a prolonged RTS period which was named the vulnerable stage. According to the results, although antioxidant priming severely curtailed the resistant stage for seeds with a long plateau in the survival curve, it decelerated viability loss for seeds in the vulnerable stage. In complement to seed storage, priming potentially retains high seed GP which would decrease without seed enhancement. To maximize the benefits of priming for high-GP seeds, two time points are advised as the start of a time window for priming: (1) just at the end of the resistant stage without notable viability loss, which is hard to grasp by GP monitoring; (2) slight but identifiable GP decline.

Composition of Mitochondrial Complex I during the Critical Node of Seed Aging in Oryza sativa.

Previous studies have documented mitochondrial dysfunction during the critical node (CN) of rice (Oryza sativa) seed aging, including a decrease in the capacity of NADH dependent O2 consumption. This raises the hypothesis that changes in the activity of NADH:ubiquinone oxidoreductase (complex I) may play a role in seed aging. The composition and activity of complex I was investigated at the CN of aged rice seeds. Using BN-PAGE and SWATH-MS 52 complex I subunits were identified, nineteen for the first time to be experimentally detected in rice. The subunits of the matrix arm (N and Q modules) were reduced in abundance at the CN, in accordance with a reduction in the capacity to oxidise NADH, reducing substrate oxidation and increase ROS accumulation. In contrast, subunits in the P module increased in abundance that contains many mitochondrial encoded subunits. It is proposed that the changes in complex I abundance subunits may indicate a premature re-activation of mitochondrial biogenesis, as evidenced by the increase in mitochondrial encoded subunits. This premature activation of mitochondrial biogenesis may under-pin the decreased viability of aged seeds, as mitochondrial biogenesis is a crucial event in germination to drive growth before autotrophic growth of the seedling is established.

[Pyramiding of 3-resistant-gene to improve rice blast resistance of a restorer line, Fuhui 673].

To improve the blast resistance of elite rice restorer line Fuhui 673, 3 blast resistance genes Pi-1, Pi-9 and Pi-kh were introduced into Fuhui 673 from a good-quality restorer line Jinhui 1059 through 3 successive backcrosses followed by one selfing using the technique of marker-assisted selection. Ten near-isogenic lines (NILs) of Fuhui 673 carrying the 3 introduced resistance genes were created. Genotype analysis using 68 SSR markers evenly distributed in the genome indicated that 92.96%-98.59% of the NILs' genetic background had been recovered to Fuhui 673. Both indoor and field resistance tests indicated that the NILs and their hybrids with sterile line Yixiang A were all resistant to rice blast, with resistance levels significantly higher than those of controls Fuhui 673 and hybrid Yiyou 673 (Yixiang A  Fuhui 673). In addition, among the 10 hybrids between the NILs and Yixiang A, 2 showed significantly higher yield than and 4 displayed similar yield to that of control Yiyou 673, suggesting that most of the NILs retained the elite characteristics of Fuhui 673. Two new hybrid rice cultivars Liangyou 7283 and Jintaiyou 683 from NIL Line 9 showed high yield, good resistance to blast and moderate growth period in regional trial, suggesting that the NIL Line 9 has a good prospect for application.

The genetic diversity and relationships of cauliflower (Brassica oleracea var. botrytis) inbred lines assessed by using SSR markers.

Inbred lines are important germplasm in cauliflower breeding programs. To understand the genetic diversity and relationships of cauliflower inbred lines, the use of simple sequence repeat (SSR) markers will be of great value for parental line selection and breeding strategy design. In this study, the genetic diversity and relationships of 165 cauliflower inbred lines primarily derived from southeast China were assessed using SSR markers. Forty-three SSR markers were polymorphic across these inbred lines and generated a total of 111 alleles. The mean values of the number of alleles (Na), effective number of alleles (Ne), Shannon's Information index (I), and polymorphism information content (PIC) per locus were 2.581, 1.599, 0.517 and 0.316, respectively. Genetic distance values among all pairs of the inbred lines varied from 0 to 0.67 with an average of 0.30. On the basis of genetic distance data estimated with the SSR markers, the 165 cauliflower inbred lines were classified into four main clusters (from group Ⅰ to group Ⅳ) by cluster analysis and four subpopulations (from POP 1 to POP 4) by structure analysis. The classification patterns of most cauliflower inbred lines were not consistent with their curd maturity, curd solidity or geographic origins. These results based on estimates by the SSR markers, suggested the genetic diversity of the 165 cauliflower inbred lines was relatively narrow. Therefore, pyramiding the valuable genes among different types of the cauliflower inbred lines is important to increase the genetic diversity to obtain desirable hybridization combinations. The information generated in this report will be useful for assessing germplasm and breeding in cauliflower.

Dehiscence method: a seed-saving, quick and simple viability assessment in rice.

BACKGROUND: Seed viability monitoring is very important in ex situ germplasm preservation to detect germplasm deterioration. This requires seed-, time- and labor- saving methods with high precision to assess seed germination as viability. Although the current non-invasive, rapid, sensing methods (NRSs) are time- and labor-saving, they lack the precision and simplicity which are the virtues of traditional germination. Moreover, they consume a considerable amount of seeds to adjust sensed signals to germination percentage, which disregards the seed-saving objective. This becomes particularly severe for rare or endangered species whose seeds are already scarce. Here we propose a new method that is precise, low-invasive, simple, and quick, which involves analyzing the pattern of dehiscence (seed coat rupture), followed by embryonic protrusion. RESULTS: Dehiscence proved simple to identify. After the trial of 20 treatments from 3 rice varieties, we recognized that dehiscence percentage at the 48th hour of germination (D(48)) correlates significantly with germination rate for tested seed lots. In addition, we found that the final germination percentage corresponded to D(48) plus 5. More than 70% of the seeds survived post-dehiscence desiccation for storage. Hydrogen peroxide (1 mM) as the solution for imbibition could further improve the survival. The method also worked quicker than tetrazolium which is honored as a fast, traditional method, in detecting less vigorous but viable seeds. CONCLUSION: We demonstrated the comprehensive virtues of dehiscence method in assessing rice seed: it is more precise and easier to use than NRSs and is faster and more seed-saving than traditional methods. We anticipate modifications including artificial intelligence to extend our method to increasingly diverse circumstances and species.

Proteomic analysis of the defense response to Magnaporthe oryzae in rice harboring the blast resistance gene Piz-t.

BACKGROUND: Rice blast (caused by Magnaporthe oryzae) is one of the most destructive diseases of rice. While many blast resistance (R) genes have been identified and deployed in rice cultivars, little is known about the R gene-mediated defense mechanism. We used a rice transgenic line harboring the resistance gene Piz-t to investigate the R gene-mediated resistance response to infection. RESULTS: We conducted comparative proteome profiling of the Piz-t transgenic Nipponbare line (NPB-Piz-t) and wild-type Nipponbare (NPB) inoculated with M. oryzae at 24, 48, 72 h post-inoculation (hpi) using isobaric tags for relative and absolute quantification (iTRAQ) analysis. Comparative analysis of the response of NPB-Piz-t to the avirulent isolate KJ201 and the virulent isolate RB22 identified 114 differentially expressed proteins (DEPs) between KJ201-inoculated NPB-Piz-t (KJ201-Piz-t) and mock-treated NPB-Piz-t (Mock-Piz-t), and 118 DEPs between RB22-inoculated NPB-Piz-t (RB22-Piz-t) and Mock-Piz-t. Among the DEPs, 56 occurred commonly in comparisons KJ201-Piz-t/Mock-Piz-t and RB22-Piz-t/Mock-Piz-t. In a comparison of the responses of NPB and NPB-Piz-t to isolate KJ201, 93 DEPs between KJ201-Piz-t and KJ201-NPB were identified. DEPs in comparisons KJ201-Piz-t/Mock-Piz-t, RB22-Piz-t/Mock-Piz-t and KJ201-Piz-t/KJ201-NPB contained a number of proteins that may be involved in rice response to pathogens, including pathogenesis-related (PR) proteins, hormonal regulation-related proteins, defense and stress response-related proteins, receptor-like kinase, and cytochrome P450. Comparative analysis further identified 7 common DEPs between the comparisons KJ201-Piz-t/KJ201-NPB and KJ201-Piz-t/RB22-Piz-t, including alcohol dehydrogenase I, receptor-like protein kinase, endochitinase, similar to rubisco large subunit, NADP-dependent malic enzyme, and two hypothetical proteins. CONCLUSIONS: Our results provide a valuable resource for discovery of complex protein networks involved in the resistance response of rice to blast fungus.

Allele-specific marker-based assessment revealed that the rice blast resistance genes Pi2 and Pi9 have not been widely deployed in Chinese indica rice cultivars.

BACKGROUND: The most sustainable approach to control rice blast disease is to develop durably resistant cultivars. In molecular breeding for rice blast resistance, markers developed based on polymorphisms between functional and non-functional alleles of resistance genes, can provide precise and accurate selection of resistant genotypes without the need for difficult, laborious and time-consuming phenotyping. The Pi2 and Pi9 genes confer broad-spectrum resistance against diverse blast isolates. Development of allele-specific markers for Pi2 and Pi9 would facilitate breeding of blast resistant rice by using the two blast resistance genes. RESULT: In this work, we developed two new markers, named Pi9-Pro and Pi2-LRR respectively, targeting the unique polymorphisms of the resistant and susceptible alleles of Pi2 and of Pi9. The InDel marker Pi9-Pro differentiates three different genotypes corresponding to the Pi2/Piz-t, Pi9 and non-Pi2/Piz-t/Pi9 alleles, and the CAPS marker Pi2-LRR differentiates the Pi2 allele from the non-Pi2 allele. Based on the two newly developed markers and two available markers Pi2SNP and Pi9SNP, the presence of Pi2 and Pi9 was assessed in a set of 434 rice accessions consisting of 377 Chinese indica cultivars/breeding materials and 57 Chinese japonica cultivars/breeding materials. Of the 434 accessions tested, while one indica restorer line Huazhan was identified harboring the Pi2 resistance allele, no other rice line was identified harboring the Pi2 or Pi9 resistance alleles. CONCLUSIONS: Allele-specific marker-based assessment revealed that Pi2 and Pi9 have not been widely incorporated into diverse Chinese indica rice cultivars. Thus, the two blast resistance genes can be new gene sources for developing blast resistant rice, especially indica rice, in China. The two newly developed markers should be highly useful for using Pi2 and Pi9 in marker-assisted selection (MAS) breeding programs.

Genetic analysis and mapping of gene fzp(t) controlling spikelet differentiation in rice.

A mutant of spikelet differentiation in rice called frizzle panicle (fzp) was discovered in the progeny of a cross between Oryza sativa ssp. indica cv. V20B and cv. Hua1B. The mutant exhibits normal plant morphology but has apparently fewer tillers. The most striking change in fzp is that its spikelet differentiation is completely blocked, with unlimited subsequent rachis branches generated from the positions where spikelets normally develop in wild-type plants. Genetic analysis suggests that fzp is controlled by a single recessive gene, which is temporarily named fzp(t). Based on its mutant phenotype, fzp(t) represents a key gene controlling spikelet differentiation. Some F(2) mutant plants derived from various genetic background appeared as the "middle type", suggesting that the action of fzp(t) is influenced by the presence of redundant, modifier or interactive genes. By using simple sequence repeat (SSR) markers and bulked segregant analysis (BSA) method, fzp(t) gene was mapped in the terminal region of the long arm of chromosome 7, with RM172 and RM248 on one side, 3.2 cM and 6.4 cM from fzp(t), and RM18 and RM234 on the other side, 23.1 cM and 26.3 cM from fzp(t), respectively. These results will facilitate the positional cloning and function studies of the gene.

Approximate estimation of minimal sample size required for marker-assisted backcross breeding.

Backcross breeding is a useful method to transfer favorable alleles from a donor parent into a recipient parent. Marker-assisted selection (MAS) can speed up the process. To make an appropriate plan before using MAS in a breeding program, breeders need to know the minimal sample size of the progeny generation required. A method to estimate the minimal sample size required for marker-assisted backcross breeding when both foreground selection and background selection are conducted is proposed. On the basis of a simplified assumption that the target loci are introgressed and the genetic background are independent, the probability of selecting individuals with desired genotypes in each generation is approximately estimated combining analytical approach (for foreground selection) and simulation according to the graphic genotypes of backcrossing parents (for background selection). The minimal sample size required to obtain at least one desired individual with a given probability is estimated. Application of the method is demonstrated with hypothesized examples. The method can be conveniently applied to practical backcross breeding programs.