NONSTOP GLUMES1 Encodes a C2H2 Zinc Finger Protein That Regulates Spikelet Development in Rice.

The spikelet is an inflorescence structure unique to grasses. The molecular mechanisms underlying spikelet development and evolution are unclear. In this study, we characterized three allelic recessive mutants in rice (Oryza sativa): nonstop glumes 1-1 (nsg1-1), nsg1-2, and nsg1-3 In these mutants, organs such as the rudimentary glume, sterile lemma, palea, lodicule, and filament were elongated and/or widened, or transformed into lemma- and/or marginal region of the palea-like organs. NSG1 encoded a member of the C2H2 zinc finger protein family and was expressed mainly in the organ primordia of the spikelet. In the nsg1-1 mutant spikelet, LHS1 DL, and MFO1 were ectopically expressed in two or more organs, including the rudimentary glume, sterile lemma, palea, lodicule, and stamen, whereas G1 was downregulated in the rudimentary glume and sterile lemma. Furthermore, the NSG1 protein was able to bind to regulatory regions of LHS1 and then recruit the corepressor TOPLESS-RELATED PROTEIN to repress expression by downregulating histone acetylation levels of the chromatin. The results suggest that NSG1 plays a pivotal role in maintaining organ identities in the spikelet by repressing the expression of LHS1, DL, and MFO1.

A Unique G-Quadruplex Aptamer: A Novel Approach for Cancer Cell Recognition, Cell Membrane Visualization, and RSV Infection Detection.

Surface staining has emerged as a rapid technique for applying external stains to trace cellular identities in diverse populations. In this study, we developed a distinctive aptamer with selective binding to cell surface nucleolin (NCL), bypassing cytoplasmic internalization. Conjugation of the aptamer with a FAM group facilitated NCL visualization on live cell surfaces with laser confocal microscopy. To validate the aptamer-NCL interaction, we employed various methods, including the surface plasmon resonance, IHC-based flow cytometry, and electrophoretic mobility shift assay. The G-quadruplex formations created by aptamers were confirmed with a nuclear magnetic resonance and an electrophoretic mobility shift assay utilizing BG4, a G-quadruplex-specific antibody. Furthermore, the aptamer exhibited discriminatory potential in distinguishing between cancerous and normal cells using flow cytometry. Notably, it functioned as a dynamic probe, allowing real-time monitoring of heightened NCL expression triggered by a respiratory syncytial virus (RSV) on normal cell surfaces. This effect was subsequently counteracted with dsRNA transfection and suppressed the NCL expression; thus, emphasizing the dynamic attributes of the probe. These collective findings highlight the robust versatility of our aptamer as a powerful tool for imaging cell surfaces, holding promising implications for cancer cell identification and the detection of RSV infections.

Hyperhomocysteine promotes cataract development through mTOR-mediated inhibition of autophagy and connexins expression.

AIM: Hyperhomocysteine has been recognized as an independent risk factor of multiple diseases, including several eye diseases. In this study, we aim to investigate whether increased homocysteine (Hcy) is related to cataracts, and to explore whether dysregulation of mTOR-mediated autophagy and connexin expression are underlying mechanisms. METHOD: We first developed a method of liquid chromatography tandem mass spectrometry to accurately measure serum concentrations of Hcy in 287 cataract patients and 334 healthy controls. Next, we treated human lens epithelial (HLC-B3) cells with Hcy at different concentrations and durations, and then analyzed expression of autophagy-related markers and connexins, as well as phosphorylated mTOR (p-mTOR) in these cells by Western blotting. Formation of autophagic vacuoles and intracellular Ca2+ in the Hcy-treated cells were observed by fluorescence microscopy. Further, we performed a rescue experiment in the Hcy-treated HLC-B3 cells by pre-incubation with rapamycin, an mTOR inhibitor. RESULTS: The serum levels of Hcy in patients with cataracts were significantly increased compared to those in healthy controls. In cultured HLC-B3 cells, expression of autophagy related markers (LC3B and Beclin1) and connexins (Cx43 and Cx50) was inhibited by Hcy treatment in a dose- and duration-dependent manner. Accumulation of Ca2+ in the Hcy-treated lens epithelial cells was observed as a consequence of reduced connexin expression. Meanwhile, expression of p-mTOR increased, representing up-regulation of the mTOR pathway. Importantly, inhibition of autophagy and connexin expression due to hyperhomocysteine was rescued via mTOR suppression by pretreatment with rapamycin in HLC-B3 cells. CONCLUSION: Our results demonstrate that hyperhomocysteine might promote cataract development through two mTOR-mediated pathways in the lens epithelial cells: 1) dysregulation of autophagy and 2) accumulation of intracellular calcium via decreased connexin expression.

[Association analysis of genetic polymorphisms of TCF7L2, CDKAL1, SLC30A8, HHEX genes and microvascular complications of type 2 diabetes mellitus].

OBJECTIVE: To study the associations of single nucleotide polymorphisms (SNPs) of TCF7L2, CDKAL1, SLC30A8, HHEX with diabetic retinopathy (DR) and nephropathy (DN) in type 2 diabetes mellitus. METHODS: A total of 479 subjects with DR,248 with DN and 650 without DR or DN were recruited to assess the associations between SNPs of TCF7L2 (rs7903146, rs6585205, rs11196218), CDKAL1 (rs10946398,rs4712527), SLC30A8 (rs13266634, rs3802177, rs11558471) and HHEX (rs1111875, rs7923837) and the development of DR and DN. RESULTS: There were significant differences in genotypic and allele frequencies of rs11558471 (SLC30A8) between DR and control groups (P< 0.05), the odds ratio (OR) values of A and AA were 1.27 and 1.68. The distributions of genotype and allele frequency for rs11196218 (TCF7L2) were significantly different between DN and control group (P=0.0051,OR=1.37). However, the P value after Bonferroni correction showed no significant difference. No significant differences were found in the distributions of rs13266634 and rs3802177 (SLC30A8), rs10946398 (CDKAL1), rs6585205, rs7903146 and rs11196218 (TCF7L2) and rs7923837 (HHEX) between DR and control groups, and nor significant differences were found in distributions of rs6585205 (TCF7L2), rs4712527 (CDKAL1), rs13266634, rs3802177 and rs11558471 (SLC30A8), and 7923837 (HHEX) between DN and control groups, though for all comparison the OR values were greater than 1. CONCLUSION: Polymorphisms of SLC30A8 and TCF7L2 genes may be associated with the development of DR and DN, respectively. Association between the polymorphisms of CKDAL1, TCF7L2 and HHEX genes and DR, and between the polymorphisms of SLC30A8, HHEX and CDKAL1 genes and DN, cannot be excluded.

[Association study between age-related macular degeneration and R1210C mutation of CFH gene in Chinese population].

OBJECTIVE: A R1210C mutation of complement factor H (CFH) gene has been associated with age-related macular degeneration (AMD) in Caucasian population. This study was to verify above association in Han Chinese population. METHODS: The mutation was detected by direct sequencing in 258 patients with wet AMD and 426 matched controls. RESULTS: The R1210C mutation has not been identified in either sample. CONCLUSION: The R1210C mutation in CFH gene is not associated with AMD in Han Chinese population.

Characterization of two novel knock-in mouse models of syndromic retinal ciliopathy carrying hypomorphic Sdccag8 mutations.

Mutations in serologically defined colon cancer autoantigen protein 8 ( SDCCAG8) were first identified in retinal ciliopathy families a decade ago with unknown function. To investigate the pathogenesis of SDCCAG8-associated retinal ciliopathies in vivo, we employed CRISPR/Cas9-mediated homology-directed recombination (HDR) to generate two knock-in mouse models, Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 , which carry truncating mutations of the mouse Sdccag8, corresponding to mutations that cause Bardet-Biedl syndrome (BBS) and Senior-Løken syndrome (SLS) (c.696T>G p.Y232X and c.1339-1340insG p.E447GfsX463) in humans, respectively. The two mutant Sdccag8 knock-in mice faithfully recapitulated human SDCCAG8-associated BBS phenotypes such as rod-cone dystrophy, cystic renal disorder, polydactyly, infertility, and growth retardation, with varied age of onset and severity depending on the hypomorphic strength of the Sdccag8 mutations. To the best of our knowledge, these knock-in mouse lines are the first BBS mouse models to present with the polydactyly phenotype. Major phototransduction protein mislocalization was also observed outside the outer segment after initiation of photoreceptor degeneration. Impaired cilia were observed in the mutant photoreceptors, renal epithelial cells, and mouse embryonic fibroblasts derived from the knock-in mouse embryos, suggesting that SDCCAG8 plays an essential role in ciliogenesis, and cilium defects are a primary driving force of SDCCAG8-associated retinal ciliopathies.

Inhibition of mTOR signaling by rapamycin protects photoreceptors from degeneration in rd1 mice.

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that begins with defective rod photoreceptor function, followed by impaired cone function, and complete blindness in its late stage. To date, however, there is no effective treatment for RP. By carrying a nonsense mutation in the Pde6b gene, rd1 mice display elevated cGMP in conjunction with higher intracellular Ca 2+ in their rod photoreceptors, resulting in fast retinal degeneration. Ca 2+ has been linked to activation of the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway integrates extracellular and intracellular signals to sense the supply of nutrients and plays a central role in regulating protein and lipid synthesis as well as apoptosis and autophagy. In the present study, we showed that mTOR and phosphorylated mTOR (p-mTOR, activated form of mTOR) are up-regulated in rd1 photoreceptors at postnatal day 10 (P10), a pre-degenerative stage. Moreover, the downstream effectors of mTOR, such as pS6K and S6K, are also increased, suggesting activation of the mTOR signaling pathway. Intravitreal administration of rapamycin, a negative regulator of mTOR, inhibits the mTOR pathway in rd1 photoreceptors. Consequently, the progression of retinal degeneration is slower and retinal function is enhanced, possibly mediated by activation of autophagy in the photoreceptors. Taken together, these results highlight rapamycin as a potential therapeutic avenue for retinal degeneration.

Identification and gene mapping of a novel mutant supernumerary lodicules (snl) in rice.

In order to gain a better understanding of rice flower development, a rice flower mutant supernumerary lodicules (snl), which was identified from ethyl methane sulfonate (EMS)-treated Jinhui10 (Oryza sativa L. ssp. indica) was used in the present study. In the snl mutant, the palea obtained lemma identity, additional glume-like organs formed, lodicules increased and elongated, stamens decreased, and a few aberrant carpels formed. These phenotypes suggest that SNL is involved in the entire rice flower development. SNL was mapped between two simple sequence repeat markers RM3512 and RM1342 on chromosome 2, an approximate 800 kb region, and it co-segregated with SSR215. We conclude that SNL is a novel gene involved in flower development in rice. The present study will be useful for further cloning of the SNL gene, which will contribute to the elucidation of rice flower development.

[Association study on single nucleotide polymorphisms in HTRA1 gene and rheumatoid arthritis].

OBJECTIVE: To study the association between the single nucleotide polymorphisms (SNPs) in the high-temperature requirement A-1 (HTRA1) gene and rheumatoid arthritis (RA) in Chinese Han population. METHODS: Five SNPs in the HTRA1 gene (rs2014307, rs2248799, rs2300433, rs714816 and rs2268356) were genotyped by ABI Snapshot method in Han Chinese cohort composed of 344 patients with RA and 288 healthy controls. The serum rheumatoid factor (RF) and C-reactive protein (CRP) of the patients were determined by endpoint nephelometry method. RESULTS: Genotypes of all the five SNPs in the HTRA1 gene were not significantly different between the RA patients and controls (P> 0.05). Haplotypes generated by these five SNPs did not show significantly difference between the two groups either (P> 0.05). Serum RF levels in the RA patients had no significant difference among the genotypes for four SNPs (rs2014307, rs2248799, rs714816, and rs2268356) in the HTRA1 gene, while RF levels in the RA patients with genotypes AA+AG of the rs2300433 locus were significantly higher than that in genotype GG carriers (P< 0.05). Serum CRP levels in the RA patients had no significant difference among the genotypes for all the five SNPs. CONCLUSION: Author's results suggested that although the five SNPs in the HTRA1 gene were not associated with RA in Chinese Han population, RF levels in the RA patients with genotypes AA and AG in the rs2300433 locus were significantly higher than the GG carriers. The HTRA1 role in RF regulation needs to be further investigated.

Diagnosis of the accurate genotype of HKαα carriers in patients with thalassemia using multiplex ligation-dependent probe amplification combined with nested polymerase chain reaction.

BACKGROUND: Patients carrying the HongKongαα (HKαα) allele and -α/ααα could be misdiagnosed as -α/αα by the current conventional thalassemia detection methods, leading to inaccurate genetic counseling and an incorrect prenatal diagnosis. This study was aimed to accurately analyze the genotypes of HKαα carriers and -α/ααα. METHODS: Samples were collected in our hospital from July 2017 to October 2019. Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction (Gap-PCR) and reverse dot blot (RDB). Anti-4.2 multiplex-PCR was used to confirm carriers of the ααα duplication with -α deletion. Two-round nested PCR and multiplex ligation-dependent probe amplification (MLPA) were applied to accurately identify and confirm their genotypes. For data analysis, we used descriptive statistics and Fisher's exact tests. RESULTS: Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples. The results showed that α, ß, and αß compound thalassemia were identified in 1190 (46.78%), 1286 (50.55%), and 68 (2.67%) cases, respectively. A total of 227 samples from thalassemia patients were identified as -α/αα by Gap-PCR, and the genotypes of two samples were uncertain. There was a difference between Gap-PCR and combined groups (Gap-PCR combined with nested PCR and MLPA) in detecting HKαα (P < 0.05). Among the 229 patients, 20 patients were identified as HKαα carriers and one was identified as -α/ααα by two-round nested PCR and MLPA, including 15 patients with HKαα/αα, three with HKαα/αα and ß-thalassemia coinheritance, one with HKαα/--, one with HKαα/-α and ß-thalassemia coinheritance, and one with -α/ααα and ß-thalassemia coinheritance. CONCLUSIONS: ααα and HKαα genotypes of patients carrying -α need to be detected to reduce the misdiagnosis rate of patients carrying HKαα and -α3.7/ααα alleles. More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.

[Advances in molecular genetic study of myopia].

Myopia is an important cause of blindness, in which an image is focused in front of the retina. Genetic factors have been implicated in the pathogenesis of myopia. Based on the molecular genetic study, some genetic loci linked to myopia have been mapped, but no disease-causing gene has been identified. Here authors review the genetic study on myopia, including gene mapping and candidate gene screening.

[Linkage analysis of a family with familial hypertriglyceridemia].

OBJECTIVE: To perform linkage analysis and mutation screening in a Chinese family with familial hpertriglyceridemia (FHTG). METHODS: Thirty-two family members including 12 hypertriglyceridemia patients participated in the study. Genotyping and haplotype analysis for 22 subjects were performed using short tandem repeat (STR) microsatellite polymorphism markers on 16 candidate genes and/or loci related to lipid metabolism. Two of the sixteen known candidate genes, APOA2 and USF1 were screened for mutation by direct DNA sequencing. RESULTS: No linkage was found between the candidate genes/loci of APOA5, LIPI, RP1, APOC2, ABC1, LMF1, APOA1-APOC3-APOA4, LPL, APOB, CETP, LCAT, LDLR, APOE and the phenotype in this family. The two-point Lod scores (theta =0) were all less than-1.0 for all the markers tested. Linkage analysis suggested linkage to chromosome 1q23.3-24.2 between the disease phenotype and STR marker D1S194 with a two-point maximum Lod score of 2.44 at theta =0. Fine mapping indicated that the disease gene was localized to a 5.87 cM interval between D1S104 and D1S196. No disease-causing mutation was detected in the APOA2 and USF1 genes. CONCLUSION: The above mentioned candidate genes were excluded as the disease causing genes for this family. The results implied that there might be a novel gene/locus for FHTG on chromosome 1q23.3-1q24.2.

[Mutation analysis of the PAX6 gene in a family with congenital aniridia and cataract].

OBJECTIVE: To identify the mutation in the PAX6 gene in a family with congenital aniridia and cataract. METHODS: Total genomic DNA was extracted from peripheral blood leukocytes of 12 family members including three living affected members and 96 unrelated healthy controls. The coding exons 4-13 of the PAX6 gene with intronic flanking sequences were amplified by polymerase chain reaction (PCR). By comparing sequences of the affected members with that of normal individuals, the disease-causing mutation was detected by direct DNA sequencing. RESULTS: A PAX6 mutation was identified in the 3 patients, which did not exist in the unaffected members and unrelated healthy individuals. The nonsense mutation of C to T was detected at the nucleotide 1143, which converted the Arg codon (CGA) to a stop codon(TGA) (R261X) in exon 10. CONCLUSION: The mutation (R261X) detected in the present study is considered to result in the occurrence of congenital aniridia and cataract in the Chinese family.

[Disease gene screening of known loci in a Chinese family with autosomal dominant retinitis pigmentosa].

OBJECTIVE: To map the disease-causing gene in a Chinese family with autosomal dominant retinitis pigmentosa. METHODS: Twenty-seven micro-satellite markers were randomly selected from the region around the known loci of causative genes, and haplotypes were determined by ABI3100 genetic analyzer. Two-point linkage analysis was performed using MLINK. RESULTS: The Lod score of each marker vs adRP was below 1. CONCLUSION: The phenotype of this family may not be caused by mutation of the known disease-causing genes.

[A novel GJA8 mutation in a Chinese family with autosomal dominant congenital cataract].

OBJECTIVE: To identify the mutations in the gap junction protein alpha3/alpha8 gene (GJA3 or GJA8) in the Chinese family with autosomal dominant congenital cataract (ADCC). METHODS: All subjects(5 family members and 100 unrelated control individuals)were undergone comprehensive ophthalmic examination, and genomic DNA was extracted from peripheral blood (5 mL). The exons and flanking introns of GJA3/GJA8 genes were amplified by polymerase chain reaction (PCR). Purified PCR products were then sequenced directly for screening disease-causing mutations. RESULTS: Upon bidirectional sequence analysis, a G-->A transition at nucleotide 138 (c.138G>A)in exon 2 of GJA8 was found, resulting in synonymous mutation of glycine (GGG) to glycine (GGA). An additional G-->T transvertion at nucleotide 139 (c.139G>T) in exon 2 of GJA8, resulting in a missense mutation of asparagines (GAU) to tyrosine (UAU) at codon 47 (D47Y). These two alterations were not seen in all unaffected members and 100 unrelated control individuals. Bioinformatic analyses also showed that a highly conserved region was located at Asp47. Meanwhile no sequence variations for GJA3 were detected from the 3 affected members. CONCLUSION: A novel disease-causing mutation (D47Y) of GJA8 gene in a Chinese family with ADCC is reported.

[Linkage analysis of one family with autosomal dominant high myopia].

OBJECTIVE: To map the high myopia gene in a Chinese family with autosomal dominant high myopia. METHODS: A family with autosomal dominant high myopia in three generations was collected. Eighteen short-tandem-repeat markers on previously reported loci linked to high myopia were chosen for genotyping and two-point linkage analysis was carried out. RESULTS: The spherical equivalent of affected individuals ranges from -6.00D to -20.00D and the genetic pattern is autosomal dominant. The LOD score was less than -1 in all 18 microsatellite markers, indicating that there was no linkage between these markers and the high myopia related genes in this family. CONCLUSION: A novel myopia locus for high-grade myopia may exist in the kindred. Genome-wide scan will be needed to determine this novel locus.

[Study of relationship between inflammatory response and apoptosis in perihematoma region in patients with intracerebral hemorrhage].

OBJECTIVE: To investigate the relationship between inflammatory response and cell apoptosis in the perihematoma region in patients with intracerebral hemorrhage (ICH). METHODS: Surgical specimens were obtained from the area 1 cm adjacent to the hematoma. Thirty patients with ICH were divided into five groups: 6, 7, 5, 6, 6 patients in surgery<6 hours, 6-12 hours, 12-24 hours, 24-72 hours and >72 hours groups after the onset, respectively. The control group specimens were obtained from the brain tissues distant to the hematoma in the process of craniotomy in the patients of two former groups. Sections were stained with hematoxylin and eosin (HE) for the examination of pathological changes. Immunohistochemistry, terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling (TUNEL) and reverse transcription-polymerase chain reaction (RT-PCR) were applied to determine apoptosis cells, Bax and Bcl-x protein and mRNA. RESULTS: The tissues from perihematoma region were almost normal in control group and <6 hours group. They were slightly damaged in 6-12 hours group, became worse in 12-24 hours group and most severe in 24-48 hours group, and they became better latter and were similar to the control group on 8th day. Infiltration of neutrophils, macrophages and lymphocyte appeared gradually at 6-12 hours, and became much more prominent at 12-24 hours (all P<0.01). The reactive gliosis began to appear at 24-72 hours, and enhanced after 72 hours (all P<0.01). The expression of the apoptosis and Bax protein increased gradually after 6 hours, reaching the peak at 12-24 hours (P<0.05 or P<0.01), and decreased gradually later. The changes in the levels of Bax mRNA were similar to that of the result of immunohistochemistry. Although the expression of Bcl-x protein and mRNA seemed to be increased at 12-72 hours, there was no significant difference between groups (P>0.05). The correlation analysis showed that the infiltration of neutrophils, macrophages and lymphocyte was positively correlated to the TUNEL positive cells and expression of Bax protein and mRNA (P<0.05 or P<0.01), and showed no correlation to Bcl-x protein and mRNA (all >0.05). CONCLUSION: There is a close relationship between inflammatory response and apoptosis and tissue damage in the perihematoma area in ICH.

[Mapping of two fertility-restoring gene for WA cytoplasmic male sterility in minghui63 using SSR markers].

F2 population derived from Shanyou63, F1 hybrid developed from the cross Zhenshan97 A/Zhenshan97B, was used in this study. Fertile bulk was constructed by polling equal amount of 15 highly fertile lines. Sterile bulk was obtained by pooling equal amount of 15 highly sterile lines. Minghui63 and Zhenshan97A, parents of Shanyou63, were analyzed with 302 pairs of SSR primers. 244 pairs of primers gave amplification products, of which 58 pairs of primers on 12 different chromosomes showed polymorphism between the two parents with polymorphic frequency up to 23.77%. Gene bulks were further assayed with the 5 pairs of primers. RM1 on chromosome 1 and RM258, RM304 on chromosome 10 was found to be polymorphic between the two gene bulks. In theory, there should be no difference detected between the two gene bulks except for the target traits governed by fertility-restoring genes. RM1, RM258 and RM304 were probably related to the restorer genes. Ten highly fertile and ten highly sterile lines were selected from F2 population of Shanyou63 to screen the gene bulks. The results showed that specific bands of Minghui63 were detected in all ten highly fertile lines while not observed in all the sterile lines. It indicated that the three SSR markers might be linked to fertility-restoring genes. Dominant lines were not selected due to their inalbility to distinguish recombinant lines from non-recombinant lines. Pure recessive lines were chosen to conduct mapping analysis. A total of 53 highly sterile lines were selected from 900 lines of Shanyou63 F2 population to estimate the genetic distance between three SSR markers and fertility-restoring genes respectively. The results demonstrated that recombination occurred in 2, 3, lines with RM1 and RM258 while no one with RM304. Using MAPMAKER/EXP 3.0, the genetic distance between RM1, RM258, RM304 and the related restorer genes were calculated as 1.9, 2.9 and 0.0 cM, respectively. It is possible that the fertility restoring gene(s) on chromosome 10 for three different types of cytoplasmic male sterility(WA, BT and HL) are of the same, or belong to a gene family.

[Analysis on the inheritance of inhibition and anti-inhibition of coleoptile purple line in rice and the SSR location of Ai(t) gene].

The ratio of purple line: no-purple line(13:3) was observed in six different F2 populations produced by crossing between parents with purple line and no-purple line in coleoptile. The backcross of XNA//XNA/ 21A150 (XNA, no-purple line and CMS, as the recurrent parent) resulted in a ratio of 1:1 (purple line: no-purple line). Genetic analysis showed that the expression of rice coleoptile purple line was influenced by two genes, inhibiting gene I and anti-inhibiting gene Ai(t). I gene inhibits P gene of C_A_P_ system and Ai(t) inhibits / gene, respectively. The gene pools of Ai(t) ai(t) and ai(t) ai(t) were constructed with BF1 of XNA//XNA/21A150. SSR analysis indicated that Ai(t) gene was linked with the markers of RM335, RM295, RM287 and RM21 and the genetic distance from Ai(t) to these four markers were 2.8 cM, 10.2 cM, 13.9 cM, 26.1 cM, respectively.

[The simple gain of templates of rice genomes DNA for PCR].

The solution of alkali-treated fresh rice leaves was used directly as the templates of PCR. The amplified results were stable, reliable, and had no difference compared with that amplified with rice total DNA extracted by common method. The stable results can still be obtained based on the templates kept at 25 degrees for tow weeks, at 4 degrees for three weeks, at -20 degrees for over four months. With this technique, less material and only common reagent are required, which is especially adapted to the screening of the precious transgenic rice in advance and large-scale PCR tests.