NB-LRR genes: characteristics in three Solanum species and transcriptional response to Ralstonia solanacearum in tomato.

MAIN CONCLUSION: NB-LRR genes in the three Solanum species showed specific constitution characteristics and evolved multiple clusters and duplicates. Some genes could respond to biotic stresses such as tomato bacterial wilt. Nucleotide-binding and leucine-rich repeat (NB-LRR, NLR) is a largest resistance gene family in plants, which plays a key role in response to biotic stresses. In this study, NB-LRR genes in cultivated tomato Solanum lycopersicum (Sl) and its wild relatives S. pennellii (Spe) and S. pimpinellifolium (Spi) were analyzed using bioinformatics approaches. In total, 238, 202 and 217 NB-LRR genes of 8 different types were found in Sl, Spe and Spi, respectively. The three species showed similar genomic characteristics. The NB-LRR genes were mainly distributed on chromosomes 4, 5 and 11 and located at the distal zones, forming multiple clusters and tandem duplicates. A large number of homologs appeared through gene expansion, with most Ka/Ks values being less than 1, indicating that purifying selection had occurred in evolution. These genes were mainly expressed in root and could respond to different biotic stresses. RT-qPCR analysis revealed that SlNLR genes could respond to tomato bacterial wilt, with SlNLR1 probably involved in the resistance response, whereas others being the opposite. The transcription factors (TFs) and interaction proteins that regulate target genes were mainly Dof, NAC and MYB families and kinases. The results provide a basis for the isolation and application of related genes in plant disease resistance breeding.

Teaching practice and experiences of verifying the three laws of genetics based on the SSLP marker analysis.

We explored the practical effect of the genetic analysis of simple sequence length polymorphism (SSLP) molecular markers in rice in the genetics lab course. Two parents and their F2 population were analyzed and detected with three SSLP molecular markers that located on two chromosomes of the rice genome. The markers' genotype data were used to verify the three laws of genetics, including segregation, independent assortment and linkage and crossing-over. Our practice has proved not only beneficial to deepen students' understandings about the three laws of genetics, but also conducive to cultivate students' interests in research and innovation and improve their skills and comprehensive analysis abilities. At the same time, the application scope of the experiment was discussed. This comprehensive experiment is also useful for the transformation of scientific research achievements into undergraduate experimental teaching.

[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.

[Mapping of Sanming dominant genic male sterility gene in rice].

The mutant of "Sanming Dominant Genic Male Sterile Rice" was found from an F2 population of cross "SE2lS/Basmati370" by Sanming Institute of Agricultural Science in 2001. It has proven that the male sterility of this mutant is controlled by a dominant gene (named as SMS). By multiple backcrosses, this dominant male sterile allele was introduced into the genetic background of an indica rice cultivar Jiafuzhan (which was known as Jiabuyu). In order to map SMS, a mapping population was constructed by crossing Jiabuyu with a japonica cultivar Nipponbare and further crossing the F1 with Jiafuzhan. By bulked segregant analysis and linkage analysis using SSR and INDEL markers, SMS was mapped to a 99 kb interval between INDEL markers ZM30 and ZM9 on chromosome 8. This result will facilitate cloning of SMS.

[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.

[Construction of a genetic map based on ILP markers in rice].

Intron length polymorphism (ILP) is a new type of PCR-based molecular markers with many advantages. We had previously developed 172 ILP markers in rice using the published genome sequence data of indica cultivar 93-11 and ja-ponica cultivar Nipponbare. In order to examine the reliability and the applicability of these ILP markers to genetic map-ping, we constructed a rice genetic map consisting of 172 ILP and 13 SSR markers with a total length of 1 905.7 cM using a BC1F1 (Nipponbare/93-11//Nipponbare) population. Comparison showed that the order of the markers in this map was ex-actly the same as their physical order, verifying the feasibility and efficiency of using ILP markers for genetic mapping. We also investigated the phenomenon of marker segregation distortion and found a region of serious segregation distortion in the short arm of chromosome 6.

Enhanced Plasmonic Biosensors of Hybrid Gold Nanoparticle-Graphene Oxide-Based Label-Free Immunoassay.

In this study, we propose a modified gold nanoparticle-graphene oxide sheet (AuNP-GO) nanocomposite to detect two different interactions between proteins and hybrid nanocomposites for use in biomedical applications. GO sheets have high bioaffinity, which facilitates the attachment of biomolecules to carboxyl groups and has led to its use in the development of sensing mechanisms. When GO sheets are decorated with AuNPs, they introduce localized surface plasmon resonance (LSPR) in the resonance energy transfer of spectral changes. Our results suggest a promising future for AuNP-GO-based label-free immunoassays to detect disease biomarkers and rapidly diagnose infectious diseases. The results showed the detection of antiBSA in 10 ng/ml of hCG non-specific interfering protein with dynamic responses ranging from 1.45 nM to 145 fM, and a LOD of 145 fM. Considering the wide range of potential applications of GO sheets as a host material for a variety of nanoparticles, the approach developed here may be beneficial for the future integration of nanoparticles with GO nanosheets for blood sensing. The excellent anti-interference characteristics allow for the use of the biosensor in clinical analysis and point-of-care testing (POCT) diagnostics of rapid immunoassay products, and it may also be a potential tool for the measurement of biomarkers in human serum.

[Genetic analysis and gene mapping of cold-induced seedling chlorosis in rice].

We found that the seedlings of indica rice cultivar Dular showed chlorosis but the seedlings of japonica rice cultivar Lemont remained green under natural low temperature in early spring. Using an F2 population of Lemont Dular, we found that the difference of cold tolerance at seedling stage between Dular and Lemont is controlled by a single major gene, with the chlorosis allele being recessive. We named the gene cisc(t). With the help of SSR markers, cisc(t) was mapped in a 5.5 cM interval between SSR markers RM257 and RM242 on chromosome 9.

Analysis of the phylogenetic relationships among several species of Gramineae using ACGM markers.

To study the transferability of rice (Oryza sativa L.) genome data, we used amplified consensus genetic markers to analyze the phylogenetic relationships among several species and genera in Gramineae. Ten accessions representing five grass genera (Oryza, Zea, Setaria, Triticum, and Phyllostachys) were used. According to the genetic distances, a cluster tree was constructed. The relationships among the five genera could be simply described as ((Oryza + (Zea +Setaria)) +Triticum) +Phyllostachys. The results suggest that the genetic distance between rice and maize (Z. mays L.) or rice and millet (Setaria italica L.) is closer than that between rice and wheat (Triticum aestivum L) or rice and bamboo.

[Studies of methods and exploitation of computer software for mapping interactive genes].

There are six possible types of gene interaction in qualitative traits, namely, complementary, duplicate, cumulative, dominant epistasis, recessive epistasis and inhibiting. In genetic studies, the problem of mapping interactive genes may be met sometimes, but no systematic researches on the methodology and computer software for the mapping of interactive genes have been reported up to date. In this paper, methods for the mapping of interactive genes based on maximum-likelihood estimation and corresponding computer software (IGMapping 1.0) are presented. Computer simulations have shown that the methods proposed can unbiasedly estimate the recombination frequency or linkage distance between a codominant marker and an interactive gene.

[Mapping of QTLs controlling chlorophyll content in rice].

Mapping of QTLs controlling chrolophyll content was conducted with the method of multiple-trait composite interval mapping, based on a recombinant inbred (RI) population consisting of 131 lines (F10) derived from a cross between two indica rice varieties, Acc8558 and H359, and on a corresponding genetic map comprising 147 RFLP and 78 AFLP markers. Six QTLs for contents of chlorophyll a and chlorophyll b were detected, respectively. Among them, five QTLs were the same between the two characters. These QTLs were mainly distributed on chromosomes 1 and 4, indicating that the two chromosomes were more important for chlorophyll content. qChlA1c and qChlB1b (these two QTLs were mapped at the same location) showed relatively large effects at all the four observation times and showed the largest effects at the sword leaf period. Another two QTLs (qChlA4a/qChlB4a and qChlA4b/qChlB4b) exhibited significant effects only at the second observation time, suggesting that they might act only at specific developmental stages.

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.

[The application of RAPD technology in genetic diversity detection of Jute].

The fingerprints of 10 species including 27 accessions in genus Corchorus were investigated with the technique of RAPD. Twenty-five primers were screened from 119 random primers, and a total of 329 DNA fragments were amplified ranging from 0.3-3.0 kb, 253 (87.78%), which were polymorphic. The average number of DNA band produced by each primer was 13.16. UPGMA cluster analysis and Nei's similarity coefficients were carried out and a dendrogram was constructed using software Biol D++. The results showed as follows: (1) There were abundant genetic diversities among 15 wild species and 12 cultivated species in Corchorus with genetic similarity coefficients ranging from 0.49-0.98. (2) The accessions could be clustered into three groups at cultivated species, and their close wild species were obviously different from wild species genetically. (3) At the level of D = 0.850, 27 accessions of Jute could be classified into ten groups, including C. sestuans, C. tridens, C. fascicularis, C. psendo-olitorius, C. psendo-capsularis, C. tilacutaris, Tian Jute (untitled), C. capsularis, C. olitorius and C. uriticifolius. Among which C. capsularis presented closer relationship with C. olitorius and further relationship with C. uriticifolius. The results matched well with that of the morphologic classification. (4) According to the molecular cluster tree, C. uritifolius, Chinese Tina Jute (untitled) and C. aestuans were at the basic level, revealing that these three species could be the primary wild species of Jute. (5) The tree also showed that C. tilacularis 21C from Africa could be a ecological subspecies of C. tilacularis, whilst niannian cai, ma cai and zhu cai collected different ecological types of C. aestuans, C. capsularis from Hainan was a close wild species of round fruit Jute cultivated species, and three species of C. olitorius collected from zhangpu, Henan and Mali were close wild species of long fruit Jute cultivated species. (6) within two cultivated species, the genetic similarity coefficients in round fruit cultivated species was higher than that of in long fruit cultivated species.

[Towards the positional cloning of a spikelet identity gene frizzle panicle (FZP) in rice (Oryza sativa L.)].

FZP is a key gene for spikelet differentiation in rice. Mutation of the gene blocks the differentiation of spikelets and makes rachis branches develop unlimitedly. A mutant of the gene named frizzle panicle (fzp) was previously found from the high-generation progeny of a cross between two Oryza sativa ssp. indica rice varieties, V20B and Hua1B. With the mutant, FZP had been mapped to a chromosomal region of about 26.4 cM in width between two SSR (Simple Sequence Repeat) markers, RM172 and RM18, on chromosome 7. In this study, high-resolution mapping of the gene was carried out for the positional cloning of the gene. Two flanking SSR markers, NRM6 and NRM8, were identified, which are 0.2 cM and 1.0 cM apart from the target gene, respectively, bracketing the target gene within an interval of 1.2 cM or 144 kb. An APETALA2 (AP2)-domain like gene was found at the expected position of FZP. As AP2 is known to play an important role in the floral development, we took it as the most possible candidate of FZP. PCR analysis showed that the mutant allele of the AP2-domain like gene contains an insert of about 4 kb in length, suggesting that the gene is very likely FZP.

[Plant retrotransposons and their application].

Retrotransposons are one of the most unstable genetic elements in the plant kingdom, they have the potential to dramatically affect gene function and host genome structure. The current status of their types and structure, expression regulation, transposition, and evolution are reviewed. Their potential as genetic tools are also discussed.

[Construction of a microsatellite linkage map in a DH population].

A doubled haploid population (DH) consisting of 86 lines derived by anther culture of Peiai64s/E32, a two-line hybrid rice variety with high heterosis, was used to construct a microsatellite or SSLP linkage map of rice chromosomes. A total of 302 PCR primers for SSLP analysis on these chromosomes were chosen from a map published by Cornell University (designated CUMAP) and 127 (42.05%) of them were found polymorphic between the two parents. Those polymorphic PCR primers were used for population genotyping. The map (designated PEMAP) comprises 122 microsatellite maker loci,covering a total length of 1213.4 cM. The PEMAP is highly comparable with the CUMAP. Most of the markers were mapped onto the same chromosomes and aligned in the same order. Serious segregation distortion was observed in this DH population,with 34 (27.8%) markers showing significant deviation. It is noted that all markers on chromosomes 1,3,10 and 11 were biased to Peiai64s, while those on chromosomes 4,6,7,8 and 9 were opposite.

[Progress of the SUPERMAN epigenetic mutation in Arabidopsis].

The SUPERMAN gene in Arabidopsis has its epigenetic mutants (the clark kent alleles,clk). The phenotype of clk and its genotype and methylated patterns and the epi-mutation mechanisms of SUPERMAN were summarized in the review. Heritable but unstable sup epi-alleles are associated with nearly identical patterns of excess cytosine methylation within the SUP gene and a decreased level of SUP RNA. The methylation of cytosine at CpG and CPXPG is controlled by METHYLTRANSFERASE1(MET1) and CHROMOMETHYLASE3 (CMT3) which is regulated by KRYPTONITE gene, respectively.

[Genetic analysis on poly-row-and-branched spike mutant in barley].

In order to study the inheritance of poly-row-and-branched spike (PRBS) in barley, the row types of F1s, F2s, F3s of four crosses ( six-row x two-row, PRBS x two-row, PRBS x six-row and six-row x PRBS ) were investigated and analyzed. The results showed that one pair of recessive genes controls the PBRS mutant trait, which has recessive epistasis on the genes controlling two-row vs. six-row.