[Transcriptome profiling and analysis of Panax japonicus var. major].

The rhizome of Panax japonicus var. major have been used as the natural medicinal agent by Chinese traditional doctors for more than thousand years. Most of the therapeutic effects of P. japonicus var. major had been reported due to the presence of tetracyclic or pentacyclic triterpene saponins. In this study, Illumina pair-end RNA-sequencing and de novo splicing were done in order to understand the pathway of triterpenoid saponins in this species. The valid reads data of 15. 6 Gb were obtained. The 62 240 unigenes were finally obtained by de novo splicing. After annotation, we discovered 19 unigenes involved in ginsenoside backbone biosynthesis. Additionally, 69 unigenes and 18 unigenes were predicted to have potential function of cytochrome P450 and UDP-glycosyltransferase based on the annotation results, which may encode enzymes responsible for ginsenoside backbone modification. This study provides global expressed datas for P. japonicus var. major, which will contribute significantly to further genome-wide research and analysis for this species.

[A strategy based on comparative genomics to align ESTs of maize].

In this study, a new strategy to locate ESTs on maize linkage groups was described. In the strategy, the rice (Oryza sativa L.) genomic sequence database of was employed to locate maize EST on rice linkage groups, and then to locate on maize linkage group by comparative genetics mapping between rice and maize genome. The aligned ESTs information should available for further study on genomics and gene cloning. As an example, 139 ESTs of maize were assayed, and 96 maize ESTs (69%) were homologous with rice genomic sequence, 55% (77/139) ESTs were located on maize linkage groups based on the strategy, indicating that the locating approach of ESTs is feasible and available.

[QTL mapping of five agronomic traits in maize].

Agronomic traits have significant influence on stability and adaptability in maize production. In this investigation, using a population with 266 F2:3 families from Yuyu22 (Zong3 x 87-1), two-location field tests were conducted in Wuhan and Xiangfan in 2001, with a randomized complete block design, to characterize five agronomic traits: ear height, tassel branch number, stalk diameter, days to pollen, and days to silk. Correlation analysis of field performance indicated that ear height, tassel branch number and stalk diameter were significantly positive correlative with single-plant yield, days to pollen and days to silk were highly positive correlative with each other, and tassel branch number was significantly positive correlative with stalk diameter too. Utilizing data of field tests and molecular markers, Composite Interval Mapping (CIM) method was used to localize the quantitative trait loci of these traits and 500 times permutation test was conducted to have proper LOD threshold value. As the results, total seven QTL of ear height, nine QTL of tassel branch number, eight QTL of stalk diameter, nine QTL of days to pollen, and seven QTL of days to silk were mapped on 10 chromosomes of maize; all of these QTL distributed unevenly on chromosomes and trended to cluster together. According to analysis of this investigation, the phenotype correlations of quantitative traits may result from the correlations of QTL controlling those traits. Those will be helpful to further understand genetic basis of agronomic traits in maize.

[Isolation of maize genes related to aluminum tolerance].

To investigate gene expression profile in response to aluminum stress and to cloning the key genes related to aluminum tolerance, are crucial to genetic improvement of plant aluminum tolerance. In this study, suppression subtractive hybridization method was adopted to construct SSH-cDNA libraries at seedling stage of two maize inbred lines (Fig. 1), of which Mo17 is sensitive to aluminum toxicity and TL94B is tolerant. As a result, a forward SSH-cDNA library including 762 clones and a reverse SSH-cDNA library including 382 clones were constructed for Mo17. In the same way, a forward SSH-cDNA library including 760 clones and a reverse SSH-cDNA library including 380 clones were constructed for TL94B. Identification of these SSH-cDNA libraries shows that the length of inserted fragments ranges from 250 bp to 1.0 kb (Fig. 2), of which nearly 18% are positive clones. Through differential hybridization screening (Fig. 3), 124 and 47 positive clones were screened from forward and reverse SSH-cDNA libraries of Mo17 respectively; 103 and 64 positive clones from forward and reverse SSH-cDNA libraries of TL94B respectively. Total 338 positive clones from four SSH-cDNA libraries were sequenced, and all of the sequences of inserted fragments were analyzed using bioinformatical methods. A total of 232 kinds of EST sequences were obtained. Among these ESTs, 70.2% had significant homology with known genes, and the remaining 29.8% were function-unknown including 21 kinds of newly found ESTs (Table 1). An aluminum tolerant gene, GDP dissociation inhibitor gene, was detected its expression character by Northern hybridization (Fig. 4). These results indicate that the responses of maize to aluminum stress involve the interactions among different signal/metabolism pathways, such as signal transduction of stress-related factors, transcription and regulation of responsive genes, synthesis and transport of substances, changes in cell structures and functions.

[Construction of physical map and polymorphism analysis of mtDNA region R from Mo17CMS-J of maize].

Total DNA from twenty-six CMS lines of maize under Mo17, 77 and W23 nuclear background were used for PCR amplification, including N, T, C, S four groups of cytoplasms. The primers was prefabricated according the sequence of R region published by Zebala (1997). Through these amplifications, mitochondrial DNA fragments were obtained from maize total DNA. Generally the results in one group are identical. And they are different from the others. The amplified fragments were sequenced and also give us much more information about the structure of mitochondrial genes that may lead to CMS. In order to isolate and identify the CMS genes, we developed a new platform to construct physical map of chromosome DNA by means of restriction enzyme double-digestion. The elongation of contigs is based on Southern hybridization. Having retrieved DNA from agarose gel after electrophoresis by beta-agarase, we labelled it with 32P-dCTP as a probe. We detected the positive clones in the gene library. Two contigs were revealed. And a restriction map covering 40 kb was constructed, including R region.

[Genetic analysis of segregation distortion of molecular markers in maize F2 population].

A genetic linkage map of maize was constructed using 150 SSR and 24 RFLP markers, with F2 population from an elite hybrid (Zong3 x 87-1). Among 174 markers, covering whole maize 10 chromosomes, 49 markers (28.1%) showed the genetic distortion (P < 0.05). Of the total segregation distortion markers, 11 markers (22.5%) deviated toward male parent, Zong3, while 12 markers (24.5%) deviated toward female parent, 87-1, besides 25 markers (51.0%) distorted to heterozygote. Only one marker distorted to both parents. Totally, 14 segregation distortion regions (SDRs) were detected among 9 different chromosomes. Four of them were located in near regions where gametophyte genes were mapped, indicating that segregation distortion may be caused by gametophyte genes partially. Two segregation distortion regions, SDR6-1 and SDR7-1, detected in this study, seemed to be new segregation distortion regions. In this paper, reasons for segregation distortion and effects of segregation distortion on genetic mapping and QTL analysis were discussed. Regarding to QTL analysis with single locus, segregation distortion would not affect QTL mapping, but regarding to analysis of digenic interactions for epistasis, the fewer distortion markers and larger size population would be needed.

Expression analysis of orf77 and R region in mitochondrial DNA of S-type CMS maize.

It is generally recognized that S type of CMS in maize associated with the recombination region R in mitochondria. Complicated DNA recombination and changes of transcripts of R are observed in several subgroups of S cytoplasm. R region includes two open reading frames (orf355 and orf77) and they are all chemeric. Among the sequence of orf77, there are three stretches similar to atp9 of mitochondria. Many different S cytoplasms are found in maize in China and researches on them show that similar R region and sequence are also found in the mitochondrial DNA in Tangxu, Shuang and J (cytoplasm. Northern analysis of Tangxu, J cytoplasm with Probe R and orf77 indicates that orf77 co-transcribes with R region and the nuclear restorer gene Rf3 affects their expression. In all tested S cytoplasm plants with the genotype of s-rf3rf3, both probe orf77 and R can detect six transcripts of 2.8, 1.6, 1.1, 0.9, 0.7 and 0.4 kb. In the presence of the nuclear restorer-of-fertility gene Rf3, previous transcripts of 2.8 kb and 1.6 kb are disappeared, but the other four transcripts are not changed. In T and C cytoplasm of CMS, only four transcripts of 1.1, 0.9, 0.7 and 0.4 kb appear when hybrid to probe R. Further Northern analysis of probe atp9 proved that all the four transcripts of 1.1, 0.9, 0.7 and 0.4 kb were actually transcripts of gene atp9, so only the transcripts of 2.8 kb and 1.6 kb are particular to R region of S. In addition, atp9, atp6 and cox II each has the same transcription pattern in tassel with different genotypes respectively. It can be included that R region and orf77 are the most important candidate gene for S-CMS.

[Comparative analyses of QTL for important agronomic traits between maize and rice].

The objective of this study was to assess syntenic relationships of quantitative trait loci (QTL) for important agronomic traits between maize and rice based on the comparative genomic map of maize and rice using two F(2:3) populations. Through the comparisons, it was observed that there were extensive conserved relationships of maize QTL affected plant height, row number, and kernels per row with rice QTL affected plant height, tillers per plant, and grains per panicle respectively. Sixteen of 45 QTL affecting five different maize traits were conserved compared with 12 of 38 QTL affecting five different rice traits, which provided some useful information for locating, isolating and cloning maize QTL by using the rice genomic data. In this study, one QTL in rice usually had two conserved QTL in maize, further supporting the hypothesis that there is a polyploidization event during maize evolution. It was interested in observing that there were QTL rich regions on chromosomes in maize and rice, where QTL affecting different traits were usually clustered. These results revealed that the QTL affected the same or similar traits in maize and rice may have the common origin. These results will be helpful to map, isolate and clone QTL in large genome crops, such as maize, by using rice genome information, as well as to understand the evolutionary forces that structured the organization of the grass genomes.

Genetic analysis of the low critical sterility temperature point in photoperiod-thermo sensitive genic male sterile rice.

It has been a long haul but photoperiod- and thermo-sensitive genic male sterile (PTGMS) rice has not been freely used in hybrid rice production because there are two perplexing problems corresponding to the critical sterility temperature point (CSTP): the uncertainty of the CSTP segregating pattern and the instability of CSTP for every originally useful line. N5088S, the most widely commercialized japonica-type PTGMS line in China, also saw that its CSTP variants have been isolated but with all other agronomic characteristics unchanged. In this report we analyzed the genetic basis of CSTP, by employing the iterated expectation and conditional maximization (IECM) algorithm on four tiller-splitting-formed sets of seven generations from N5088S and its CSTP-variant H5088S, each set treated with one temperature regime. The main results indicated that there are two dominant major genes and polygene, as well as their respective epistasis conditioning the CSTP in the 23.5 degrees C regime. Based on the results obtained, the strategy for breeding of PTGMS lines with stable low CSTP was outlined.

[Progress of molecular biology of CMS in maize].

In the paper,we have summarized the molecular biological accomplishment acquired and accepted by most of maize researchers on CMS of maize. A brief review of current molecular biological progress of CMS of maize are displayed in the paper. These progresses concern in the positioning,cloning and maker-assisted selection of nucleic genes associated with fertility,expression and cloning of cytoplasmic genes associated with male sterility. In order to elucidate the molecular mechanism of CMS of maize, the areas about cloning and expression profiling of male sterile nucleic genes,and functional genomics of mitochondria,and interaction cytoplasmic genes with nucleic genes will need to be researched in the future.