Construction of a high density integrated genetic map for cucumber (Cucumis sativus L.).

The high-density consensus map was constructed based on the GY14 × PI 183967 map from an inter-subspecific cross and the extended S94 × S06 map from an intra-subspecific cross. The consensus map was composed of 1,369 loci, including 1,152 SSR loci, 192 SRAP loci, 21 SCAR loci and one STS locus as well as three gene loci of fruit external quality traits in seven chromosomes, and spanned 700.5 cM, of which 682.7 cM (97.5%) were covered by SSR markers. The average genetic distance and physical interval between loci were 0.51 cM and ~268 kbp, respectively. Additionally, the physical position of the sequence-associated markers aligned along the assembled cucumber genome sequence established a relationship between genetic maps and cucumber genome sequence and to a great extent validated the order of markers in individual maps and consensus map. This consensus map with a high marker density and well-ordered markers is a saturated and reliable linkage map for genetic analysis of cucumber or the Cucurbitaceae family of plants.

Testifying the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAgamma1.

The recessive gene xa5 for resistance to bacterial blight resistance of rice is located on chromosome 5, and evidence based on genetic recombination has been shown to encode a small subunit of the basal transcription factor IIA (Iyer and McCouch in MPMI 17(12):1348-1354, 2004). However, xa5 has not been demonstrated by a complementation test. In this study, we introduced the dominant allele Xa5 into a homozygous xa5-line, which was developed from a cross between IRBB5 (an indica variety with xa5) and Nipponbare (a japonica variety with Xa5). Transformation of Xa5 and subsequent segregation analysis confirmed that xa5 is a V39E substitution variant of the gene for TFIIAgamma on chromosome 5 (TFIIAgamma5 or Xa5). The rice has an addition gene for TFIIAgamma exists on chromosome 1 (TFIIAgamma1). Analysis of the expression patterns of Xa5 (TFIIAgamma5)/xa5 and TFIIAgamma1 revealed that both the genes are constitutively expressed in different rice organs. However, no expression of TFIIAgamma1 could be detected in the panicle by reverse transcriptase-polymerase chain reaction. To compare the structural difference between the Xa5/xa5 and TFIIAgamma1 proteins, 3-D structures were predicted using computer-aided modeling techniques. The modeled structures of Xa5 (xa5) and TFIIAgamma1 fit well with the structure of TFIIA small subunit from human, suggesting that they may all act as a small subunit of TFIIA. The E39V substitution in the xa5 protein occurs in the alpha-helix domain, a supposed conservative substitutable site, which should not affect the basal transcription function of TFIIAgamma. The structural analysis indicates that xa5 and Xa5 potentially retain their basic transcription factor function, which, in turn, may mediate the novel pathway for bacterial blight resistance and susceptibility, respectively.

[Generation of selectable marker-free and vector backbone sequence-free Xa21 transgenic rice].

The dominant gene Xa21 with broad-spectrum and high resistance to Xanthomonas oryzae pv. oryzae (Xoo) was transferred into C418, an important restorer line of japonica hybrid rice in China using double right-border (DRB) T-DNA binary vector through Agrobacterium-mediated transformation. 17 transgenic lines were Xa21-positive with high resistance to the race P6 of Xoo through PCR analysis and resistance identification, among the total 27 independent primary transformants (T0) obtained. The subsequent analysis of the T1 progenies of these 17 T0 lines through PCR-assisted selection and resistance investigation showed that four Xa21 transgenic T0 lines could produce selectable marker-free (SMF) progenies. The frequency of primary transformants producing SMF progenies was 15%. In addition, PCR analysis also revealed these SMF progenies did not contain vector backbone sequence, and they were named as SMF and vector backbone sequence-free (SMF-VBSF) Xa21 transgenic plants. The further molecular and phenotypic analysis of the T2 and T3 progenies testified the homozygous SMF-VBSF Xa21 transgenic plants were obtained with high resistance to Xoo.

[Cloning and analysis of a new NBS-LRR resistance gene family in rice].

Sequence-based gene isolation has been a practical approach for plant resistance gene cloning. In this study, RS13, a cloned rice sequence with the NBS (nucleotide-binding site) domain of resistance genes, was used as a probe to screen a bacterial artificial chromosome (BAC) library of rice variety IR64,and four positive clones were obtained. Of them the clone 14E19 covered the other three clones and was sequenced through a shotgun approach. The whole sequence of the insert fragment of 14E19 was assembled into approximately 73 kb in length. Genes on the whole assembled sequence were predicted,and four genes encoding NBS and LRR (leucine-rich repeat) domains were found, named as NL-A, B, C and D respectively. For further analysis, another longer BAC clone,106P13, covering 14E19 on the same chromosome position was identified from a BAC library of IRBB56 which had the same genome background with IR64. Ten NL-homologous copies were discovered on the sequence of the BAC clone 106P13, and four copies were identical with those on 14E19. The similar homologous sequences were also found in the genomic sequences of Nipponbare,93-11 and Guangluai4. However, NL sequences were less homologous with the known NBS-LRR resistance genes. This result indicated that NL was a new NBS-LRR gene family and was composed of ten members at least. RT-PCR and cDNA screening displayed that NL-B expressed in a bacterial blight-resistant rice variety IRBB4, indicating the gene was possibly involved in resistance reactions.

[Identification and marker-assisted selection of two major quantitative genes controlling rice sheath blight resistance in backcross generations].

We constructed an F2 clonal population of intercross,Teqing/Lemont, and identified two quantitative trait loci (QTLs) contributing to rice sheath blight resistance on chromosome 9 and 11. The two QTLs were qSB-9 and qSB-11, respectively. From the population, three clonal lines were selected by markers' band types on both sides of these two QTLs, qSB-9 and qSB-11. Two were double-susceptible parent with homozygous susceptible alleles of these two loci,and the other was named as double-resistant parent,of which these two loci were all homozygous resistant alleles. These parents were separately backcrossed to recurrent parents, Teqing or Lemont. From BC2F1, marker-assisted selection was conducted in each proceeding generation and all back-crossed plants in BC2F1 and BC4F1 were inoculated by short toothpicks incubated with a strain, RH-9 of the fungus for identification of the resistance. Results suggested that these two QTLs were selected effectively in each backcross generation and their positions were also verified in identification of resistance to rice sheath blight. In seedling nursery of BC3F2 population, plants were selected through marker-assisted selection, and were separately mixed as homozygous lines of double-susceptible alleles on the background of Teqing, double-susceptible and double-resistant on the background of Lemont. The homozygous lines and their recurrent parents were simultaneously planted on experiment fields of Agriculture Collage of Yangzhou University and Lixiahe District Institute of Agricultural Science. The inoculation was performed by a random-block test with two replicates at each site. The results indicated that 1) The difference of sheath blight disease development was highly significant among materials under the same genetic background,and the order of disease seriousness among different homozygous lines were: double-susceptible line on the background of Lemont > double-susceptible line on the background of Teqing > Lemont > Teqing > double-resistant line on the background of Lemont; 2) When the resistant allele of qSB-9 or qSB-11 solely existed in a plant, its disease rating was reduced about 1.2 score, and 2.0 score when they simultaneously existed on the background of Lemont; 3) No significant interaction between the two QTLs controlling sheath blight resistance and environments was found. These studies have laid a strong groundwork in operation and application, of these QTLs contributing to rice sheath blight resistance.

Cloning, sequence and expression analysis of a zinc finger protein gene in wheat.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed on total RNA of resistant-line TAM104R and susceptible-line TAM104S using 33 primers designed according to conservative domain of plant R-genes and the sequence of barley powdery mildew resistance genes Mlo, Mla1, Mla6 and wheat leaf rust resistance gene Lrk10. A polymorphic cDNA fragment TaZF was cloned and sequenced. The Open Reading Frame (ORF) of TaZF is comprised of 822 base pairs which encodes a zinc finger-like DNA or RNA-binding protein with 273 amino acids and molecular weight of 31 kD. TaZF is more than one copy gene in TAM104R genome based on southern blot. It is constitutively expressed, but level of expression was enhanced in tissue infected by Erysiphe Graminis. There is no intron in TaZF genome DNA.

Genetic dissection for leaf correlative traits of rice (Oryza sativa L.) under drought stress.

Water is becoming a restricted factor of agricultural development owing to the global shortage of water resources. Screening and improving drought tolerant rice cultivars would be helpful for increasing and stabilizing yield, economizing water and reducing environmental pollution. In this study, 127 rice lines of DII population derived from an indica variety Zhaiyeqing 8 (ZYQ8) and a japonica variety Jingxi 17 (JX17) were used to locate QTLs for leaf rolling, relative water content and rate of electric conductivity under drought stress. The results showed that significant differences between the parents were detected for all measured traits. The tremendous transgressive segregations for these traits were observed in the population. The frequency of all traits in the population was approximately normally distributed with slight skew. A total of six QTLs for the three traits were detected with molecular linkage map of 234 markers, including three QTLs (qLR-1, qLR-5 and qLR-11) for leaf rolling, two QTLs (qRWC-1 and qRWC-6) for relative water content and one QTL (qREC-6) for rate of electric conductivity. Visual measurement for leaf rolling can be used to screen a large number of rice germplasm resources or varieties, which is of importance to screening and utilization of drought tolerant rice varieties.

[Genetic dissection of cooked rice elongation in rice (Oryza sativa L.)].

QTLs for milled rice length (MRL), cooked rice length (CRL) and cooked rice elongation (CRE) were identified by using a population of 127 DH lines derived from a cross between ZYQ8 and JX17. Totally, 14 QTLs for rice elongation traits were detected on chromosomes 1, 2, 3, 5, 6, 7, 10, 11 and 12. Two putative QTLs for MRL were mapped on chromosomes 2. Seven putative QTLs for CRL were mapped on chromosomes 1, 6, 7, 10, 11 and 12. Five putative QTLs for CRE were mapped on chromosomes 5, 6, and 10. The regions of G249-G164 on chromosome 3, G30-RZ516 on chromosome 6 and G1082-GA223 on chromosome 10 were detected simultaneously for affecting cooked rice length and cooke rice elongation. LODs of the QTLs related to rice elongation varied from 2.26 to 9.25, and their explained variations from 5.31% to 17.21% . It is indicated that cooked rice elongation was controlled by polygene and Wx-gene located on the same region with qCRE-6 was important to cooked rice elongation.

[Marker-assisted selection and pyramiding for three blast resistance genes, Pi-d(t)1, Pi-b, Pi-ta2, in rice].

G46B is a promising holding line used for three-lines breeding strategy in hybrid rice, but it is susceptible to blast disease caused by Pyricularia grisea. To improve its blast resistance, three rice varieties, Digu, BL-1, and Pi-4, with blast resistance genes, Pi-d(t), Pi-b, and Pi-ta2, respectively, were used to be crossed with G46B, and 15 plants with these three blast resistance genes, Pi-d(t)1, Pi-b, and Pi-ta2, were selected from their F2 and B1C1 populations via a marker-aided crossing procedure. Among them, four plants were heterozygotes in the three resistance genes, with the genotype of Pi-d(t)1 pi-d(t)/Pi-b pi-b/ Pi-ta2 pi-ta2; ten plants were heterozygotes in two of the three resistance genes, of which six with the genotype of Pi-d(t)1 Pi-d(t)1/Pi-b pi-b/Pi-ta2 pi-ta2, three with the genotype of Pi-d(t)1 pi-d(t)1/Pi-b pi-b/Pi-ta2 Pi-ta2, and one with the genotype of Pi-d(t)1pi-d(t)1/Pi-b Pi-b/Pi-ta2 pi-ta2; and only one plant was homozygote in two of the three resistance genes with the genotype of Pi-d(t)1 Pi-d(t)/Pi-b pi-b/Pi-ta2 Pi-ta2. These results demonstrate the capacity of maker-assisted selection (MAS) in gene pyramiding for rice blast resistance and its enhancement for the efficiency in rice resistance breeding.

[Analysis of the transgenic rice plants derived from transformed anther calli].

Rice calli derived from anther culture were used as recipient to transfer a rice blight resistance gene, Xa21, into a japonica rice variety, Taipei 309, via Agrobacterium-mediated transformation. Seven green transgenic plants, including one mixoploid, two haploid, and four diploid plants, were regenerated. PCR, Southern blot, FISH and blight resistance analysis all indicated that Xa21 gene has been integrated into the T0 plant genomes. T1 generations of the four diploid T0 plants were further investigated for resistance segregation. Chi2 test showed that two T1 populations segregated with a ratio of 3:1, indicating that a single copy of Xa21 gene was integrated into the genome, whereas the segregation ratios of the other two T1 populations were non-Mendelian. Therefore, the four diploid transgenic plants should be heterozygous diploids.

[The thirty years of Acta Genetica Sinica].

Acta Genetica Sinica (AGS) is sponsored by the Genetics Society of China and the Institute of Genetics and Developmental Biology of Chinese Academy of Sciences, and is published by Science Press. The journal is a leading national academic periodical and one of the Chinese key periodicals of natural sciences. Currently, AGS is being indexed by several well-known domestic and international indexing systems, such as the American Chemical Digest (CA), BIOSIS database, Biological Digest (BA), Medical Index and Russian Digest (P [symbol: see text]). Papers in the areas of genetics, developmental biology, cell molecular biology and evolution are regularly published by AGS.

Characterization of QTLs for harvest index and source-sink characters in a DH population of rice (Oryza sativa L.).

A DH population containing 81 DH lines from an indica-japonica cross of rice and an RFLP linkage map consisting of 232 markers were used to map quantitative trait loci(QTLs) for harvest index, biomass, grain yield, sink capacity and plant height by a computer program QTLMapper1.0 based on mixed linear models. A total of 21 significant main-effect QTLs and 9 pairs of epistatic loci were detected. Of these, three detected QTLs for grain yield collectively accounted for 42% of the phenotypic variation with a LOD of 7.10. These three grain yield QTLs were corresponded either to QTLs for harvest index or QTLs for biomass in both locations and directions of additive effects, which sheds light on the genetic basis of the formula 'grain yield = biomass x harvest index'. Four detected QTLs for harvest index collectively explained 46% of the total phenotypic variation and four QTLs for biomass jointly accounted for 64% of the trait variation. No coincidence of harvest index QTLs with any biomass QTLs was found, therefore indicating the possibility of pyramiding favorable alleles for both traits through gene recombination so as to obtain a genotype possessing both high harvest index and heavy plant biomass. Five QTLs for plant height were detected that cumulatively explained 64% of the phenotypic variation with a LOD of 11.62. Among these, three with smaller effects respectively co-located with some of the QTLs for biomass, sink capacity and/or grain yield, but not with any of harvest index QTLs, thus suggesting that plant height was to some extent directly associated with 'source' and 'sink' but not with 'transportation' of the 'source-transportation-sink' concept, at least in this genetic background and environment. In view of a somewhat low resolution of the genetic map used in the study and the fact that when plant height QTLs co-located with those for yield and/or yield related traits, these co-located QTLs were all in the same directions of additive effects, it is more likely that these QTLs co-located in a same chromosomal region might be a single QTL which have effects on multiple traits. If this is true, the above observation have led us to assume that QTLs which have pleotropic effects on yield and/or yield related traits and plant height are very different from those which had relatively large effects only on plant height. The former contribute strongly to yield and/or yield related traits but weakly to plant height while the later contribute mainly to plant height. Obviously, due to that an increase of plant height is always coupled with an increase in lodging susceptibility, discriminating between above two types of QTLs is critical in breaking the traits' undesired association in breeding for improved yield potential of rice. In addition, based on the co-location analysis of main-effect QTLs for the studied traits, five genomic regions were found to be highly associated with harvest index, biomass, sink capacity and grain yield.

[Genetic analysis and molecular tagging of a gene for non-palea in rice].

The mutants involved in the development of floral organ are good material for understanding the molecular genetic mechanisms of floral development. A rice mutant, that lacks palea in its florets, was derived from a spontaneous mutation in an indica line, SAR III-93-369. Genetic analyses in three F2 populations from the mutant crossed with three rice varieties, Sheng 47, N625 and CDR22, respectively, showed that the mutant trait is controlled by a single recessive gene. In the F2 population from npa-1/Sheng47 the gene for the non-palea trait was mapped between two restriction fragment length polymorphism markers, C498 and RZ450, with distances of 7.5 cM and 2.4 cM, respectively. The tagged recessive non-palea gene is temporarily designated npa-1.

[Analysis of gene loci and epistasis for drought tolerance in seedling stage of rice (Oryza sativa L.)].

Drought tolerance of rice is important because a considerable proportion of the world rice area is not irrigated and is prone to water deficit. In this study, an indica variety, Zhai Ye Qing 8 (ZYQ8), and a japonica variety, Jing Xi 17 (JX17), and their double haploid (DH) population were used for genetic study of drought tolerance. Water supply was stopped in seedling period for 15 days and then drought tolerance of the DH population and their parents were investigated. Mapping quantitative trait loci (QTLs) was undertaken base on the constructed molecular linkage map of this population. Two QTLs (qDR-5 and qDR-12) for drought tolerance were identified, they were in the region of GA41-GA257 on chromosome 5 and RG457-Y12817R on chromosome 12, respectively. The tolerance alleles of both QTLs were from the indica parent, ZYQ8. In the meantime two genes for drought tolerance near GA257 and Y12817R were detected too by using Epistat software, that is in accordance with the result by using Mapmaker/QTL. In addition, three loci (RG541, G318 and G192 on chromosome 1, 4 and 8, respectively) were found interacting with GA257 by Epistat software, while one locus (CT234 on chromosome 3) found interacting with Y12817R were also detected by Epistat software.

Molecular linkage maps of the Populus genome.

We report molecular genetic linkage maps for an interspecific hybrid population of Populus, a model system in forest-tree biology. The hybrids were produced by crosses between P. deltoides (mother) and P. euramericana (father), which is a natural hybrid of P. deltoides (grandmother) and P. nigra (grandfather). Linkage analysis from 93 of the 450 backcross progeny grown in the field for 15 years was performed using random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), and inter-simple sequence repeats (ISSRs). Of a total of 839 polymorphic markers identified, 560 (67%) were testcross markers heterozygous in one parent but null in the other (segregating 1:1), 206 (25%) were intercross dominant markers heterozygous in both parents (segregating 3:1), and the remaining 73 (9%) were 19 non-parental RAPD markers (segregating 1:1) and 54 codominant AFLP markers (segregating 1:1:1:1). A mixed set of the testcross markers, non-parental RAPD markers, and codominant AFLP markers was used to construct two linkage maps, one based on the P. deltoides (D) genome and the other based on P. euramericana (E). The two maps showed nearly complete coverage of the genome, spanning 3801 and 3452 cM, respectively. The availability of non-parental RAPD and codominant AFLP markers as orthologous genes allowed for a direct comparison of the rate of meiotic recombination between the two different parental species. Generally, the rate of meiotic recombination was greater for males than females in our interspecific poplar hybrids. The confounded effect of sexes and species causes the mean recombination distance of orthologous markers to be 11% longer for the father (P. euramericana; interspecific hybrid) than for the mother (P. deltoides; pure species). The linkage maps constructed and the interspecific poplar hybrid population in which clonal replicates for individual genotypes are available present a comprehensive foundation for future genomic studies and quantitative trait locus (QTL) identification.

[Genome analysis of transgenic homozygous line "Minghui 63-Xa21"].

By using rice SSRP, RAPD and AFLP molecular markers, the genome of rice transgenic line "Minghui 63-Xa21" was analyzed. 32 SSRP primers, 42 RAPD primers and 8 AFLP primers could produce obvious PCR bands in the analysis of at least 12 individual plants selected randomly from "Minghui 63-Xa21" T3 generation. Totally 550 PCR bands, equivalent to 550 genomic sites, were detected. Different individual plants of the transgenic homozygous line displayed almost the same PCR pattern. Compared with the control "Minghui 63", no difference was found in their PCR patterns. This indicated that the introduction of Xa21 into the genome of "Minghui 63" did not change these 550 genome sites and their heredity. Very few variant PCR bands were observed in some individual plants from both "Minghui 63-Xa21" and "Minghui 63". However, the variant percentage was equivalent between the transgenic line and the non-transgenic control line.

[QTL analysis of sheath blight resistance in rice (Oryza sativa L.)].

In this study, an indica variety Zhai Ye Qing 8 (ZYQ8) and a japonica variety Jing Xi 17(JX17) and their double haploid (DH) population were used. Sheath blight resistance of the DH population and their parents was investigated using a syringe inoculation method. Quantitative trait loci (QTL) analysis was undertaken based on a constructed molecular linkage map of this population in Hangzhou and Hainandao respectively. A total of four sheath blight resistant QTLs (qSBR-2, qSBR-3, qSBR-7 and qSBR-11) were identified, and located on chromosome 2, 3, 7 and 11, respectively. Resistant alleles of qSBR-2, qSBR-3, and qSBR-7 were derived from the resistant parent ZYQ8, whereas resistant allele of qSBR-11 from susceptible parent JX17. qSBR-2, qSBR-3 and qSBR-7 were detected both in Hangzhou and Hainandao, whereas qSBR-11 was detected only in Hangzhou. Sheath blight response was significantly correlated with culm length and heading date in Hangzhou's test. Among the QTLs for culm length and heading date, only qCL-3 for culm length, was in the same chromosome region of qSBR-3, the other QTLs were not linked to the QTLs for sheath blight resistance.

[Progress of molecular floral development research in rice].

Rice (Oryza sativa L.) is not only one of the most important food crops in the world,but also a model plant for study of molecular developmental biology in monocots. In addition, the rice floral organs provide the basis for grain formation. Study of rice floral development has become a new focus of plant molecular genetics. Recently, notable progress has been made in study of gene regulation in rice floral development. In the review, genetic and molecular mechanisms of floral induction, floral meristem formation, and floral organ development in rice are summarized.

[Identification of markers linked to resistance locus of Marssonina leaf spot in poplars by bulked segregant analysis(BSA)].

DNA markers linked to resistance locus of Marssonina leaf spot in poplars were found by bulked segregant analysis(BSA). The bulks consisted of individual with a extreme phenotype taken from a population of 91 F1 clones,which is a progeny of Populus deltoides Bartr.cv."Lux"(I-69/55)(Resistance) and P.euramericana cv.I-45(Susceptible). Out of 114 RAPD primers, four markers showed polymorphisms between the resistance-bulk and the susceptible-bulk.By using selective genotype linkage analysis,OPAI17-1550 and OPAI13-900 were found linked to the resistance locus. The genetic distances between the two markers and the resistance locus were 29.9cM and 37.4cM,respectively.

[Molecular mapping of quantitative trait loci (QTL) for characters of vascular bundles in peduncle related to indica-japonica differentiation in rice (Oryza sativa L.)].

A doubled-haploid population, consisting of 81 DH lines derived from the F1 hybrid of a cross between an indica cultivar and a japonica cultivar, was used to map quantitative trait loci (QTL) controlling numbers of vascular bundles in peduncle, primary rachis branches and the ratio of vascular bundles to primary rachis branches (V/R ratio). For vascular bundles, three QTL were detected. Among them, the QTL qVB-8 with the largest effect individually accounted for 31.1% of the total variation. Two QTL controlling primary rachis branches were identified and they were co-located with 2 of the 3 QTL for vascular bundles respectively. Three QTL for the V/R ratio were mapped on chromosome 1, 2 and 8, respectively. Of the three QTL, the QTL qV/R-1 with the largest additive effect, explained 25.3% of the total variation, was located on chromosome 1 and found to be closely linked to the gene sh-2, a major gene underlying grain-shattering ability. In addition, four and two pairs of significant epistatic QTL were detected for vascular bundles and the V/R ratio, respectively, but none for rachis branches. Our results suggested that the numbers of vascular bundles and primary rachis branches were independently controlled by different polygenic systems, but the two polygenic systems shared a fraction of quantitative trait loci. It was also shown that the chromosome region carrying the QTL qV/R-1 on chromosome 1 might play an important role in the processes of indica-japonica differentiation in rice.