Molecular verification of the integration of Tripsacum dactyloides DNA into wheat genome through wide hybridization

RAPD and RFLP analyses of double haploid lines which derived from hybridization between hexaploid wheat (Triticum aestivum L.2n=42) and eastern gamagrass (Tripsacum dactyloides L.2n=4x=72) are reported.Two of the 340 Operon primers have been screened,which stably amplified Tripsacum dactyloides (male parent) specific bands in the double haploid lines.These results confirm the fact that Tripsacum dactyloides DNA has been integrated into wheat genome by sexual hybridization at molecular level.This idea has been further testified by RFLP analysis.Application and potentials of transferring Tripsacum dactyloides DNA into wheat genome by sexual hybridization in wheat breeding are discussed.     

Breeding and molecular cytogenetic identification of wheat-Thinopyrum intermedium addition lines resistant to powdery mildew

Wheat-related species Th. intermedium was used to cross with common wheat Yannong 15. In the self progenies of the hybrid, two addition lines, Ⅱ-1-7-1 and Ⅱ-3-3-2, stable in cytology, were developed by cytology and powdery mildew resistance identification. Their chromosome number were 2n = 44 and formed 22 bivalents at PMC MI. In F₁ of the two addition lines crossing with Yannong 15, there appeared about one univalent at PMC MI, respectively. Resistance identification in greenhouse and field using the No. 15 and mixed strains of E. gramnis f. sp. tritici showed that they were immune to powdery mildew. Chromosome number and resistance identification using the F² single plants of the addition line crossing with Yannong 15 indicated that the resistant gene was located on the alien chromosomes. In situ hybridization using St and E genomic DNA as probe showed that the added chromosome in the two addition lines probably came from the E genome of Th. intermedium, which indicated that a pair of E genome chromosomes carried a new resistant gene to powdery mildew.     

Physical location ofHelminthosporium carbonum susceptibility gene hm1 by FISH of a RFLP marker umc119 in Maize

A fluorescencein situ hybridization (FISH) procedure was adopted to physically map a RFLP marker, umc119 near the centromere of the long arm of linkage group1 in maize. The hm1 gene (Helminthosporium carbonum susceptibility gene) was linked closely with the marker umc119. RFLP markers are very good landmarks for mapping genes. Therefore, we also determined the position of the gene hm1 on the chromosome based on the physical location of umc119. The disease induced by infection ofHelminthosporium carbonum is one of the serious maize diseases and it distributes in many countries including China. Hybridization sites were showed on 1 L (long arm of chromosome1) and 5 L. The percentage distance from centromere to the hybridization site was 22.86 on 1 L and 58.23 on 5 L the detection rate was about 12% for mitotic cells. In interphase nuclei five hybridized sites were detected. It demonstrated that umc119 was multiplicated sequences. FISH has more advantages overin situ hybridization (ISH) detected by DAB for increasing the detection ratio and contrast between chromosomes and hybridization signals. The ability to detect the hybridization signal of a small low copy DNA sequence is a very important key towards wide application of FISH for plant genome mapping. Supported by the National Natural Science Foundation and Doctorate Vesting Point Foundation of the Education Committee of China Li Lijia: born in 1967. Ph. D.     

Haploidy or diploidy: which is better?

Although the evolutionary advantages of sexual reproduction have been extensively discussed, much less attention has been paid to haploid and diploid phases of the sexual life cycle. The relative lengths of these phases differ greatly in various taxa, including as extremes those with one or the other phase reduced to a single cell. Here we consider the efficiency of elimination of deleterious mutations as an evolutionary force and compare the mutation loads under haploid and diploid selection, Ln and L2n. With truncation-like selection, partial dominance, and heterozygous effect of a mutation less than about 1/4 its hemizygous effect, L2n less than Ln; otherwise L2n greater than Ln. The difference becomes important when the genomic deleterious mutation rate exceeds about 1 per genome. This suggests that the mutation rate, degree of dominance and mode of selection can be important in life-cycle evolution.     

Molecular cloning, chromosomal mapping and expression analysis of disease resistance gene homologues in rice (Oryza sativa L.)

Resistance-like sequences have been amplified from first strand cDNA and genomic DNA of rice by PCR using oligonucleotide primers designed from sequence motifs conserved between resistance genes of tobacco andArabidopsis thaliana. 3 PCR clones, designatedOsr1, Osr2 andOsr3 which were 98% identical in nucleotide sequence level, have been found to be significantly homologous to known plant resistance genes and all contained the conserved motifs of NBS-LRR type resistance genes, such as P-loop, kinase2a, kinase3a and transmembrane domain.Southern hybridization revealed that rice resistance gene hornologueswere organized as a cluster in the genome. RFLP mapping using a DH population derived from anindica/japonka cross (Zhaiyeqing 8/Jingxi 17) and an RFLP linkage map assigned two copies ofOsrl and one copy ofOsr3 to the distal position of chromosome 12 where a blast resistance QTL has been mapped previously. Northern blot analysis showed thatOsrl gene was constitutively transcribed in rice leaves, shoots and roots. Further study concerning isolation of full-length cDNAs would be conducive to elucidating the functions of these genes.     

Breeding of T. aestivum-Ag. intermedium translocation lines with T. timopheevii cytoplasm and characterization by GISH

A male-sterileT. aestivum-Ag. intermedium partial amphiploid with cytoplasm ofT. timopheevii as a female parent was crossed to common wheat. The hybrid was backcrossed to the male parent several times continually and setf-crossed at last. Two stable lines with common wheat phenotype, H96269-2 and H96278, have been obtained. The chromosome numbers of the two lines are all2n = 42 in somatic cetls. By inoculation test, the two lines show a high levet of resistance to yetlow rust. Through genomicin situ hybridization (GISH) withAg. intermedium total genomic DNA as a probe, it is demonstrated that the two stable lines are all small segmental translocation lines, and the translocated chromosome segments fromAg. intermedium are located on the short arm terminals of wheat chromosomes. Genetics analysis suggests that the yetlow rust resistance gene(s) are probably located on the translocated chromosome segments ofAg. intermedium.     

Analysis of DNA methylation variation in wheat genetic background after alien chromatin introduction based on methylation-sensitive amplification polymorphism

During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH （genome in situ hybridization） and AFLP （amplified fragment length polymorphism） on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism （MSAP） analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines （CN12, 20.15%; CN17, 20.91%; CN18, 22.42%）, but the ratios of hemimethylated sites were significantly lowered （CN12, 21.41%; CN17, 23.43%; CN18, 22.42%）, whereas 16.37% were fully-methylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns （33.74%）, 9 demethylation patterns （22.76%） and 7 uncertain patterns （4.07%）. In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.     

Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

The use of plants as bioreactors for the expression oftherapeutic proteins has developed into a new field in biotechnology research[1]. Plant systems provide simple genetic manipulation, low production costs, and low risk of contamination by animal viruse…     

Starch RVA profile parameters of rice are mainly controlled byWx gene

Quantitative trait locus (QTLs) mapping for rapid visco analyser (RVA) profile parameters has been carried out by using a double haploid (DH) population derhred from a cross betweenindica variety Zhai-Ye-Qing 8 andjaponica variety Jing-Xi 17 and its genetic linkage map. The results indicate that the segregation of the RVA profiles is continually distributed among the DH lines, and some DH lines show transgressive segregation for all the parameters. A major QTL,Waxy (Wx) gene on chromosome 6 which controls the amylose synthesis, has been detected significantly for 5 traits: hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), consistency viscoslty (CSV) and setback viscoslty (SBV). Therefore, the RVA profile parameters are mainly controlled byWx gene. Other 3 and 2 QTLs have also been identified for BDV and SBV, respectively, and two of them share the same region on chromosomes 1 and 5. However, the peak viscosity (PKV) is controlled by a minor QTL on chromosome 12, qPKV-12.     

Microdissection of chromosome 7B of common wheat and cloning of low-copy specific DNA sequences

The 7B chromosome of common wheat was microdissected from pollen mother cells of the 7B monosomic line of common wheat cv. Chinese Spring (CS). After proteinase K and DNA topoisomerase Ⅰ treatments, the isolated chromosomes were subjected to 1-3 rounds of DOP-PCR amplification, which produced continuous DNA fragments ranging from 150 to 700 bp. Ge-nomic Southern hybridization confirmed that the PCR products were originated from the wheat genome. Cloning of portion ( > 200 bp) of the 3rd round DOP-PCR products (50 μL) could generate about 20 000 recombinant clones. Characterization of 50 randomly chosen clones indicated that 21 clones produced discrete PCR products with the size of 240-600 bp. Dot-blot hybridization showed that among the 21 clones, 11 ( ～ 55%) were of low-copy nature while 10 ( ～ 45%) were repetitive. Southern hybridization with the complete set of the CS "nullisomic-tetrasomic (NT)" lines demonstrated that all the 6 low-copy clones were specific to either chromosome 7B or the 7th homoeologous group, whereas the 3 arbitrarily chosen repetitive clones were non-specific, disperse sequences.     

Construction of a BAC library from cucumber (Cucumis sativus L.) and identification of linkage group specific clones

A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94; derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen markers gave at least two positive clones. As a result, 32 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.     

Isolation and characterization of soybean NBS analogs

Isolation of plant resistance genes is greatly helpful to crop resistance breeding and the insight of resistance mechanism. The cloned plant resistance genes are classified into four classes according to their putative structural domain, of which the majority possesses nucleotide-binding site (NBS) domain that consists of P-loop, kinase2a and kinase3a. The conservation of this domain affords the potential possibility of cloning the plant resistance genes, which is homology-based cloning technique. In the present study, the degenerate oligonucleotide primers were designed according to the tobaccoN andArabidopsis RPS2, and 358 clones were isolated from the genomic DNA of resistance soybean cultivar Kefengl, resistant to soybean mosaic virus, and 4 open-reading NBS analogs were finally characterized and designated asKNBS1, KNBS2, KNBS3 andKNBS4. Southern hybridization suggested that they were present with multicopy in the soybean genome;KNBS4 was mapped to F linkage group andKNBS2 co-located J linkage group with the SCAR marker ofRsa resistant to soybean mosaic virus by RFLP analysis. Northern analysis suggested thatKNBS2- related sequence was low and constitutively expressed in the root, stem and leaves of soybean. The detailed characterization of NBS analogs is very helpful to ultimately cloning the soybean resistance gene.     

Construction of a BAC library from cucumber (Cucumis sativus L.) and identification of linkage group specific clones

A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94; derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each linkage group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen markers gave at least two positive clones. As a result, 32 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.     

Breeding and molecular cytogenetic identification of wheat-Thinopyrum intermedium addition lines resistant to powdery mildew

Wheat-related species Th. intermedium was used to cross with common wheat Yannong 15. In the self progenies of the hybrid, two addition lines, Ⅱ-1-7-1 and Ⅱ-3-3-2, stable in cytology, were developed by cytology and powdery mildew resistance identification. Their chromosome number were 2n = 44 and formed 22 bivalents at PMC MI. In F1 of the two addition lines crossing with Yannong 15, there appeared about one univalent at PMC MI, respectively. Resistance identification in greenhouse and field using the No. 15 and mixed strains of E. gramnis f. sp. tritici showed that they were immune to powdery mildew. Chromosome number and resistance identification using the F2 single plants of the addition line crossing with Yannong 15 indicated that the resistant gene was located on the alien chromosomes. In situ hybridization using St and E genomic DNA as probe showed that the added chromosome in the two addition lines probably came from the E genome of Th. intermedium, which indicated that a pair of E genome chromosomes carried a new resistant gene to powdery mildew.     

Size variation in chromosomes from independent cultured isolates of Plasmodium falciparum

The complexity of the life cycle of the protozoan malaria parasite Plasmodium falciparum has hindered genetic analysis; even the number of chromosomes in P. falciparum is uncertain. The blood stages of rodent malaria parasites are haploid and hybridization with cloned complementary DNAs similarly suggests a haploid genome in P. falciparum blood stages (ref. 4 and our unpublished results). A novel approach to karyoptic and linkage analysis in P. falciparum has been provided recently by the technique of pulsed-field gradient (PFG) gel electrophoresis, which allows the fractionation of DNA molecules of 30-3,000 kilobases (kb), a range including the sizes of intact chromosomal DNA molecules from eukaryotes such as yeast and trypanosomatids. We describe here the fractionation by PFG electrophoresis of chromosomal DNA molecules from P. falciparum into at least seven discrete species which vary in size by up to 20% between different isolates. Several genes for P. faciparum antigens which contain repetitive sequences are located on different chromosomes. Surprisingly, two of the chromosomes seem to contain the same sequences.     

Construction of a genetic map and location of quantitative trait loci for dwarf trait in maize by RFLP markers

Using F₂ population derived from the cross of tall inbred 7922 by dwarf inbred 5003, an RFLP linkage map of maize has been constructed, on which 85 markers are distributed among 10 linkage groups and span maize genome about 1827.8 cM with an average distance (24.4 cM) between markers. 106 F_2:3 lines of the population were grown in a 10 × 11 simple rectangular lattice design of one-raw plots with two replications and evaluated for plant height (PH). With interval mapping procedure, 5 QTLs controlling plant height have been identified and their genetic effects and gene action determined. 2 major QTLs with opposite effect have been discovered. One for increasing plant height isph1 which is located at chromosome 2 and accounts for 51.8% of the total phenotypic variation; the other for decreasing plant height isph3 which is located at chromosome 5 and accounts for 38.6% of the total phenotypic variation. The chromosomal location ofph3 might be the same as or close to the position ofbv1, a dwarf mutant of maize.     

Analysis of F1 hybrid and BC1 monosomic alien addition line plants from Brassica oleracea × Sinapis alba by GISH

Sterile and semi-fertile F₁ plants were obtained by intergeneric sexual hybridization between paternal Brassica oleracea var. alboglabra (genome CC, 2n=18) and maternal Sinapis alba (genome SS, 2n=24), BC₁ plants were obtained by backcrossing between paternal B. oleracea and maternal semi-fertile F₁ plants. Genomic in situ hybridization (GISH) combined with dual-colour fluorescence in situ hybridization (dcFISH) showed that sterile F₁ plants contained 21 chromosomes consisting of one B. oleracea chromosome set and one S. alba chromosome set, belonging to expected hybrids, and semi-fertile F₁ plants contained 30 chromosomes consisting of two B. oleracea chromosome sets and one S. alba chromosome set. It is obvious that the semi-fertile F₁ plants belong to unexpected hybrids. 1―3 trivalents were detected at meiotic metaphase I of semi-fertile F₁ pollen mother cells (PMCs). Different separation ratios of S chromosomes were detected at anaphase I. A monosomic alien addition line (MAAL) was identified by GISH-dcFISH from BC₁ plants; it contained 19 chromosomes consisting of 18 C chromosomes and 1 S chromosome. At meiotic metaphase I, 9 divalents from B. oleracea and one univalent from S. alba could be detected. Sometimes, one putative C-S trivalent could also be detected. The achievement of B. oleracea-S. alba monosomic alien addition lines lays a foundation for gene introgression, location and cloning.     

浅谈叶绿体基因组分析技术在植物系统学中的应用

本文对植物叶绿体基因组做一简介,包括叶绿体基因组的发现、结构与特点、编码基因、进化特点和遗传方式。综述了DNA杂交、DNA限制酶谱分析、RFLP分析、PCR-RFLP分析、微卫星序列分析、SSCP分析和核酸序列分析等叶绿体DNA分析技术的原理、特点及应用。阐述了叶绿体DNA在植物系统学研究中广泛应用的利弊。     

Testes and chromosomes in interspecific hybrids betweenHelicoverpa armigera (Hübner) andHelicoverpa assulta (Guenée)

The interspecific hybridization betweenHelicoverpa armigera females andHelicoverpa assulta males yieldedF ₁ hybrids (RS), fertile males and sterile individuals with abnormal genitals. The reverse hybridization betweenH. assulta females andH. armigera males yielded F₁ hybrids (SR)-fertile males and fertile females. The morphology of testes and the karyotype of chromosomes of larvae in the hybrids were investigated. Among the 2d old fifth-instar SR larvae, individuals without testes were fertile females and those with testes were fertile males. The length and breadth of testes between SR and parental species were not significantly different (p>0.05). Among the 2d old fifth-instar RS larvae, the testes were observed in all the individuals, but it could be classified into two types. The length and the breadth of testes in Type 1 larvae were not significantly different from those of their parental species (p>0.05), while those in Type 2 were significantly less than those of their parental species (p<0.01). Mitotic metaphase I of brain cells showed the diploid chromosomes number of both reciprocal hybrids was 2n=62, as many as their parents. The haploid number of 31 was confirmed by counts from spermatocytes at meiotic metaphase from SR male larvae and Type 1 larvae of RS. Meiosis was not observed in spermatocytes of Type 2 larvae of RS. Considering the characteristics of adult hybrids of RS, it was concluded that Type 1 individuals in RS were fertile and those of Type 2 were sterile. The sterility of Type 2 individuals in RS is attributed to the abnormity in development of testes and the failing meiosis of spermatocytes. As a result, the normal spermatozoon could not been produced.     

Construction and characterization of a bacterial artificial chromosome library of rice 5460F

Thermo-sensitive genie male sterile (TGMS) rice has a number of desirable characteristics for hybrid rice production. Many studies have demonstrated that the sterility of TGMS rice is controlled by a single recessive gene. It has been mapped for the first time on chromosome 8 and namedtms 1. Several AFLP markers which tightly linked to thetms 1 gene have been identified recently. In order to develop a detailed physical map of thetms1 gene-encompassing region and finally clone thetms1 gene, a bacterial artificial chromosome (BAC) library of rice 5460F (the fertile mutant line of TGMS rice 5460S) using a modified vector pECBAC1 has been constructed. The constructed 5460F BAC library consists of 16 896 clones with an average insert size of 119 kb, which represents about 4.7 times rice haploid genome equivalents. Neither chloroplast nor mitochondrial DNA was detected from the library. The library was screened with three single copy sequence amplified fragment length polymorphism (AFLP) markers which tightly linked totms1 gene as probes and eight positive clones were identified.