Molecular Marker Assisted Selection for Fusarium Wilt Resistance Breeding in Watermelon(Citrullus lanatus)

Molecular identification on diploid and tetraploid watermelon breeding lines which were resistant to Fusarium wilt was carried out with the published dCAPS marker ＂4451_fon＂ which was closely linked with resistance gene of Fusarium wilt race 1. The results showed that all the diploid and tetraploid lines expressed as re- sistant genotype, which were defined as Fusarium wilt-resistant materials. The re- sults were consistent with that of artificial inoculation identification. Molecular identifi- cation results also indicated that the resistant lines were homozygote, and the Fusarium wilt-resistant gene would not separate or lose during the future self- crossed purification. Therefore, resistance selection would not be necessary in their progeny populations. The study results thought that dCAPS marker ＂4451_fon＂ could be applied on molecular marker assisted selection for Fusarium wilt resistance breeding in watermelon to increase breeding selection efficiency and accelerate breeding progress.

Morphological comparison and molecular marker screening of three Skeletonema species found in Changjiang(Yangtze)River Basin

In our recent investigations of diatom diversity,we studied three species,namely,Skeletonema costatum,Skeletonema subsalsum,and Skeletonema potamos.Although they have been found frequently in Changjiang(Yangtze)River Basin,their morphological and molecular identification is difficult in taxonomy.Therefore,to integrate morphological and molecular biological approaches,we compared systematically their morphological characters and performed phylogenetic analysis.Twelve strains of Skeletonema were collected and isolated from Shanghai and Jiangsu,China,and their morphological characteristics were examined by light microscopy(LM)and the scanning electron microscopy(SEM).Based on morphological comparison,we determined that S.potamos is easy to distinguish from the other two species.The heavily silicified areolae,undulated or cleft distal ends of terminal fultoportula processes(TFPPs),absence of basal pores of fultoportula processes(FPPs),the rootlike protrusions of FPPs,and no interlocking connection are the stable characteristics that can be used to identify S.potamos.However,there are only two features that can distinguish S.costatum from S.subsalsum,namely the location of terminal rimoportulae(TRPs)and the distal shape of TFPPs.In addition,we amplified and sequenced nine common genetic markers from the strains,from which 101 sequences were obtained,constructed phylogenetic trees based on the nine genes and evaluated that seven genes can be used to identify S.potamos,and revealed that S.subsalsum is the closest known relative of S.costatum,and only ATP synthetase beta-subunit gene(atp B)is able to distinguish them from each other,which strongly support that it is an effective molecular marker for Skeletonema.This work provided a theoretical basis for the taxonomic study of Skeletonema.

Karyotype establishment and development of specific molecular markers of Aegilops geniculata Roth based on SLAF-seq

The constant evolution of pathogens poses a threat to wheat resistance against diseases,endangering food security.Developing resistant wheat varieties is the most practical approach for circumventing this problem.As a close relative of wheat,Aegilops geniculata,particularly accession SY159,has evolved numerous beneficial traits that could be applied to improve wheat.In this study,we established the karyotype of SY159 by fluorescence in situ hybridization(FISH)using the oligonucleotide probes Oligo-pTa535 and Oligo-pSc119.2 and a complete set of wheat–Ae.geniculata accession TA2899 addition lines as a reference.Using specific-locus amplified fragment sequencing(SLAF-seq)technology,400 specific markers were established for detecting the SY159 chromosomes with efficiencies reaching 81.5%.The SY159-specific markers were used to classify the different homologous groups of SY159 against the wheat-Ae.geniculata addition lines.We used these specific markers on the 7Mg chromosome after classification,and successfully confirmed their suitability for studying the different chromosomes of SY159.This study provides a foundation for accelerating the application of SY159 in genetic breeding programs designed to improve wheat.

Assessment of molecular markers and marker-assisted selection for drought tolerance in barley(Hordeum vulgare L.)

This review updates the present status of the field of molecular markers and marker-assisted selection(MAS),using the example of drought tolerance in barley.The accuracy of selected quantitative trait loci(QTLs),candidate genes and suggested markers was assessed in the barley genome cv.Morex.Six common strategies are described for molecular marker development,candidate gene identification and verification,and their possible applications in MAS to improve the grain yield and yield components in barley under drought stress.These strategies are based on the following five principles:(1)Molecular markers are designated as genomic‘tags’,and their‘prediction’is strongly dependent on their distance from a candidate gene on genetic or physical maps;(2)plants react differently under favourable and stressful conditions or depending on their stage of development;(3)each candidate gene must be verified by confirming its expression in the relevant conditions,e.g.,drought;(4)the molecular marker identified must be validated for MAS for tolerance to drought stress and improved grain yield;and(5)the small number of molecular markers realized for MAS in breeding,from among the many studies targeting candidate genes,can be explained by the complex nature of drought stress,and multiple stress-responsive genes in each barley genotype that are expressed differentially depending on many other factors.

Research Progress on Application of Molecular Markers in Breeding of Camellia oleifera

Camellia oleifera is an important woody oil tree species unique to China.It is known as the world s four major woody oil crops along with olive,oil palm and coconut.It is known as the‘king of oil’because of its high oil content.With the increase of people's attention to the yield of Camellia oleifera,its high yield has become the focus.In traditional breeding model,judgment is performed by phenotypic traits,but this method is single and easily affected by the environment,and can no longer meet the demand.In contrast,molecular marker breeding is not affected by the environment,and is stable and efficient and capable of accurately mapping target genes,so it has attracted much attention.In this paper,the research progress on C.oleifera germplasm resources diversity,DNA fingerprinting construction,genetic linkage map construction and QTL mapping was summarized,and the application of SSR molecular marker technique combined with association analysis in C.oleifera breeding in recent years was discussed,in order to provide new ideas for high-yield breeding of C.oleifera.

Introduction of T-DNA into Watermelon by Stigma Smeared Method and Its Molecular Marker Research

[Objective]The aim was to introduce T-DNA into watermelon for its molecular marker research.[Method]Based on the method of foreign DNA introduced to Arabidopsis thaliana via dipping flowers,the stigma smear was used to transfer T-DNA into watermelon and its molecular marker research was carried out.[Result]The ideal transformed species was ZXG01078 for the highest fruit setting rate and the most deviant seedlings.The best concentration of kanamycin for treating watermelon seeds was 500-700 mg/L with differences among the species.The best position was spire leaf or young leaf and the best concentration of kanamycin for treating the watermelon leaf was 4 000-8 000 mg/L with no significant difference among species.The steadily variation appearing of growing pointless and conjoined twin seedlings indicated that the normal growth had been interfered by foreign DNA in the progeny.[Conclusion]This study had provided basis for the further research on watermelon.

ACC Synthase Gene (ACSG) as a Possible Molecular Marker for Female Lines in Cucumber

利用本实验室根据已知序列分离得到的ACC合酶基因 (ACSG)为探针对不同黄瓜 (CucumissativusL .)品种(系 )的基因组DNA进行Southern杂交 ,初步分析了该基因与黄瓜性别表型之间的相关性。发现在所检测的 10个不同品系中 ,ACSG与雌性系表型之间存在明显的相关关系 ,而且这种相关关系在不同的实验中具有良好的重复性。ACSG基因可能是鉴定黄瓜雌性系的一个分子标记。

The Construction of Heat-resistant Near Isogenic Line (NIL) of Bombyx mori and the Development of Molecular Marker Breeding Technique

[Objective] The study aimed to provide theoretical basis for development and application of molecular marker breeding technique to obtain Bombyx mori near-isogenic lines （NILs）. [Method] Thermotolerance gene was introduced into sensitive variety Ou17 by developing NILs and recurrent backcross,then through six generations of backcross,thermotolerance-assisted selection,and two generations of self-cross. [Result] Bombyx mori NILs carrying thermotolerance gene （new germplasm） were produced. Meanwhile,thermotolerance level of progenies of each backcross and molecular markers of NILs were determined,and then attempts were made to produce practical thermotolerance hybrids by using thermotolerance varieties whose thermotolerance gene is linked to SSR markers. [Conclusion] The study successfully construct thermotolerance NILs,monitor thermotolerance level and breeding results of progenies of each backcross,and determine molecular marker of NILs.

Development and Utilization of 1S^1 Chromosomearm-specific Molecular Markers of Aegilops longissima

Introducing the 1S^1 chromosome of Aegilops longissima into wheat genome can significantly improve wheat grain quality and contents of iron and zinc. Therefore, the development of molecular markers specific to 1S^1 chromosome of A. longissima is of important significance for breeding high-quality wheat with high contents of iron and zinc in grains. In this study, nine molecular markers specific to 1S^1 chromosome of A. longissima were developed, including two 1S^1S specific markers,six 1S^1L specific markers and one 1S^1 specific marker which was located on both short and long arms. The practicability of these molecular markers were verified using hybrid population as materials. The results showed that hybrid population could be effectively screened and identified, which indicated that the developed 1S^1 chromosome-specific molecular markers could be used for screening and identification of hybrid population and could be used in marker-assisted breeding of high-quality wheat with high contents of Fe and Zn in grains.

Genetic Relationships Among Soluble Carbohydrates, Anthocyanins and Growth Characteristics in Leymus (Gramineae) Detected with Molecular Markers

Low-temperature soluble carbohydrate accumulations are commonly associated with anthocyanin coloration, attenuated growth and cold adaptation of cool-season grasses. The vrn-1 gene has potent effects on vernalization requirement, growth, and soluble carbohydrate accumulations of the winter-annual Triticeae species. Two hundred and four unmapped AFLP markers and genome-specific DNA markers genetically linked to the vrn-1 gene were used to detect QTL controlling soluble carbohydrate accumulations, anthocyanin coloration and growth characteristics in a segregating population derived from open pollinated Leymus cinereus x L. triticoides hybrids. These perennial Triticeae grasses are distinguished by adaptation and growth habit. As expected, positive trait correlations and pleiotropic gene effects were detected for soluble carbohydrate accumulations and anthocyanin coloration. Likewise, positive trait correlations and pleiotropic gene effects were detected for tillering, leaf development, leaf growth, regrowth and rhizome spread. However, soluble carbohydrate accumulations were not associated with attenuated growth. In fact, several DNA marker alleles, including one near vrn-Ns1, had positive effects on soluble leaf carbohydrate concentrations and low temperature growth. The corresponding DNA marker near vrn-Ns1 had more specific effects on tillering. We speculate that vrn-1 exerts quantitative effects on low-temperature soluble leaf carbohydrate accumulations and growth habit of the perennial Leymus. However, a number of other DNA markers displayed highly significant effects on soluble carbohydrate accumulations and various growth characteristics. Findings indicate that anthocyanin coloration may be a useful phenotypic marker for soluble carbohydrate accumulation. Although variation for soluble carbohydrates was not associated with attenuated growth in this population, this trait was under genetic control.

Advances in Molecular Marker Techniques in Pomegranate(Punicagranatum L.)

At present, there is a need to develop a universal standard for the classification of pomegranate（Punica granatum L.） species, which facilitates the collection,sorting, classification and application of pomegranate resources. This review introduces recent research progress in pomegranate classification（in particular with molecular markers）, and finally the potential application of molecular markers in future research about pomegranate.

Screening and Application of Molecular Markers for Starch Synthesis-related Genes in Rice

In this study, the genotypes of starch synthesis-related genes were systematically screened from different rice varieties using molecular markers. The results showed that starch synthesis-related genes were highly polymorphic between indica and japonica varieties, as they greatly variated among indica varieties, but were conserved among japonica varieties. The genotypes of two indica varieties9311 and Minghui 63 were more similar to that of japonica varieties. Two or three alleles of six starch synthesis-related genes were found in 28 japonica parental varieties. Four genotypes of two soluble starch synthase genes, SSIIa and SSIIIa,were detected in 88 stable lines derived from the cross of Kanto 194/ Wujing 13 using molecular markers.

Genetic Inheritance and Molecular Marker of Clubroot Resistance Genes in Brassica campestris ssp. chinensis

Objective] This study was conducted to investigate the genetic inheritance of clubroot resistance in Chinese non-heading cabbage （Brassica campestris ssp. chinensis）. [Method] The clubroot resistance gene was introduced from a Brassica campestris ssp. pekinensis cultivar to non-heading Chinese cabbage, and the inheri-tance and molecular markers of clubroot resistance gene in parental lines, F1, F2 and BC1 of non-heading Chinese cabbage were studied through pathogen inoculation at seedling stage and ISSR-PCR. [Result] Clubroot resistance in non-heading Chi-nese cabbage was control ed by a single dominant gene. ISSR molecular markers with Bulk segregant analysis （BSA） found that primer-873 was linked to resistance gene, named CR-873, and the genetic distance between the marker and the resis-tance gene was 9.72 cM. [Conclusion] The results provide references for the molecular marker assisted breeding of non-heading Chinese cabbage.

Specific molecular markers in hepatocellular carcinoma

BACKGROUND: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multistep and complex process. Its prognosis is poor, and early diagnosis and monitoring metastasis of HCC is of the utmost importance. Circulating diagnostic and prognostic biomarkers could be used in proper postoperative treatment of patients at an early stage of HCC development. This review summarizes recent studies of the specific biomarkers in diagnosis and monitoring metastasis or postoperative recurrence of HCC. DATA SOURCES: An English-language literature search was conducted using MEDLINE (June 1998 to Spetember 2006) on researches of some valuable specific biomarkers in diagnosis and monitoring metastasis or postoperative recurrence of HCC. RESULTS: Hepatoma tissues can synthesize various tumor-related proteins, polypeptides, and isoenzymes, such as alpha-fetoprotein (AFP), hepatoma-specific gamma-glutamyl transpeptidase (HS-GGT), etc, and then secrete into blood. The valuable early diagnostic and prognostic biomarkers could predict the development an metastases of HCC. Recent researches have confirmed that circulating hepatoma-specific AFP subfraction, transforming growth factor (TGF)-beta 1, HS-GGT, and free insulin-like growth factor (IGF)-II may be more specific markers than total AFP level for early diagnosis for HCC. The circulating genetic markers such as AFP-mRNA, TGF-beta 1-mRNA, IGF-II-mRNA, etc from peripheral blood mononuclear cells of HCC patients have been most extensively used in monitoring distal metastasis or postoperative recurrence of HCC. CONCLUSIONS: Hepatoma tissues synthesize and secrete valuable molecular markers into blood. The analyses of circulating hepatoma-specific biomarkers are useful to early diagnosis of HCC or monitoring metastasis or postoperative recurrence of HCC.

Assessment on Evaluating Parameters of Rice Core Collections Constructed by Genotypic Values and Molecular Marker Information

Eleven evaluating parameters for rice core collection were assessed based on genotypic values and molecular marke＇ information. Monte Carlo simulation combined with mixed linear model was used to eliminate the interference from environment in order to draw more reliable results. The coincidence rate of range （CR） was the optimal parameter. Mean Simpson index （MD）, mean Shannon-Weaver index of genetic diversity （M1） and mean polymorphism information content （MPIC） were important evaluating parameters. The variable rate of coefficient of variation （VR） could act as an important reference parameter for evaluating the variation degree of core collection. Percentage of polymorphic loci （p） could be used as a determination parameter for the size of core collection. Mean difference percentage （MD） was a determination parameter for the reliability judgment of core collection. The effective evaluating parameters for core collection selected in the research could be used as criteria for sampling percentage in different plant germplasm populations.

Identification and Purity Test of Super Hybrid Rice with SSR Molecular Markers

Five super hybrid rice combinations, i.e. HYS-1/R105, Pei'ai 64S/E32, Liangyoupeijiu (Pei'ai 64S/9311), 88S/0293, and J23A/Q611, and their parental lines were tested by means of SSR analysis. A total of 144 SSR primer pairs distributed on 12 rice chromosomes were used, out of which 47 detected polymorphism among the tested rice lines. Among all these primers, RM337 and RM154 produced polymorphic patterns in four or more of the tested experimental materials respectively, and they could distinguish among most rice genotypes tested. Twenty-four primer pairs, two on each rice chromosome, were selected to make a reference SSR marker-based fingerprinting for the rice lines. For most of the primer pairs, F1 hybrids mainly showed complementary pattern of both parents, which could be very useful to distinguish the F1 from its parental lines. In addition, 5 primer pairs were selected as special primer pairs for five hybrid rice combinations respectively. By combining the rapid, simple method on DNA extraction, it is suggested that SSR technique has wide prospective in variety authentication and purity identification.

Databasing Molecular Identities of Sugarcane (Saccharum spp.) Clones Constructed with Microsatellite (SSR) DNA Markers

This paper reports the development of the first SSR marker-based sugarcane (Saccharum spp.) molecular identity database in the world. Since 2005, 1,025 sugarcane clones were genotyped, including 811 Louisiana, 45 Florida, 39 Texas, 130 foreign, and eight consultant/seed company clones. Genotyping was done on a fluorescence-capillary electrophoresis detection platform involving 21 highly polymorphic SSR markers that could potentially amplify 144 distinctive DNA fragments. Genotyping data were processed with the GeneMapper? software to reveal electrophoregrams that were manually checked against the 144 fragments. The presence (A) or absence (C) of these 144 fragments in any sugarcane clone was recorded in an affixed sequence order as a DNAMAN? file to represent its molecular identity being achieved into a local molecular identity database. The molecular identity database has been updated annually by continued genotyping of newly assigned sugarcane clones. The database provides molecular descriptions for new cultivar registration articles, enables sugarcane breeders to identify mis-labeled sugarcane clones in crossing programs and determine the paternity of cross progeny, and ensures the desired cultivars are grown in farmers’ fields.

Evolutionary history and phylogeography of Scots pine (Pinus sylvestris L.) in Europe based on molecular markers

In this review we summarized recent historical records and molecular studies on evolutionary history and phylogeography of Scots pine with focus on the European highly fragmented distribution area of the species. Fossilized pollen, plant micro- and macrofossil records provided evidences on the large-scale species’ range shifts and demographic changes during the Quaternary. Populations of Scots pine were documented both in the glacial (incl. full glaciation) and interglacial periods. Recolonization of Europe after the glaciation originated from the (Sub) Mediterranean areas like the Balkan Peninsula but also from around the Eastern Alps and the surroundings of the Danube plain. Fennoscandia and northern European Baltic regions were most probably colonized from two main directions, from Western Europe and from the Russian Plain. Modern history of Scots pine was hardly affected by anthropogenic activities that started to strengthen in the Bronze and Iron Age. Along with the fossil records, molecular genetic tools were used to infer the origin and putative history including migration, differentiation and demography of the species. In this paper we compiled the major publications (30) of molecular genetic studies of the past 20 years derived from distinctly inherited organelle genomes (mitochondrial, chloroplast, nuclear) revealed by different marker systems (mtDNA-cox1, -nad1, -nad3, -nad7, ISSR, cpSSR, nSSR, B-SAP, SNP). It is important to consider that different phylogeographic patterns can be drawn by the analysis of different DNA marker types. Accordingly the use of more than one marker simultaneously outlines the most sophisticated phylogeographical pattern on the genetic lineages and can reveal high differentiation of the European distribution. Combined marker systems and markers derived from coding sequences have also been used to detect species’ phylogeographic patterns, but these were rarely applied to Scots pine. Although new molecular techniques can provide higher resolution data for populations, the reviewed results can shape the direction of further studies.

Molecular Marker Assisted Selection for Yield-Enhancing Genes in the Progeny of Minghui63 x O. rufipogon

Two yield-enhancing genes (yld1.1 and yld2.1) are located on chromosomes 1 and 2 respectivelyin a weedy relative of cultivated rice, Oryza rufipogon. SSR markers RM9 and RM166 are closelylinked with the two loci respectively. Minghui63 (MH63) has been a widely used restorationline in hybrid rice production in China during the past two decades. The F1 of cross 'MH63O.rufipogon' was backcrossed with MH63 generation by generation. RM9 and RM166 were used toselect the plants from the progeny of the backcross populations. The results were as follows:(1) In BC2F1 population, the percentage of the individuals which have RM9 and RM166 amplifiedbands simultaneously was 12.2%, while in the BC3F1 population, that was 16.3%. (2) Among 400individuals of BC3F1, four yield-promising plants were obtained, with yield being 30% more thanthat of MH63. (3) The products amplified by primer RM166 in O. rufipogon and MH63 weresequenced. It was found that the DNA fragment sequence amplified by RM166 from MH63 was 101 bpshorter than that from O. rufipogon. The 101bp sequence is a part of an intron of the PCNA(proliferating cell nuclear antigen) gene.

Identification of Molecular Markers for a Aphid Resistance Gene in Sorghum and Selective Efficiency Using These Markers

In this study, an F2 segregated population obtained by hybridization between the aphid-sensitive sorghum strain Qiansan and aphid-resistant cultivar Henong 16 was used to establish an aphid-resistant pool and an aphid-sensitive pool. 192 pairs of AFLP （amplified fragment length polymorphism） marker primers were screened in these pools using BSA （bulked segregant analysis）. Three pairs of EcoR I-CTG/Mse I-CCT, EcoR I-CTG/Mse I-CAT, and EcoR I-AGT/Mse I-CCC showed linkage with aphis resistance. EcoR I-CTG/Mse I-CCT-475, EcoR I-CTG/Mse I-CAT-390, and EcoR I-AGT/Mse I-CCC- 350 （E42/M52-350） were mapped within 6, 10, and 13 cM distances with the aphid-resistant gene by using Mapmaker 3.0 software. The bands amplified by EcoR I-CTG/Mse I-CCT-475 and EcoR I-CTG/Mse I-CAT-390 were extracted, cloned, and sequenced. Specific primers of SCAR （sequence characterized amplified regions） were then designed from these bands. A specific band of 300 bp was amplified by a pair of SCAR primers designed based on the sequence obtained from the EcoR I-CTG/Mse I-CAT-390 marker. The SCAR marker was named SCAS0. The marker was used to detect the F2, BC1, and F2：3 populations. The selective efficiency was 86.8, 91.1, and 86.3% in the BC1, F2, and F2：3 populations, respectively. The average selective efficiency was 88.2%.