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.

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.

Fast tracking alien gene discovery by molecular markers in a late flowering Chinese cabbage-cabbage translocation line‘AT7–4'

Flowering time is an important agronomic trait of Chinese cabbage with late flowering being a primary breeding objective.In our previous work,we obtained Chinese cabbage-cabbage translocation lines that contained several beneficial cabbage genes.Cabbage-specific molecular markers show that these genes were coming from chromosome C01 of cabbage.In this study,we investigated the inheritance of flowering time in a couple of translocation lines and analyzed the transmission rate of molecular markers in the offspring.Consequently,we obtained the late flowering Chinese cabbage-cabbage translocation line‘AT7–4’in which the flowering time was later than that of‘85–1’by about 7 days under 4-week vernalization.Based on previous studies of the genomes of Chinese cabbage and cabbage,we located the cabbage-specific molecular markers that were closely linked at the top of the chromosome A01 in the F2mapping population generated by self-crossing F1s derived from a cross between the translocation line‘AT7–4’and Chinese cabbage‘14–36’.Five flowering-related genes in the alien fragment were found by functional annotation and their molecular markers were developed.This study lays the foundation for the future improvement of Chinese cabbage varieties using A-C translocation lines.

Development of Molecular Marker Linked with Cercospora Leaf Spot (CLS) Disease Resistance in Vigna radiata, Cloning, and Expression for Evaluating Antifungal Activity against Cercospora canescens

We developed a molecular marker for MAS of mungbean resistant varieties against CLS from the consensus sequence(MB-CLsRG)of identified RGAs(MB-ClsRCaG1 and MB-ClsRCaG2).The MB-CLsRG sequence-specific primer pair was used to screen Cercospora leaf spot(CLS)resistant varieties of mungbean in genomic analysis that showed congruency with phenotypic screening.Validation of molecular marker linkage with CLS resistance was performed using rtPCR in transcriptomic analysis.The sequenced PCR products showed 100%homology with MB-CLsRG sequence and putative disease resistance proteins that confirmed the linkage of molecular marker with CLS resistance in mungbean.The antifungal potential of MB-CLsRG gene encoding protein was assessed.The MB-CLsRG gene sequence was cloned in the E.coli expression vector for recombinant protein production.The recombinant protein was then investigated for its in vitro antifungal potential against Cercospora canescens.The in vitro investigation showed strong antifungal activity of recombinant protein as it restricted the growth of fungal mycelial mass.The results validated the linkage of developed marker with CLS-resistant mungbean varieties;therefore,it can be used to screen resistant varieties from a large population in MAS.Moreover,the recombinant protein of the MB-CLsRG gene sequence revealed antifungal potential,which proved the gene sequence could be suitable to use in transgenic plants technology to develop fungal-resistant transgenic crops.

Large scale genetic landscape and population structure of Ethiopian sesame (Sesamum indicum L.) germplasm revealed through molecular marker analysis

Sesame(Sesamum indicum L.) plays a crucial role in Ethiopian agriculture,serving both subsistence and commercial purposes.However,our understanding of the extensive genetic diversity and population structure of Ethiopian sesame remains limited.To address this knowledge gap,we genotyped 368 Ethiopian sesame germplasms,categorizing into four distinct breeding groups:Accessions,landraces,improved varieties,and wild types,using a comprehensive set of 28 polymorphic markers,including 23 simple sequence repeat(SSR) and five Insertion-Deletion(InDel) markers.These markers ensured robust genomic representation,with at least two markers per linkage group.Our results unveiled substantial genetic diversity,identifying a total of 535 alleles across all accessions.On average,each locus displayed 8.83 alleles,with observed and expected heterozygosity values of 0.30 and 0.36,respectively.Gene Diversity and Polymorphic Information Content(PIC) were recorded at 0.37 and 0.35.The percentage of polymorphic loci varied significantly among breeding groups,ranging from8.00% to 82.40%,indicating high diversity in accessions(82.4%),moderate diversity in improved varieties(31.20%) and landraces(29.60%),and limited diversity in wild types(8.00).Analysis of Molecular Variance(AMOVA) results emphasized significant genetic differentiation among populations,with substantial diversity(P<0.001) within each population.Approximately 8% of the entire genetic diversity could be attributed to distinctions among populations,while the larger proportion of genetic diversity(92%) resided within each individual sesame population,showcasing heightened diversity within each group.Our study’s findings received support from both Bayesian clustering and Neighbor-joining(NJ) analysis,reaffirming the credibility of our genetic structure insights.Notably,Population structure analysis at its highest Δk value(k=2) revealed the existence of two primary genetic clusters,further subdivided into four sub-populations at k=4.Similarly,NJ analysis identified two prominent clusters,each displaying additional sub-clustering.In conclusion,our research provides a comprehensive understanding of genetic groups,subpopulations,and overall diversity within Ethiopian sesame populations.These findings underscore the significant genetic diversity and population structure within Ethiopian sesame germplasm collections.This genetic richness holds promise for breeding and conservation efforts,highlighting the importance of preserving genetic diversity to ensure adaptation to changing environments and meet the needs of farmers and consumers.

SDC1 acts as a novel molecular marker and prevents rheumatoid arthritis and modulates inflammatory response by targeting the miR-4531/SDC1 axis

Objective:Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by chronic erosive arthritis.Due to the lack of effective biomarkers for diagnosis and treatment,RA patients have many complications in the later stage,seriously affecting their quality of life.Thus,this study was conducted to investigate new therapeutic targets and to discover diagnostic biomarkers in RA.Methods:In this study,the expression profiles of GSE55235 and GSE55457 were downloaded from the Gene Expression Omnibus database to obtain DEGs between RA and healthy samples.Genetic Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on the common genes existing in the RA-related modules.Additionally,we used the STRING database to construct the protein‒protein interaction network.Furthermore,we established the interaction analysis of Hub Genes and microRNA(miRNA)and verified the 10 Hub genes through the GSE77298 dataset and quantitative real-time polymerase chain reaction Results:276 and 69 DEGs were screened from the GSE55235 dataset and GSE55457 dataset,respectively.Then,we obtained 42 up-regulated genes in two chip datasets intersection.Genetic Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the 42 up-regulated genes showed that they were mainly concentrated in immune response-activating cell surface receptor signaling pathway,etc.Furthermore,the protein-protein interaction network indicated that 10 hub genes are closely related to RA,including MS4A1,CD27,LCK,CD79A,SDC1,CXCL9,CXCL10,CXCL13,IGLL5,and IGJ.In addition,we found that miR-4531 is the same target miRNAs between MS4A1 and SDC1 through messenger RNA-miRNA co-expression network.Finally,the GSE77298 gene chip and quantitative real-time polymerase chain reaction verified the expression of 10 Hub genes.The six Hub genes of CD27,SDC1,CXCL9,CXCL10,CXCL13,and IGJ are significantly increased.Conclusions:We found that SDC1 may be a novel molecular marker for the prevention and treatment of RA.The miR-4531/SDC1 regulatory axis may play a key role in this process.In conclusion,our study not only provides potential biomarkers for the diagnosis and treatment of RA,but also provides a basis and new targets for further revealing the potential mechanism of RA occurrence and development and discovering targeted drugs.

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.

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.

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.

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.

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.

Molecular Marker Techniques Using Single Primers and Their Advances

Molecular marker techniques have been widely applied in the fields of genetic diversity analysis,germplasm resources identification,molecular fingerprint and genetic linkage map construction,QTL mapping and molecular assisted breeding.On the basis of stating the concept of molecular marker techniques based on single primer amplification reactions,this study focused on the sorting and induction of single-primer molecular marker techniques,and expounded their derivative development.Finally,the application prospect and future expectation of single-primer molecular marker techniques were described in detail.The purpose of this study was to clarify the types of molecular marker techniques based on single primer amplification reactions,so that researchers can quickly and conveniently select molecular marker techniques according to their own specific scientific research conditions.

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.

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.