AFLP Molecular Markers for Leaf Rust Resistance Genes in Foxtail Millet

Foxtail millet is not only a very useful experimental crop, but also a fodder crop and a staple food of people who live on the marginal agricultural lands, and it supplies high-quality protein for food products and industrial materials. However, leaf rust greatly reduces foxtail millet yields in susceptible varieties when weather conditions favor rust spread and development. Therefore, it is necessary to use molecular markers detecting and marking resistance genes. Compared with other molecular markers, AFLP maker is more efficient and powerful in the study of foxtail millet. In this study, AFLP analysis of 131 F2 individuals was performed and three AFLP makers were found to be linked to the rust resistance gene, with the genetic distances of 9.2, 9.8 and 12.4 cM, respectively. Cloning of rust re- sistance genes is of great importance and meaning in studying rust resistance of foxtail millet in the future. Furthermore, we may convert specific AFLP markers into single locus PCR markers, such as sequence characterised amplified region （SCAR） markers or cleaved amplified polymorphic site （CAPS） markers.

Development of Molecular Marker Linked to Cf-10 Gene Using SSR and AFLP Method in Tomato

The leaf mould resistance gene Cf-10 on tomato confered resistant or immune to all prevalent physiological races of Cladosporium fulvum presented in three northeastern provinces of China in inoculation test. In order to better utilize Cf-10 gene in a marker-assisted selection program and to permit the pyramiding of one or several resistance genes in a cultivar, tightly linked SSR and AFLP markers were obtained by the bulked segregant analysis method. One SSR marker and three AFLP markers were identified linked to Cf-10 gene, with the distance of 9.73, 5.8, 8.5, and 10.6 cM, respectively. These markers will facilitate the selection of resistant tomato germplasm containing Cf-10 gene.

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.

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.

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.

Population Genetic Diversity in Chinese Pomegranate (Punica granatum L.) Cultivars Revealed by Fluorescent-AFLP Markers

Eighty-five pomegranate （Punica granatum L.） cultivars from six geographical populations located at Shandong, Anhui, Shaanxi, Henan, Yunnan, and Xinjiang Provinces were studied for its population genetic diversity by means of fluorescent-AFLP markers. The results indicated that 135-185 polymorphic loci were amplified by eight pairs of primers at species level. An average of 158.25 polymorphic loci was amplified for each primer combination. The polymorphism percentage ranged from 62.5% to 86.11%, and the average polymorphism percentage was 73.26%. This indicated that there was plentiful genetic diversity in pomegranate cultivars. The genetic diversity at the species level was higher than that at the population level. The order of the genetic diversity was Henan population 〉 Xinjiang population 〉 Shaanxi population 〉 Anhui population 〉 Shandong population 〉 Yunnan population. Variance analysis showed that there was significant difference between populations in genetic diversity. The genetic differentiation coefficient between populations （Gsr） was 0.2018, which indicated that gene differentiation was mainly within the population, and between populations, it accounted for 20.18% of the total variation. Gene flow （Nm） between the populations measured was 1.9027 based on the genetic differentiation coefficient between populations, indicating that there was mild gene flow between populations. The UPGMA cluster analysis showed that most accessions from the same population were clustered together, but there was partly gene exchange. All genetic parameters indicated that there was plentiful genetic diversity in pomegranate cultivars in China, of which Henan population was significantly higher than the other populations, and it had wide application foreground in pomegranate breeding in China.

Population Genetic Structure in Apricot (Prunus armeniaca L.) Cultivars Revealed by Fluorescent-AFLP Markers in Southern Xinjiang,China

Population-wide genetic structure was studied using fluorescent-AFLP markers on 85 apricot （Prunus armeniaca L.） cultivars collected from Kuche, Kashi, Hetian in the Tarim Basin, southern Xinjiang Uygur Autonomous Region of China. The purpose of this study was to determine the genetic structure and genotypic diversity among the different eco-geographical populations. Based on the results from this study, 8 pairs of fluorescent-AFLP primers showed clear electrophoregram and high polymorphism amongst the 64 pairs of EcoR Ⅰ/Mse Ⅰ （Mse Ⅰ - a FAM fluorescent marked primer） primers screened. There was a significant polymorphic difference for the same primer pair in different populations and for the same population with different primer pairs. The percentage of polymorphic loci （P） at species level was higher than Kuche, Hetian, Kashi population levels, respectively. The Nei＇s gene diversity index （H） and Shannon＇s information index （I） at species level were higher than those of Kuche, Hetian, and Kashi at population level, respectively. H and I of Kuche population were the highest amongst the three populations. Apricot population genetic diversity was found mainly within the population, Genetic differentiation coefficient between populations （GST） was 0.0882. Gene flow Nm between the populations was 5.1689. Population genetic identity was between 0.9772-0.9811 and genetic distance was between 0.0191-0.0232. These results further indicated that the similarity between populations was higher and the genetic distance between populations was smaller. The UPGMA cluster analysis indicates that the geographical populations at Kuche, Kashi, Hetian were relatively independent Mendelian populations. Concurrently, there was also partial gene exchange between the populations. All the evidences indicated that the genetic diversity in Kuche population was the highest, suggesting that it could be a transition population from wild apricot to cultivated apricot. There were abundant genetic diversities in apricot cultivar populations in southern Xinjiang, China, which provide promising germplasm for further breeding and theoretical basis for biodiversity conservation and utilization for apricot population in this area.

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 Diversity Within a Jackfruit (Artocarpus heterophyllus Lam.) Germplasm Collection in China Using AFLP Markers

Jackfruit is cross-pollinated and mostly seed propagated, a wide range of variation exists in fruit quality. With the development of efficient vegetative propagation methods, excellent genotypes selected from these seed propagated seedlings will gradually replace other genotypes in jackfruit producing areas. In this study, genetic diversity of 50 jackfruit accessions from three provinces in China was analyzed based on amplified fragment length polymorphic （AFLP） markers. A total of 320 unambiguous bands were produced by eight primer combinations, and 65 （20.3%） of them were polymorphic. Genetic similarity coefficients ranged from 0 to 0.9841, with an average of 0.5000, indicating a moderate genetic diversity in this collection. The dendrogram derived by unweighted pair group method with arithmetic mean algorithm （UPGMA） analysis revealed five groups, and no correlation between genetic relationship and geographical origin were found. Accessions of soft and firm flesh type were not clustered into distinct groups, neither could yearly bearing once, or twice fruit accessions. This study has provided useful information for collection and preservation of jackfruit germplasm worldwide.

Inheritance of Powdery Mildew Resistance in Cucumber (Cucumis sativus L.) and Development of an AFLP Marker for Resistance Detection

Cucumber powdery mildew is one of the most destructive diseases of cucumber throughout the world. In the present study, inheritance of powdery mildew resistance in three crosses, and linkage of resistance with amplified fragment length polymorphism （AFLP） markers are studied to formulate efficient strategies for breeding cultivars resistant to powdery mildew. The joint analysis of multiple generations and AFLP technique has been applied in this study. The best model is the one with two major genes, additive, dominant, and epistatic effects, plus polygenes with additive, dominant, and epistatic effects （E-l-0 model）. The heritabilities of the major genes varied from 64.26% to 97.82%, and susceptibility was incompletely dominant for the two major genes in the three crosses studied. The additive effects of the two major genes and the dominant effect of the second major gene were high, and the epistatic effect of the additive-dominant between the two major genes was the highest in cross I . In cross II, the absolute value of the additive effect, dominant effect, and potential ratio of the first major gene were far higher than those of the second major gene, and the epistatic effect of the additive-additive was the highest. The genetic parameters of the two major genes in cross III were similar to those in cross II. Correlation and regression analyses showed that marker E25/M63-103 was linked to a susceptible gene controlling powdery mildew resistance. The marker could account for 19.98% of the phenotypic variation. When the marker was tested on a diverse set of 29 cucumber lines, the correlation between phenotype and genotype was not significant, which suggested cultivar specialty of gene expression or different methods of resistance to powdery mildew. The target DNA fragment was 103 bp in length, and only a small part was found to be homologous to DNA in the other species evaluated, which indicated that it was unique to the cucumber genome.

Identification of AFLP Markers Linked to Lr19 Resistance to Wheat Leaf Rust

AFLP analyses were carried out on Thatcher, 23 near-isogenic lines and F2 generation of TcLr19 × Thatcher, to develop molecular markers for gene Lr19 resistance to wheat leaf rust. Seven markers linked to Lr19 resistance trait were obtained, which were P-AGT/M-GAG289 bp （3.3 cM）, P-ACA/M-GGT102 bp （4.1 cM）, P-ACA/M-GGT106 bp （4.1 cM）, P-AAC/M-CAG123 bp （4.9 cM）, P-AAC/M-GGT203bp （5.0 cM）, P-ACA/M-GGT290bp （5.7 cM）, and P-ATC/M-GAG293bp （9.6 cM）. All of these specific fragments were isolated from the polyacrylamide gels, reamplified, cloned, and sequenced. The research may facilitate genetic mapping, physical mapping, and the eventual cloning of Lr19.

Identification of Heat Tolerance Linked Molecular Markers of Chinese Cabbage(Brassica campestris L.ssp.pekinensis)

Genetically stable population of recombination inbred line (RIL) was derived from a cross between a heat tolerant line 177 and a heat sensitive line 276 of Chinese cabbage (Brassica campestris L. ssp. pekinensis) by single seed descent. The RILs were analyzed using isozyme, RAPD and AFLP techniques in order to find molecular markers that are linked to heat tolerance quantitative trait loci (QTL). The results of variance analysis of single factor indicated that there were 9 molecular markers closely linked with heat tolerance QTL, including 5 AFLP markers, 3 RAPD markers and 1 PGM isozyme marker. Total genetic contribution of these makers to heat tolerance was 46.7%. Five of the nine markers distributed in one linkage group, the remaining 4 markers were located in separate groups. Thus the 9 heat tolerance linked markers distributed in 5 independent locations in the genome of Chinese cabbage.

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.

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.

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.