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.

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.

Development of branchless watermelon near isogenic lines by marker assisted selection

Pruning is time-consuming and laborious in watermelon cultivation,which can not meet the needs for simplified cultivation in the future.The development of branchless lines will provide important germplasms for breeding watermelon varieties and is an important method for genetic improvement.In this study,the watermelon accession,Wu Cha Zao(WCZ)is a branchless inbred line that carries the branchless gene Clbl,which was used as the donor parent to develop branchless near isogenic lines(NILs).To construct the NILs of Clbl,WCZ crossed with the normal branching watermelon inbred line WT20 which was used as the recurrent parent.The co-segregating markers dCAPS10 and Indel1 with Clbl were used for foreground selection,and a total of 108 SSR markers was selected with good polymorphism between two parental lines for background selection which had relatively uniform distribution across 11 chromosomes.Using these markers to select individuals from the BC_(1)F_(1),BC_(2)F_(1),and BC_(2)F_(2) generations,three NILs with a proportion of recurrent parent genome(PRPG)>99%were finally obtained.The lateral branch and plant height phenotypes did not significantly differ between the NILs and WCZ,indicating that the NILs of Clbl under the genetic background of WT20 has been successfully developed.These results provide ideal materials for further in-depth analysis of the genetic mechanisms of lateral branch development and ideal plant architecture breeding in watermelon.

Green Rice Leafhopper Resistance Gene Transferring Through Backcrossing and CAPS Marker Assisted Selection

Grh2, a green rice leafhopper resistant gene from an indica cultivar DV85, was located on chromosome 11, and two RFLP markers C189 and G1465 were found to be linked to this gene. In order to transfer Grh2 into Taichung65, a japonica cultivar with elite characters, backcross method with Taichung65 as the recurrent parent was used and the two RFLP markers were converted into CAPS markers for marker assisted selection (MAS). In the BC6F3 population, both phenotypic evaluation and MAS were conducted to screen the resistant plants with Taichung65 background. The linkage distance between CAPS markers and Grh2 was calculated and the efficiency of MAS was analyzed.

SCAR Markers Assisted Selection for a Bentazon Susceptible Lethality Gene (ben) in Rice

In progenies resulting from crosses involving rice cultivar Norin 8m susceptible to bentazon as the donor of ben gene, SCARs tightly linked to ben were utilized for selection of ben. The homozygous and heterozygous genotypes with ben could be identified with the SCARs. The molecular markers offer a powerful tool for indirect selection of ben and can accelerate the introgression of ben into current rice cultivars.

Analysis of short fruiting branch gene and Marker-assisted selection with SNP linked to its trait in upland cotton

Background： With the rapid development of genomics, many functional genes have been targeted. Molecular marker assisted selection can accelerate the breeding process by linking selection to functional genes. Methods： In a study of upland cotton （Gossypium hirsutum L.）, the F2 segregated population was constructed by crossing X1570 （short branches） with Ekangmian 13 （long branches） to identify the short fruiting branch gene and marker assisted selection with SNP（Single Nucleotide Polymorphisms, SNP） linked to its trait. Result： The result demonstrated that linked SSR marker BNL3232 was screened by BSA（Bulked segregant analysis, BSA） method; one SNP locus was found, which was totally separated from the fruiting branches trait in upland cotton. Conclusion： It was verified that this SNP marker could be used for molecular assisted selection of cotton architecture

Using Marker Assisted Selection to Hasten Screening of Cassava Cultivars Developed through Introgression of Cassava Mosaic Disease （CMD） Resistance into Cassava Landraces in Ghana

Cassava mosaic disease （CMD）, the most important disease of cassava （Manihot esculenta Crantz） is a potential threat to Africa＇s cassava production. The disease is embedded in most landraces resulting in low yields. Host plant resistance has been found to be the best control strategy. A breeding programme using genetic hybridisation and Marker Assisted Selection was initiated in 2007 to improve the resistance levels of farmer-preferred landraces and reduce the long breeding cycle for developing improved cassava varieties. Thirty farmer-preferred landraces were selected and crossed with a high yielding and mosaic resistant cultivar （TMEI l） from International Institute of Tropical Agriculture （IITA）. The resultant progenies found to have a reasonable level of resistance （score, 1-3） to the CMD were AW 18, 273 Nyamebekyere, NK 43, AW 3, NK 26, K 25, Dabodabo, Ahwengyankwa, 674 Debor, Degarti, Agric Bankye, and NK 57. These resultant progenies were backcrossed to the resistant cultivar. The backcross one （BC l） progenies totaling 224 were screened with molecular markers that are associated to the CMD 2 gene. De-oxyribonucleic Acid （DNA） was extracted from leaves of the 224 BC1 progenies and 13 parents. Two Simple Sequence Repeats （SSR） markers （SSY28 and NSl58） and one Sequenced Characterized Amplified Region （SCAR） marker RME1, were used to screen and select for the resistant BC l progenies. Marker Assisted Selection （MAS） revealed that 82% of the genotypes had at least a marker allele for the CMD2 gene, indicating resistance. The study further revealed that by using MAS, the breeding cycle of cassava in the generation of varieties could be reduced from 8 years to 2 years. The resistant genotypes identified will be evaluated for yield and starch cooking quality in future breeding work.

Genome Mapping to Enhance Efficient Marker-Assisted Selection and Breeding of the Oil Palm (<i>Elaeis guineensis</i>Jacq.)

The oil palm (Elaeis guineensis Jacq.) is one of the major cultivated crops among the economically important palm species. It is cultivated mainly for its edible oil. For a perennial crop like oil palm, the use of Marker Assisted Selection (MAS) techniques helps to reduce the breeding cycle and improve the economic products. Genetic and physical maps are important for sequencing experiments since they show the exact positions of genes and other distinctive features in the chromosomal DNA. This review focuses on the role of genome mapping in oil palm breeding. It assesses the role of genome mapping in oil palm breeding and discusses the major factors affecting such mapping. Generating a high-density map governed by several factors, for instance, marker type, marker density, number of mapped population, and software used are the major issues treated. The general conclusion is that genome mapping is pivotal in the construction of a genetic linkage map. It helps to detect QTL and identify genes that control quantitative traits in oil palm. In perspective, the use of high-density molecular markers with a large number of markers, a large number mapping population, and up-to-date software is necessary for oil palm genome mapping.

Haplotype variation and KASP markers for SiPSY1-A key gene controlling yellow kernel pigmentation in foxtail millet

Carotenoid biosynthesis and accumulation are important in determining nutritional and commercial value of crop products.Yellow pigmentation of mature kernels caused by carotenoids is considered a vital quality trait in foxtail millet,an ancient and widely cultivated cereal crop across the world.Genomic regions associated with yellow pigment content(YPC),lutein and zeaxanthin in foxtail millet grains were identified by genome-wide association analysis(GWAS),and SiPSY1(Phytoene synthase 1 which regulates formation of the 40-carbon backbone of carotenoids)was confirmed as the main contributor to all three components by knockout and overexpression analysis.SiPSY1 was expressed in seedlings,leaves,panicles,and mature seeds,and was subcellularly localized to chloroplasts.Transcription of SiPSY1 in 15 DAP immature grains was responsible for YPC in mature seeds.Selection of SiPSY1 combined with increased YPC in mature grains during domestication of foxtail millet was confirmed.Haplotype analysis suggested that expression level of SiPSY1 could be a selection target for future breeding programs,and a KASP marker was developed for selection of favorable SiPSY1 alleles in breeding.The results of this work will benefit nutritional and commercial improvement of foxtail millet varieties,as well as other cereal crops.

Screening and Application of SSR Markers of Resistant Gene against Rice Stripe Virus

[Objective] New SSR primers were designed and screened to apply in the backcross breeding for modified resistance against rice stripe virus.[Method] The conventional late japonica rice varieties including 502 with high resistance to stripe virus,Xiushui 09 with high susceptibility to stripe virus and their derived strains were adopted as the test materials,SSR and SAPR markers were used to locate RSV1 gene with high resistance against stripe virus,and three pairs of SSR markers （M-11-1,M-11-2,M-11-3） were further designed.Through screening and analysis,M-11-3 was selected as the RSV1 detection marker gene for tracking RSV1 gene,thus RSV1 gene was successfully introduced to the backcross breeding of late japonica rice varieties such as Xiushui 09,and the resistance expression of different strains was identified.[Result]The resistance of improved strains against stripe virus was significantly higher than Xiushui 09,the resistance of most strains was close to the level of donor,and the expression of resistance among years was stable.Therefore,the resistance effect of RSV1 gene used in the test was very obvious,which was accurate with the assisted selection of RSV1 gene linked markers M-11-3.[Conclusion]The study certified the feasibility of molecular markers application in resistance improvement against rice stripe virus,which also showed that optimization and development of new marker genes could effectively improve the efficiency of marker-assisted selection.

Applications of Molecular Markers in Fruit Crops for Breeding Programs—A Review

Selection and use of molecular markers for evaluation of DNA polymorphism in plants are couple of the most important approaches in the field of molecular genetics.The assessment of genetic diversity using morphological markers is not sufficient due to little differentiating traits among the species,genera or their individuals.Morphological markers are not only highly influenced by environmental factors but skilled assessment is also prerequisite to find the variations in plant genetic resources.Therefore,molecular markers are considered as efficient tools for detailed DNA based characterization of fruit crops.Molecular markers provide new directions to the efforts of plant breeders particularly in genetic variability,gene tags,gene localization,taxonomy,genetic diversity,phylogenetic analysis and also play an important role to decrease the time required for development of new and excellent cultivars.The success of molecular markers technology in genetic improvement programs depends on the close relationship among the plant breeders,biotechnologists,skilled manpower and good financial support.The present review describes application and success of molecular markers technology used for genetic improvement in different fruit crops.

Rice molecular markers and genetic mapping:Current status and prospects

Dramatic changes in climatic conditions that supplement the biotic and abiotic stresses pose severe threat to the sustainable rice production and have made it a difficult task for rice molecular breeders to enhance production and productivity under these stress factors. The main focus of rice molecular breeders is to understand the fundamentals of molecular pathways involved in complex agronomic traits to increase the yield. The availability of complete rice genome sequence and recent improvements in rice genomics research has made it possible to detect and map accurately a large number of genes by using linkage to DNA markers. Linkage mapping is an effective approach to identify the genetic markers which are co-segregating with target traits within the family. The ideas of genetic diversity, quantitative trait locus（QTL） mapping, and marker-assisted selection（MAS） are evolving into more efficient concepts of linkage disequilibrium（LD） also called association mapping and genomic selection（GS）, respectively. The use of cost-effective DNA markers derived from the fine mapped position of the genes for important agronomic traits will provide opportunities for breeders to develop high-yielding, stress-resistant, and better quality rice cultivars. Here we focus on the progress of molecular marker technologies, their application in genetic mapping and evolution of association mapping techniques in rice.

Molecular Characterization of Cowpea Breeding Lines for Striga Resistance Using SCAR Markers

S. gesnerioides （Willd） Vatke is a major biological constraint to cowpea production in the dry savanna of sub-Saharan Africa. Yield losses caused by S. gesnerioides in these regions are estimated in millions of tons annually, and prevalence of Striga soil infestation is steadily increasing. The availability of molecular markers tightly linked to S. gesnerioides resistance genes opens up the possibility of applying Marker-Assisted Selection （MAS） to cowpea and would fast track the process of developing resistance varieties to the parasite. In the present study, we report the use of Fast Technology for Analysis （FTA） also known as PlantSaver Cards （Whatman~ FTA）, developed by Flinder Technology associate to retrieve DNA from plant tissue for molecular analysis. A total of 100 F2 individual plants derived from two crosses were validated for SG3 resistance using two different SCAR markers （MahSe2 and C42B） linked to Striga race 3 （SG3） and 5 （SG5） resistance in other segregating populations. Genomic DNA was successfully recovered from leaf tissues of cowpea pressed onto FTA classic card and the DNA obtained from the FTA papers was found to be suitable for molecular analysis by PCR-based techniques. The marker efficiency of SCAR MahSe2 and C42B in detecting SG3 resistance was 98.5% and 93% respectively. This result revealed the utility of SCAR markers in cowpea breeding programme. Therefore, the application of MAS using FTA technology has the potential to increase efficiency of selection and for molecular characterization of cowpea lines for Striga resistance..

Search for a Microsatellite Marker Linked with Resistance Gene to Xanthomonas axonopodis pv.malvacearum in Brazilian Cotton

The cotton cultivar DELTAOPAL is resistant under field as well as under glasshouse conditions to the Brazilian isolates of Xanthomonas axonopodis pv. malvacearum (Xam). Segregating populations derived from the cross between this cultivar and one susceptible cv. BRS ITA 90, were utilized to identify molecular marker linked with the resistance gene to Xam by “Bulk Segregant Analysis (BSA)”. Two hundred and twenty microsatellite (Single Sequence Repeat—SSR) primers were tested. The amplification products were visualized in polyacrylamide gels stained with silver nitrate. Only one primer was informative and showed polymorphism between the DNA of the parents and their respective bulks of homozygous F2 populations contrasting for resistance and susceptibility, and hence was used to analyze DNA of 120 F2 populations. The microsatellite primer yielded one band of 80 bp linked with the resistance locus, which was absent in the susceptible parent as well as in the bulk of the homozygous susceptible plants of the cross. The segregation ratio as determined by phenotypic analysis was 3R:1S. It is believed that the microsatellite marker was linked with the resistance locus and hence may offer new perspectives for marker assisted selection against the angular leaf spot disease of cotton. It is however, felt necessary to repeat the microsatellite analysis and make sure that the primer is tightly linked with the resistance locus and at the same time verify the genetic distance between the marker and the resistance locus.

Validation of SSR Markers Linked to Flowering Time QTLs in Sorghum through Progeny Test

Flowering time is critically important for crop yield, and detection of its genetic factors with strongly associated DNA markers is necessary in breeding programs. This study was undertaken to validate the quantitative trait loci （QTLs） underlying flowering time of sorghum based on the association between genotypes at SSR marker loci and flowering time in F3 family lines from self-pollinated heterozygous F2 plants developed by crossing between ＂SC112＂---an early flowering variety from Ethiopia and ＂Kikuchi Zairai＂--a late flowering variety from Japan. The results showed that the SSR markers linked to the QTLs on sorghum chromosomes 1, 2, 3, 5b, 7 and 8b were significantly （P 〈 0.05） associated with flowering time, and these markers and the QTLs reported previously are valid. On the other hand, the genotypes at the marker locus SB596 of qFT1-2 on chromosome 1 was not significantly associated with flowering time. The valid DNA markers, SB258 in qFTI-1, SB 1512 in qFT2, SB 1839 in qFT3, SB3369 in qFT5b, SB4096 in qFT7 and SB4540 and SB4660 in qFT8b, might be useful for DNA-marker assisted breeding.

A Pyramid Breeding of Eight Grain-yield Related Quantitative Trait Loci Based on Marker-assistant and Phenotype Selection in Rice(Oryza sativa L.)

1000-Grain weight and spikelet number per panicle are two important components for rice grain yield. In our previous study, eight quantitative trait loci （QTLs） conferring spikelet number per panicle and 1000-grain weight were mapped through sequencing-based genotyping of 150 rice recombinant inbred lines （RILs）. In this study, we validated the effects of four QTLs from Nipponbare using chromosome segment substitution lines （CSSLs）, and pyramided eight grain yield related QTLs. The new lines containing the eight QTLs with positive effects showed increased panicle and spikelet size as compared with the parent variety 93-11. We further proposed a novel pyramid breeding scheme based on marker-assistant and phenotype selection （MAPS）. This scheme allowed pyramiding of as many as 24 QTLs at a single hybridization without massive cross work. This study provided insights into the molecular basis of rice grain yield for direct wealth for high-yielding rice breeding.

A Simple Method for Preparation of Rice Genomic DNA

The extraction of DNA is often the most time consuming and laborious step in high-throughput molecular genetic analysis and marker assisted selection （MAS） programs. A simple method for preparation of rice genomic DNA was developed. A small amount （1~50 mg） of leaf tissue of rice seedling, 500 pL of extraction buffer, and one steel bead were put into a 2-mL microcentrifuge tube. After vigorously mashing for 2 min, 5 μL of supernatant was directly applied to PCR amplification. Otherwise, the supematant was precipitated with two times volume of ethanol to obtain high quality genomic DNA. This method is simple, rapid, low cost, and reliable for PCR analysis. One person can manipulate as many as 96 samples for PCR in 10 min. It is especially suitable for genotyping of large number of samples.

qRgls1.06, a major QTL conferring resistance to gray leaf spot disease in maize

Gray leaf spot(GLS)caused by Cercospora zeae-maydis and C.zeina is an extremely devastating leaf disease that limits maize production annually.The use of GLS-resistant maize hybrids is the most cost-effective approach for reducing losses.Resistance to GLS is quantitatively inherited in maize(Zea mays L.)and further sources of resistance remain to be analyzed.Here,we detected qRgls1.06,a major quantitative trait locus for GLS resistance in bin 1.06 that explained approximately 55%of the phenotype variance.Fine mapping over 2 consecutive years localized qRgls1.06 to a 2.38-Mb region.Homozygous qRgls1.06^(WGR/WGR) plants in DZ01 background displayed higher GLS resistance and 100-grain weight than DZ01 plants.The GLS responses of several susceptible elite inbred lines were improved by the introduction of qRgls1.06 by marker-assisted backcrossing.Our findings extend the understanding of the genetic basis of resistance to GLS and provide a set of resistant germplasm for genetic improvement of resistance to GLS in maize.

Meta-Analysis of 100-Seed Weight QTLs in Soybean

100-seed weight is a very complicated quantitative trait of yield. The study of gene mapping for yield trait in soybean is very important for application. However, the mapping result of 100-seed weight was dispersed, the public map should be chosen which was suitable for the published results integrated, and to improve yield. In this research, an integrated map of 100-seed weight QTLs in soybean had been established with soymap2 published in 2004 as a reference map. QTLs of 100-seed weight in soybean were collected in recent 20 yr. With the software BioMercator 2.1, QTLs from their own maps were projected to the reference map. From published papers, 65 QTLs of 100-seed weight were collected and 53 QTLs were integrated, including 17 reductive effect QTLs and 36 additive effect QTLs. 12 clusters of QTLs were found in the integrated map. A method of meta-analysis was used to narrow down the confidence interval, and 6 additive QTLs and 6 reductive QTLs and their corresponding markers were obtained respectively. The minimum confidence interval （C.I.） was shrunk to 1.52 cM. These results would lay the foundation for marker-assisted selection and mapping QTL precisely, as well as QTL gene cloning in soybean.

Identification and Molecular Mapping of the Rs Dm R Locus Conferring Resistance to Downy Mildew at Seedling Stage in Radish(Raphanus sativus L.)

Downy mildew （DM）, caused by the fungus Peronospora parasitica, is a destructive disease of radish （Raphanus sativus L.） worldwide. Host resistance has been considered as an attractive and environmentally friendly approach to control the disease. However, the genetic mechanisms of resistance in radish to the pathogen remain unknown. To determine the inheritance of resistance to DM, F1, F2 and BC1F1 populations derived from reciprocal crosses between a resistant line NAU-dhp08 and a susceptible line NAU-qtbjq-06 were evaluated for their responses to DM at seedling stage. All F1 hybrid plants showed high resistance to DM and maternal effect was not detected. The segregation for resistant to susceptible individuals statistically iftted a 3：1 ratio in two F2 populations （F2（SR） and F2（RS））, and 1：1 ratio in two BC1F1 populations, indicating that resistance to DM at seedling stage in radish was controlled by a single dominant locus designated as RsDmR. A total of 1 972 primer pairs （1 036 SRAP, 628 RAPD, 126 RGA, 110 EST-SSR and 72 ISSR） were screened, and 36 were polymorphic between the resistant and susceptible bulks, and consequently used for genotyping individuals in the F2 population. Three markers （Em9/ga24370, NAUISSR826700 and Me7/em10400） linked to the RsDmR locus within a 10.0 cM distance were identiifed using bulked segregant analysis （BSA）. The SRAP marker Em9/ga24370 was the most tightly linked one with a distance of 2.3 cM to RsDmR. These markers tightly linked to the RsDmR locus would facilitate marker-assisted selection and resistance gene pyramiding in radish breeding programs.