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.

Marker-assisted selection to pyramid Fusarium head blight resistance loci Fhb1 and Fhb2 in the high-quality soft wheat cultivar Yangmai 15

Fusarium head blight(FHB)is one of the most detrimental wheat diseases which greatly decreases the yield and grain quality,especially in the middle and lower reaches of the Yangtze River of China.Fhb1 and Fhb2 are two major resistance loci against Fusarium graminearum.Yangmai 15(YM15)is one of the most popular varieties in the middle and lower reaches of the Yangtze River,and it has good weak gluten characters but poor resistance to FHB.Here we used Fhb1 and Fhb2 to improve the FHB resistance of YM15 by a molecular marker-assisted selection(MAS)backcrossing strategy.The selection of agronomic traits was performed for each generation.We successfully selected seven introgressed lines which carry homozygous Fhb1 and Fhb2 with significantly higher FHB resistance than the recurrent parent YM15.Three of the introgressed lines had agronomic and quality characters that were similar to YM15.This study demonstrates that the pyramiding of Fhb1 and Fhb2 could significantly improve the FHB resistance in wheat using the MAS approach.

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-b＇ and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region （SCAR） marker tightly linked with Stv-b＇ and a cleaved amplified polymorphic sequence （CAPS） marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.

Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato

The resistance in tomato plants to bacterial speck caused by Pseudomonas syringae pv. tomato is triggered by the interactions between the plant resistance protein Pto and the pathogen avirulence proteins AvrPto or AvrPtoB. Fen is a gene encoding closely related functional protein kinases as the Pto gene. To investigate the status of resistance to the pathogen and natural variation of Pto and Fen genes in tomato, 67 lines including 29 growing in China were subject to disease resistance evaluation and fenthion-sensitivity test. Alleles of Pto and Fen were amplified from genomic DNA of 25 tomato lines using polymerase chain reaction （PCR） and sequences were determined by sequencing the PCR products. The results indicated that none of the 29 cultivars/hybrids growing in China were resistant to bacterial speck race 0 strain DC3000. Seven of eight tomato lines resistant to DC3000 were also fenthion-sensitive. Analysis of deduced amino acid sequences identified three novel residue substitutions between Pto and pto, and one new substitution identified between Fen and fen. A PCR-based marker was developed and successfully used to select plants with resistance to DC3000.

Breeding Rice Restorer Lines with High Resistance to Bacterial Blight by Using Molecular Marker-Assisted Selection

Two bacterial blight （BB） resistance genes, Xa21 and Xa4, from IRBB24 were introduced into hybrid rice restorer line Mianhui 725, which is highly susceptible to BB, by using hybridization and molecular marker-assisted selection technology. Four homologous restorer lines were obtained through testing the R target genes with molecular markers and analyzing parental genetic background. Inoculation of the four lines and their hybrids with the specific strains of Xanthomonas oryzae pv. oryzae, P1, P6 and seven representative strains of Chinese pathotype, C Ⅰ -CⅦ, showed that all of the four lines and their hybrids were highly resistant and presented broad resistance-spectrum to BB. The hybrids of G46A / R207-2 displayed good agronomic characters and high yield potential, and R207-2 was named Shuhui 207.

Marker-assisted Selection of ZmC_4Ppc in Rice Breeding and Yield Trait Performances of Advanced Lines

The full-length of intact Zea mays gene for phosphoenolpyruvate carboxylase gene （ZmC4Ppc） is 6 781 bp. The products of PCR for this gene were not clear with poor repeatability, resulting in that it was difficult for marker-assisted selection （MAS） both in rice and maize. For selecting the markers for MAS, sequences presented only in maize rather than in rice were identified by BLAST, and used for primer design using Primer Premier 5.0. A pair of specific primer termed MRpc （Forward： 5＇ AAGCAGGGAAGCGAGACG 3＇, Reverse： 5＇ GATTGCCGCCAGCAGTAG 3＇） was used for selection of transformed rice, and ZmC4Ppc could be highly and constitutively expressed at each tested developmental stages in the transformed rice selected by using MRpc. Thus, MRpc was used for MAS of progenies carrying ZmC4Ppc gene in rice and some restorer lines with ZmC4Ppc （e.g. FPM881） derived from ZmC4Ppc-transformed Kitaake backcrossed with a restorer line Shuhui 881 were obtained. The analyses on genetic background, PEPCase activity, net photosynthetic rate, general combining ability （GCA） and specific combining ability （SCA） of FPM881 showed that similarity of genetic background reached above 95%, the PEPCase and net photosynthetic rate were higher than those of the control, and some of the progenies carrying ZmC4Ppc gene had better GCA and SCA for grain yield per plant, number of panicles per plant, and 1000-grain weight than those of the control. This suggested that the introduction of maize ZmC4Ppc gene via MAS and its stable expression could increase grain yield of rice and would likely provide a pathway for rice varietal improvement.

Improving Blast Resistance of a Thermo-Sensitive Genic Male Sterile Rice Line GD-8S by Molecular Marker-Assisted Selection

The broad-spectrum blast resistance gene Pi-1, from donor line BL122, was introduced into a thermo-sensitive genic male sterile rice line GD-8S, which possessed good grain quality but high susceptibility to rice blast, by using backcross breeding and molecular marker-assisted selection. Five elite improved male sterile lines, RGD8S-1, RGD8S-2, RGD8S-3, RGD8S-4 and RGD8S-5, were selected based on the results of molecular marker analysis, spikelet sterility, recovery rate of genetic background and agronomic traits. Thirty-three representative blast isolates collected from Guangdong Province, China were used to inoculate the improved lines and the original line GD-8S artificially. The resistance frequencies of the improved lines ranged from 76.47% to 100%, much higher than that of the original line GD-8S （9.09%）. On the agronomic characters, there were no significant differences between the improved lines and GD-8S except for flag leaf length and panicle number per plant. The improved lines could be used for breeding hybrid rice with high blast resistance.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

Breeding of R8012,a Rice Restorer Line Resistant to Blast and Bacterial Blight Through Marker-Assisted Selection

Genetic improvement is one of the most effective strategies to prevent rice from blast and bacterial blight （BB） diseases, the two most prevalent diseases jeopardizing rice production. Rice hybrids with dural resistance to blast and BB are needed for sustainable production of food. An incomplete diallele design resulted in 25 crosses between five blast and five BB resistant germplasm accessions. Only one pair of parents, DH146 ×TM487, showed polymorphism for all the markers to identify one blast resistance gene Pi25 and three BB resistance genes, Xa21, xa13 and xa5, thus it was used in the marker-assisted selection （MAS）. F2 individuals of DH146× TM487 were genotyped using flanking markers of RM3330 and sequence tagged site （STS） marker SA7 for Pi25. The resistant F2 plants with Pi25 were used for pyramiding BB resistance genes Xa21, xa13 and xa5 identified by the markers pTA248, RM264 and RM153, respectively in subsequent generations. Finally, after selection for agronomic traits and restoration ability among 12 pyramided lines, we acquired an elite restorer line, R8012 including all four target genes （Pi25＋Xa21＋xa13＋xa5）. Hybrid Zhong 9NR8012 derived from the selected line showed stronger resistance to blast and BB, and higher grain yield than the commercial checks uniformally in experimental plots, 2007 state-wide yield trial and 2008 nation-wide yield trial. This study provides a paradigmatic example to show that MAS is a practically feasible tool in effectively pyramiding multiple resistance genes. The resultant restoring line and its hybrid would play an important role in securing rice production in China.

Pyramiding the disease resistant genes to southern rust and stalk rot in maize(Zea mays L.) with marker-assisted selection

Southern corn rust(SCR) caused by Puccinia polysora Underw and maize stalk rot caused by Pythium inflatum Matthews(MSR-2) are two destructive diseases of maize(Zea mays L.) in China.Our previous studies indicated that maize inbred line Qi319 is highly resistant to SCR but susceptible to MSR-2,while inbred line 1145 is highly resistant to MSR-2 but susceptible to SCR.The SCR resistant gene(RppQ) in Qi319 and MSR-2 resistant gene(Rpi1) in 1145 have been mapped on chromosome 10 and 4 respectively.In this research,through marker-assisted selection(MAS) with the molecular markers,bnlg1937 tightly linked to Rpi1 and phi041 tightly linked to RppQ,pyramid breeding of the two kinds of disease resistant genes were carried out from the year of 2003 to 2007.Two homozygotic inbred lines of F5 generation,DR94-1-1-1 and DR36-1-1-1 were identified.MAS result suggested DR94-1-1-1 and DR36-1-1-1 contained the two resistance genes RppQ and Rpi1.Field inoculation tests confirmed their high resistance to the two diseases.In addition,field investigation indicated that the two selected inbred lines,particularly DR94-1-1-1,had excellent agronomic traits such as plant height,ear height and yield-relating traits including ear length,ear diameter,ear weight,kernels per ear,kernels per row and kernel weight per ear.The two selected inbred lines DR94-1-1-1 and DR36-1-1-1 can either be directly developed into commercial variety or used as immediate donors of SCR and MSR resistance breeding programs in maize.

Lowering Grain Amylose Content in Backcross Offsprings of indica Rice Variety 057 by Molecular Marker-Assisted Selection

To lower the amylose content （AC） of the indica rice restorer line 057 with high AC, backcrosses were made respectively by using four indica varieties （R367, 91499, Yanhui 559, Hui 527） as low AC donor parents and 057 as the recurrent parent. A molecular marker （PCR-Acc Ⅰ） was used to identify the genotypes （GG, TT and GT） of the waxy （Wx） gene. Plants with GT genotype were selected and used as female parent and crossed with 057 to advance generation. The ACs of rice grains harvested from plants with different Wx genotypes were measured and compared to analyze the efficiency of marker-assisted selection. The ACs of the rice grain, harvested from the plants of Wx genotypes GG, GT and TT, were higher than 20%, in the range of 17.7-28.5%, and less than 18%, respectively. The PCR-Acc Ⅰ marker could be used for efficiently lowering the AC of 057 through backcrossing, and there were some influence of parental genetic background on the AC of rice grains with the same Wx genotype.

Breeding of Maintainer Line and Sterile Line Brown Planthopper Resistant Rice Using Marker-assisted Selection

Guangxi common wild rice variety brown planthopper highly resistant introgression line HS204 was taken as antigen donor material,hybridization,backcrossing,and molecular marker-assisted selection methods were adopted,to select maintainer line and sterile line materials,so as to provide excellent material foundation for resistance breeding of hybrid rice. Through the marker-assisted selection,it obtained 4 pieces of homozygous resistance gene maintainer line intermediate materials( 100B,101B,102B and 103B),and 2 pieces of resistant sterile line materials( 100A and 103A). All 10 combinations that have testcross with highly resistant sterile 100A showed higher level of brown planthopper resistance: 100A/R2586,100A/KR838,and 100A/KR527 had high resistance level,the others had low to intermediate resistance;100A/KR527,100A/R2586,100A/Minghui 63,100A/Fuhui 838 and 100A/Gui 99 combinations had yield per plant significantly higher than the control group( Teyou 7118),increasing by 14. 45%-49. 26%. The obtained resistant lines are expected to provide a better gene source for the breeding of resistant sterile lines of hybrid rice and the obtained resistant sterile lines can be directly used in the selection of three-line hybrid rice.

Stimulation Study of Gene Pyramiding inAnimals by Marker-Assisted Selection

This gene pyramiding strategy is based on the idea of efficiently pyramiding genes of interest by crosses and selection to obtain a population with favorable alleles from different breeds or lines, which is called an ideal population. We investigate impacts of some factors on the pyramiding efficiencies by simulation. These factors include selection strategies （the breeding value selection, the molecular scores selection and the index selection）, proportion selected （2, 10 and 20%）, recombination rates between adjacent target genes （0.1, 0.3 and 0.5） and different mating types （the random mating and the positive assortative mating avoiding sib mating）. The results show that： （1） The more recombination rate and the lower proportion male selected, the better pyramiding efficiency; （2） the ideal population is obtained via various selection strategies, while different selection strategies are suitable for different breeding objectives. From the perspective of pyramiding target genes merely, the molecular scores selection is the best one, for the purpose of pyramiding target genes and recovering genetic background of the target trait, the index selection is the best one, while from the saving cost point of view, the breeding value selection is the best one; （3） the positive assortative mating is more efficient for gene pyramiding compared with the random mating in the terms of the number of generations of intercross for getting the ideal population.

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

Molecular Marker-assisted Selection for Verticillium Wilt Resistance in Upland Cotton (Gossypium hirsutum)

Verticillium wilt is a global important disease ofcotton,which threatens the development ofcotton production seriously.Recent years,because of the change in climate and croppingpattern,Verticillium wilt was broke out incotton production areas in China,which

Marker-Assisted Selection of Xa21 Conferring Resistance to Bacterial Leaf Blight in indica Rice Cultivar LT2

Bacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive diseases in Asian rice fields. A high-quality rice variety, LT2, was used as the recipient parent.IRBB21, which carries the Xa21 gene, was used as the donor parent. The resistance gene Xa21 was introduced into LT2 by marker-assisted backcrossing. Three Xoo races were used to inoculate the improved lines following the clipping method. Eleven BC_3F_3 lines carrying Xa21 were obtained based on molecular markers and agronomic performance. The 11 lines were then inoculated with the three Xoo races. All the 11 improved lines showed better resistance to BLB than the recipient parent LT2. Based on the level of resistance to BLB and their agronomic performance, five lines (BC_3F_3 5.1.5.1, BC_3F_3 5.1.5.12, BC_3F_3 8.5.6.44, BC_3F_3 9.5.4.1 and BC_3F_3 9.5.4.23) were selected as the most promising for commercial release. These improved lines could contribute to rice production in terms of food security.

Molecular mapping of a novel wheat powdery mildew resistance gene Ml92145E8-9 and its application in wheat breeding by marker-assisted selection

Powdery mildew, caused by Blumeria graminis f. sp. tritici(Bgt), is one of the most devastating diseases of common wheat(Triticum aestivum L.). The wheat line 92145 E8-9 is immune to Bgt isolate E09. Genetic analysis reveals that the powdery mildew resistance in 92145 E8-9 is controlled by a single dominant gene, temporarily designated Ml92145 E8-9. Bulkedsegregant analysis(BSA) with simple sequence repeat(SSR) markers indicates that Ml92145 E8-9 is located on chromosome 2 AL. According to the reactions of 92145 E8-9,VPM1(Pm4 b carrier), and Lankao 906(PmLK906 carrier) to 14 Bgt isolates, the resistance spectrum of 92145 E8-9 differs from those of Pm4 b and PmLK906, both of which were previously localized to 2 AL. To test the allelism among Ml92145 E8-9, Pm4 b and PmLK906, two F2 populations of 92145 E8-9 × VPM1(Pm4 b) and 92145 E8-9 × Lankao 906(PmLK906) were developed in this study. Screening of 784 F2 progeny of 92145 E8-9 × VPM1 and 973 F2 progeny of 92145 E8-9 × Lankao 906 for Bgt isolate E09 identified 37 and 19 susceptible plants, respectively. These findings indicated that Ml92145 E8-9 is non-allelic to either Pm4 b or PmLK906. Thus, Ml92145 E8-9 is likely to be a new powdery mildew resistance gene on2 AL. New polymorphic markers were developed based on the collinearity of genomic regions of Ml92145 E8-9 with the reference sequences of the International Wheat Genome Sequencing Consortium(IWGSC). Ml92145 E8-9 was mapped to a 3.6 c M interval flanked by molecular markers Xsdauk13 and Xsdauk682. This study also developed five powdery mildew-resistant wheat lines(SDAU3561, SDAU3562, SDAU4173, SDAU4174, and SDAU4175)using flanking marker-aided selection. The markers closely linked to Ml92145 E8-9 would be useful in marker-assisted selection for wheat powdery mildew resistance breeding.

Fine mapping and marker-assisted selection (MAS) of a low glutelin content gene in rice

Rice with low glutelin content is suitable as functional food for patients affected with diabetes and kidney failure. The fine mapping of the gene(s) responsible for low glutelin content will provide information regarding the distribution of glutelin related genes in rice genome and will generate markers for the selection of low glutelin rice varieties. Following an SDS-PAGE screen of rice germplasm from Taihu Valley of China, Japonica selection W3660 is identified to be a novel mutant characterized with low glutelin content. For fine mapping the mutant gene for low glutelin content, F2 and F3 populations were derived from a cross between W3660 and Jingrennuo. SDS-PAGE analysis of the total endosperm protein showed that the low glutelin content trait was controlled by a single dominant nuclear gene. Genetic mapping, using SSRs, located this gene to chromosome 2, in the region between SSR2-001/SSR2-004 and RM1358. The dis- tances of the two markers to the target gene were 1.1 cM and 3.8 cM respectively. By semi-quantitative RT-PCR analysis, the transcripts of GluB4/GluB5 genes located within the region do not change. However, GluB5 gene located proximal to SSR2-001/SSR2-004 was specifically reduced. SSR profiles of seven Japonica varieties were compared with that of W3660 for loci in the relevant genetic region. The markers SSR2-004 and RM1358 were used for marker- assisted selection. The selection efficiencies of SSR2-004 and RM1358 were 96.8% and 92.7% respectively. This provides a standard starting point for the breeding of low glutelin content rice varieties in China.

Comparison between Marker-assisted Selection and Phenotypical Selection for Fiber Strength and Resistance to Helicoverpa armigera in Upland Cotton

We conducted this study to determine if marker-assisted mass recurrent selection for fiberstrength and resistance to Helicoverpa armigerawithin an upland cotton complex populationcould he more efficient than conventionalphenotype selection.Two cycles of marker-assisted selection,phenotypic selection,andmarker and phenotype concurrent selection,

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.