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.

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

Hybrid Rice Resistant to Bacterial Leaf Blight Developed By Marker Assisted Selection

Through recurrent backcrossing in combination with molecular marker-assisted selection (MAS), restorer lines R8006 and Rl176 carrying Xa-21, a gene having broad-spectrum resistance to rice bacterial leaf blight, were selected. By crossing the two lines to CMS line Zhong 9A, two new hybrid rice combinations, Zhongyou 6 and Zhongyou 1176 were developed. The hybrids showed high resistance to diseases, good grain quality and high yielding potential in national and provincial adaptability and yield trials.

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.

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.

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.

Pyramiding Breeding by Marker-Assisted Recurrent Selection in Upland Cotton: Selected Effects on Resistance to Helicoverpa armigera

The coincidence rates were more than 96% among the instar-weighted average of bioassaysin the lab, the percentage of resistance to Km in the field and the percentage of plantscontaining Bt gene. So, the performance of resistance to Km in the field can be used torepresent the transgenic Bt gene for selecting the resistance to bollworm. The instar-weighted averages were 30.585, 24.182, 16.615, 10.601, 10.123, 7.440 and 7.215 for theC0, P1, M1, M2, MP1, P2 and MP2 populations, respectively. The variance analysisindicated that the instar-weighted average in C0 was greatly significantly higher thanthat in all other populations, i.e., the performance of resistance to bollworm in C0 washighly significantly lower than all other populations. And the resistance in P1 wasgreatly lower than that of M1, M2, MP1, P2 and MP2, and M1 greatly lower than that of M2,MP1, P2 and MP2. There were no significant differences among M2, MP1, P2 and MP2. Withinthe populations of the first cycle selection, MP1 and M1 were greatly significantlyhigher than P1, and MP1 significantly higher than M1. The populations of the second cycleselection were significantly higher than their initial population M1, but no significantdifference among them. The boll size, seed index, the percent of the first harvest yield,fiber length, strength and elongation of the resistant plants to bollworm were significantlylower than that of sensitive plants to bollworm. And the yield of seed and lint cottonof the resistant plant to bollworm were lower than that of the sensitive to bollworm, butno significant difference between them. The boll numbers per plant, lint percent andmicronaire of the resistant plants to bollworm were significantly higher than that of thesensitive plant to bollworm.

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.

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.

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.

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.

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.

Genome Maps, Genetic Diversity and Marker-Assisted Selection for Soybean Improvement

Soybean [Glycine max (L.) Merr.] is a major legume used for human and livestock consumption. It has protein quality and oil contents that closely meet the dietary requirements for both humans and animals (Lusas, 2004).

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.