Survey of the Bru1 gene for brown rust resistance in Brazilian local and basic sugarcane germplasm

Bru1 is currently the major gene conferring brown rust resistance in sugarcane, and diagnostic markers are available. A survey for the presence of this gene was conducted on 391 genotypes including Brazilian cultivars, clones and basic germplasm. The efficiency of these markers for identifying resistant cultivars and artificially inoculated basic germplasm was also evaluated. The Bru1 frequency among cultivars (73.5%) suggests this gene is the prevalent source of brown rust resistance in Brazilian sugarcane breeding programmes. Most of the cultivars known to be resistant were positive for Bru1, although other genes for resistance could be present in lines not having Bru1. Only 17.8% of the basic germplasm accessions were positive for the Bru1 gene, and a low correlation between Bru1 diagnostic markers and brown rust severity was observed for basic germplasm accessions. Overall, Bru1 diagnostic markers proved to be efficient identifying resistant cultivars and clones and have potential to be in screening brown rust resistance in Brazilian breeding programmes.     

Validation of molecular markers for pathogen resistance in potato

DNA markers have a large potential to improve efficiency and precision of conventional plant breeding programmes based on marker‐assisted selection (MAS). In our study, we have evaluated the predictive abilities of the SCAR marker RYSC3 and the CAPS marker GP122₅₆₄ with regard to the PVY resistance genes Ry_adg and Ry_sto, respectively, and of marker TG689 linked to H1 conferring resistance to Globodera rostochiensis and marker HC associated with high levels of G. pallida resistance. The evaluations were made in 28 cultivars and accessions and in 219 progeny genotypes descending from ten different crosses. We observed in all evaluated cultivars and accessions the expected marker patterns according to their phenotypic classification into resistant and susceptible genotypes. However, in part considerable discrepancies were observed when analysing progeny of controlled crosses involving these resistance sources, particularly with respect to H1. Based on these results, practical aspects for the efficient implementation of marker‐assisted selection are discussed, which consider the genetic origin of the material, costs aspects and methodology applied.     

Marker‐assisted selection for rice blast resistance genes Pi2 and Pi9 through high‐resolution melting of a gene‐targeted amplicon

The use of host resistance (R) genes is considered the most cost‐effective option to control the rice blast disease. The two allelic R genes Pi2 and Pi9 confer very broad‐spectrum resistance against blast isolates collected worldwide. However, the two genes have not yet been widely deployed in rice breeding programmes. Availability of specific markers for them would facilitate incorporating the two R genes into new rice lines through marker‐assisted selection. Herein, we report the development and utilization of a robust and specific marker for the Pi2 and Pi9. This marker was derived from polymorphisms within the target gene, and achieved simultaneously distinguish Pi2 and Pi9 from other alleles through high‐resolution melting of a small amplicon. With the marker, we were able to transfer the Pi2 into an elite restorer line through marker‐assisted backcrossing, successfully obtained effective resistance to blast disease, and we were also able to, respectively, incorporate the Pi2 and Pi9 with two other R genes. As the additive effect, blast resistance in these stacking lines harbouring three R genes were significantly improved.     

Molecular markers for late blight resistance breeding of potato: an update

Late blight is the most devastating disease of the potato crop that can be effectively managed by growing resistant cultivars. Introgression of resistance (R) genes/quantitative trait loci (QTLs) from the Solanum germplasm into common potato is one of the plausible approaches to breed resistant cultivars. Although the conventional method of breeding will continue to play a primary role in potato improvement, molecular marker technology is becoming one of its integral components. To achieve rapid success, from the past to recent years, several R genes/QTLs that originated from wild/cultivated Solanum species were mapped on the potato genome and a few genes were cloned using molecular approaches. As a result, molecular markers closely linked to resistance genes or QTLs offer a quicker potato breeding option through marker‐assisted selection (MAS). However, limited progress has been achieved so far through MAS in potato breeding. In near future, new resistance genes/QTLs are expected to be discovered from wild Solanum gene pools and linked molecular markers would be available for MAS. This article presents an update on the development of molecular markers linked to late blight resistance genes or QTLs by utilization of Solanum species for MAS in potato.     

Development and validation of a PCR-based functional marker system for the brown planthopper resistance gene Bph14 in rice

Brown planthopper (BPH) is the most damaging rice pest globally. Resistant varieties are the most effective and environmental strategy for protecting the rice crop from BPH. Functional markers (FMs) designed from polymorphic sites within gene sequences affecting phenotypic variation are highly efficient when used for marker assisted selection (MAS). Bph14 is the first and only cloned insect resistance gene so far in rice. Compared to the sequences of its non-effective alleles there are a number SNP differences. In this study, the method of allele-specific amplification (ASA) was adopted to design a simple, co-dominant, functional marker Bph14P/N for Bph14. Bph14P/N was combined with two specific dominant markers: one, named Bph14P, targets the promoter region of Bph14 and amplifies 566 bp fragments; and the other, Bph14N, targets the LRR region of bph14 and amplifies 345 bp fragments. Specificity and applicability of the functional marker system were verified in two breeding populations and a Chinese mini core collection of Oryza sativa. We recommend the use of this simple, low-cost marker system in routine genotyping for Bph14 in breeding populations.     

Application of marker‐assisted backcross to introgress Bph3, Bph14 and Bph15 into an elite indica rice variety for improving its resistance to brown planthopper

To improve brown planthopper (Nilaparvata lugens Stål; BPH) resistance of an elite indica cultivar of South China, Hemeizhan (HMZ), we applied marker‐assisted backcross (MABC) to incorporate three BPH‐resistance genes (Bph3, Bph14 and Bph15) into the genetic background of HMZ. In the third backcross (BC₃) generation, we obtained near‐isogenic lines (Bph3‐NIL, Bph14‐NIL, Bph15‐NIL and Bph14 + Bph15‐NIL) with more than 96% recovery of recurrent parent genome, and pyramided lines (Bph3 + Bph14‐PYL, Bph3 + Bph15‐PYL and Bph3 + Bph14 + Bph15‐PYL) with more than 89% recovery of recurrent parent genome. These lines showed stronger resistance against BPH than HMZ at seedling and booting stages. The rank of resistance gene effect was Bph3 + Bph14 + Bph15 ≥ Bph3 + Bph15 ≥ Bph3 +Bph14 ≥ Bph14 + Bph15 ≥ Bph3 ≥ Bph15 ≥ Bph14 > none. Compared with HMZ, only Bph3 + Bph14 + Bph15‐PYL had a significant difference in yield per plant, and the lines carrying Bph3 had higher amylose contents, indicating that Bph3 was tightly linked to Wx^a allele. These improved lines are good intermediate sources of broad‐spectrum and durable BPH resistance to improve other indica cultivars. Our results demonstrate that MABC is a very efficient approach to improve BPH resistance of elite rice cultivar.     

Development of gene‐based markers for the Turnip mosaic virus resistance gene retr02 in Brassica rapa

Turnip mosaic virus (TuMV) is responsible for a serious disease that affects the production of Chinese cabbage. Previous studies have cloned a series of TuMV resistance genes and developed molecular markers. In this study, a derived cleaved amplified polymorphism sequence (dCAPS) marker and a Kompetitive Allele Specific PCR (KASP) marker were developed based on a single recessive gene, retr02, which confers broad‐spectrum TuMV resistance in Chinese cabbage by means of an additional G at the junction of exon 1 and intron 1. The two markers were able to detect the retr02 allele in Chinese cabbage accessions used in breeding programmes. Compared with the dCAPS marker, the KASP marker was flexible, cost‐effective and quick to process, which is likely to be beneficial in establishing high‐throughput assays for marker‐assisted selection.     

Rapid survey for presence of a blast resistance gene Pi-ta in rice cultivars using the dominant DNA markers derived from portions of the Pi-ta gene

The Pi‐ta gene in rice confers resistance to strains of the blast pathogen Magnaporthe grisea (Herbert) Borr. (anamorph Pyricularia oryza Cav.) containing the corresponding avirulence gene AVR‐Pita in a gene‐for‐gene fashion. The Pi‐ta gene is a typical nucleotide‐binding site type resistance gene. Nucleotide sequences distinguishing the resistant Pi‐ta and susceptible pi‐ta alleles were previously identified and used for developing DNA markers for a resistant Pi‐ta haplotype and three susceptible pi‐ta haplotypes. In the present study, the existence of the Pi‐ta gene in 141 rice germplasm accessions was rapidly determined using these markers, and the results were confirmed by inoculating rice germplasm with an M. grisea strain containing AVR‐Pita. The Pi‐ta gene was found in accessions from several major rice producing countries, including China, Colombia, Japan, Vietnam, the Philippines, Iran and the United States. The usefulness of DNA markers for rapid determination of the genotype of rice germplasm was thus demonstrated. The Pi‐ta gene also was found in rice cultivar known to contain the Pi‐ta² gene, although the allelic relationship of these genes remains to be determined. The presence of the Pi‐ta gene in landrace cultivars in several different geographical locations, the Philippines and Vietnam, other indica rice cultivars in China and Colombia suggest that the Pi‐ta gene may have spontaneously originated in indica rice cultivars. These results are useful for incorporating the Pi‐ta gene into advanced breeding lines by marker‐assisted selection for rice breeding programmes worldwide.     

培育水稻恢复系抗稻褐飞虱基因导入系和聚合系

稻褐飞虱是水稻的主要虫害之一,培育优良的抗性基因聚合系对于防治稻褐飞虱具有重要的意义.本研究通过回交、分子标记辅助选择和接虫鉴定三者相结合的办法,将抗稻褐飞虱基因Bph3和Bph24(t)分别导入主栽杂交水稻恢复系广恢998、9311、R15、明恢63、R29中,最终获得遗传稳定的Bph3导入系32份和Bph24(t)...     

Genetic analysis of resistance in barley to barley yellow dwarf virus

The inheritance of resistance to barley yellow dwarf virus (BYDV) was studied in the selected 24 spring and winter barley cultivars that showed a high or intermediate resistance level in 1994‐97 field infection tests. The polymerase chain reaction diagnostic markers YLM and Ylp were used to identify the resistance gene Yd2. The presence of the Yd2 gene was detected with both markers in all the resistant spring barley cultivars and lines from the CIMMYT/ICARDA BYDV nurseries. The results of field tests and genetic analyses in winter barley corresponded with marker analyses only when the Ylp marker was used. Genes non‐allelic with Yd2 were detected by genetic analyses and the Ylp marker in moderately resistant spring barley cultivars ‘Malvaz’, ‘Atribut’ and ‘Madras’, and in the winter barley cultivars ‘Perry’ and ‘Sigra’. Significant levels of resistance to BYDV were obtained by combining the resistance gene Yd2 with genes detected in moderately resistant cultivars. The utilization of analysed resistance sources in barley breeding is discussed.     

RFLP/AFLP mapping of a brown planthopper (Nilaparvata lugens Stål) resistance gene Bph1 in rice

We have constructed a linkage map of the rice brown planthopper (BPH)resistance gene, Bph1. RFLP and AFLP markers were selected by thebulked segregant analysis and used in the mapping study of 262 F₂sthat were derived from a cross of `Tsukushibare', a susceptible japonica cultivar, and `Norin-PL3', an authentic japonicaBph1-introgression line. Twenty markers were mapped within a 28.9-cMregion containing the Bph1 locus on the long arm of rice chromosome12. Combining the result of segregation analysis of BPH resistance by themass seedling test and that of the markers, the Bph1 locus wasmapped within a 5.8-cM region between two flanking markers. The closestAFLP markers, em5814N and em2802N, was at 2.7 cM proximal to theBph1 locus. Together with the previously constructed high-resolutionmap of bph2 locating the locus at ca. 10 cM proximal to the Bph1 locus, this improved version of the linkage map would facilitatepyramiding these two important BPH resistance genes.     

水稻抗褐飞虱基因及其育种应用研究进展

褐飞虱是危害水稻生产最重要的害虫之一。遏制水稻褐飞虱为害的最安全、有效的措施既利用水稻自身的抗虫性。迄今为止,已经于栽培稻和野生稻中成功鉴定出30个水稻抗褐飞虱基因,其中26个主效抗性基因已被定位,显性基因Bph14已成功克隆,但仅少量主效抗性基因被育种家利用并培育出抗性品种。对褐飞虱的生物型和抗性机理、水稻抗褐飞虱的遗传基础及育种应用研究进行了综述,并就今后的抗性育种研究趋势展开了讨论。     

A new gene <Emphasis Type="Italic">Bph33</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) conferring resistance to brown planthopper (BPH), <Emphasis Type="Italic">Nilaparvata lugens</Emphasis> (Stål) in rice line RP2068-18-3-5

The rice brown planthopper (BPH) Nilaparvata lugens (Stål) is one of the major pests of rice across Asia. Host-plant resistance is the most ecologically acceptable means to manage this pest. A rice breeding line RP2068-18-3-5 (RP2068) derived from the land race Velluthacheera is reported to be resistant to BPH populations across India. We identified a new R gene [Bph33(t)] in this line using advanced generation RILs derived from TN1 × RP2068 cross through phenotyping at two locations and linkage analysis with 99 polymorphic SSR markers. QTL analysis through IciMapping identified at least two major QTL on chromosome 1 influencing seedling damage score in seed box screening, honey dew excretion by adults and nymphal survival. Since no BPH R gene has been reported on chromosome 1, we designate this locus as a new gene Bph33(t) which accounted for over 20% of phenotypic variance. Scanning the region for candidate gene suggested two likely candidates a leucine rich repeat (LRR) gene and a heat shock protein (HSP) coding gene. Expression profiling of the two genes in the two contrasting parents and RILs showed induction of the HSP gene (LOC_Os01g42190.1) at 6 h after infestation while LRR gene did not show such induction. It is likely that the HSP represented Bph33(t).     

User‐friendly markers linked to Fusarium wilt race 1 resistance Fw gene for marker‐assisted selection in pea

Fusarium wilt is one of the most widespread diseases of pea. Resistance to Fusarium wilt race 1 was reported as a single gene, Fw, located on linkage group III. The previously reported AFLP and RAPD markers linked to Fw have limited usage in marker‐assisted selection due to their map distance and linkage phase. Using 80 F₈ recombinant inbred lines (RILs) derived from the cross of Green Arrow × PI 179449, we amplified 72 polymorphic markers between resistant and susceptible lines with the target region amplified polymorphism (TRAP) technique. Marker–trait association analysis revealed a significant association. Five candidate markers were identified and three were converted into user‐friendly dominant SCAR markers. Forty‐eight pea cultivars with known resistant or susceptible phenotypes to Fusarium wilt race 1 verified the marker–trait association. These three markers, Fw_Trap_480, Fw_Trap_340 and Fw_Trap_220, are tightly linked to and only 1.2 cM away from the Fw locus and are therefore ideal for marker‐assisted selection. These newly identified markers are useful to assist in the isolation of the Fusarium wilt race 1 resistance gene in pea.     

Assessing diversity in Triticum durum cultivars and breeding lines for high versus low cadmium content in seeds using the CAPS marker usw47

Cadmium (Cd) is a toxic heavy metal that occurs naturally in soils. Durum wheat is known to accumulate generally more Cd than other cereal crops. The uptake of Cd in durum wheat is governed by the gene Cdu1, which co‐segregates with several DNA markers, such as the co‐dominant marker usw47 and the dominant marker ScOPC20. A panel of 314 durum wheat cultivars or lines originating from 16 countries or regions were assessed with usw47. The plant material was mainly comprised of cultivars and modern breeding lines. From this set, 165 durum wheat lines were classified as low‐Cd accumulators, 144 high‐Cd accumulators and five were heterogeneous. A smaller subset of 16 cultivars had previously been evaluated for Cd accumulation in replicated field trials. Lines with the high‐Cd allele showed a 2.4‐fold higher Cd content in the seeds than lines with the low‐Cd allele. We also compared the utility of markers usw47 and ScOPC20 as selection tools. Marker‐assisted selection appears as a robust and practicable tool for breeding durum cultivars with low‐Cd content.     

Mapping of quantitative trait loci for resistance to race 1 of Pyrenophora tritici‐repentis in synthetic hexaploid wheat

Tan spot, caused by a necrotrophic fungus Pyrenophora tritici‐repentis (Ptr), has become an important foliar disease of wheat worldwide. Effective control of tan spot can be achieved by deployment of resistant wheat cultivars. An F_2:3 population derived from a cross between synthetic hexaploid wheat (SHW), TA4161‐L1 (moderately resistant) and susceptible winter wheat cultivar, ‘TAM105’ was evaluated with race 1 of Ptr under controlled conditions. The population was genotyped using Diversity Arrays Technology (DArT). Presence of transgressive segregants indicated contribution of positive alleles from both parents. Two major QTLs were located on the short arm of chromosomes 1A and 6A and designated as QTs.ksu‐1A and QTs.ksu‐6A, respectively. Two additional QTLs were identified on chromosome 7A. Resistant alleles of all the QTLs were contributed by TA4161‐L1. Novel QTLs on 6A and 7A can be a valuable addition to known resistance genes and utilized in breeding programmes to produce highly resistant cultivars.     

Genetic mapping of bph20(t) and bph21(t) loci conferring brown planthopper resistance to Nilaparvata lugens St?l in rice (Oryza sativa L.)

Brown planthopper(BPH) is one of the most serious and destructive insect pests of rice in most rice growing regions of the world. In this study, two major resistance genes against BPH have been identified in an Oryza rufipogon (Griff.) introgression rice line, RBPH54. Inheritance of the BPH resistance in RBPH54 was studied by screening the resistance in parents, F1, F2 and BC1 generations against BPH biotype 2. A population of BC3F2 lines was developed and SSR markers were employed for the gene mapping, and new markers were designed for fine mapping of the resistance genes, while sequence information of BAC/PAC clones was used to construct physical maps of the genes. The results showed that the BPH resistance in RBPH54 was governed by recessive alleles at two loci, tentatively designated as bph20(t) and bph21(t). The locus bph20(t) was fine mapped to the short arm of chromosome 6 about 2.7 cM to the upper marker RM435 and 2.5 cM to lower marker RM540 and in a 2.5 cM region flanked by two new SSR markers BYL7 and BYL8 which were developed in the present study. The other BPH resistance locus bph21(t) was initially mapped to a region 7.9 cM to upper marker RM222 and 4.0 cM to lower marker RM244 on the short arm of chromosome 10. For physical mapping, the bph20(t)-linked markers were landed on BAC/PAC clones of the reference cv., Nipponbare, released by the International Rice Genome Sequencing Project. The bph20(t) locus was physically defined to an interval of about 75 kb with clone P0514G1. Identification and location of these two genes in the present study have diversified the BPH resistance gene pool, which give benefit to the development of resistant rice cultivars, and the linkage PCR-based SSR markers for the bph20(t) and bph21(t) genes would help realize the application of the genes in rice breeding through marker-assisted selection.     

Multiplex PCR genotyping for five bacterial blight resistance genes applied to marker‐assisted selection in rice (Oryza sativa)

Bacterial blight (BB) is the most economically damaging disease of rice in Asia and other parts of the world. In this study, a multiplex PCR genotyping method was developed to simultaneously identify genotypes of five BB resistance genes, Xa4, xa5, Xa7, xa13 and Xa21. The resistance R alleles were amplified using five functional markers (FMs) to generate amplicons of 217, 103, 179, 381 and 595 bp in IRBB66. Amplicons of 198, 107, 87, 391 and 467 bp corresponded to susceptible alleles in Taiwanese japonica rice cultivars. In backcross breeding programmes, the multiplex PCR assay was integrated into selection from a population using BB resistance donor IRBB66 crossed to rice cultivar ‘Tainung82’. Two plants with homozygosity for Xa4, xa5, Xa7, xa13 and Xa21 were selected from 1100 BC₂F₂ plants. In addition, the five BB resistance genes were also accurately identified in F₂ populations. This multiplex PCR method provides a rapid and efficient method for detecting various BB resistance genes, which will assist in pyramiding genes to improve durability of BB resistance in Taiwanese elite rice cultivars.