Molecular screening for identification of blast resistance genes in North East and Eastern Indian rice germplasm (Oryza sativa L.) with PCR based makers

Molecular screening and genetic diversity of major rice blast resistance (R) genes were determined in 32 accessions of rice germplasm from North East and Eastern India with ten gene based single nucleotide polymorphisms and sequence tagged sites (STS) markers, namely z56592, zt56591, k39512, k3957, candidate gene marker, Pita3, YL155/YL87, YL183/YL87, Pb28, 195R-1 which showed close-set linkage to nine major rice blast resistance (R) genes, Piz, Piz-t, Pik, Pik-p, Pik-h, Pita/Pita-2, Pib and Pi9 and one susceptible pita gene. Among the 32 accessions, 13 were positive for Piz gene and six for Piz-t gene. Six accessions were positive for Pik gene, seven for Pik-p and 16 for Pik-h gene. One accession, Atte thima, was positive for three of Pik multiple genes. Out of 32, only two germplasm, Dudhraj and Nepali dhan, were detected with both Pita3 and YL155/YL87 marker for Pita/Pita-2 gene. The Pib gene appeared to be omnipresent and was detected in 31 of 32 germplasm with marker Pb28. The gene specific STS marker, 195R-1, for Pi9 gene produced positive bands in only two germplasm, Kalchatti and Bachi thima. The Uniform Blast Nursery (UBN) analysis showed that out of 32, six germplasm was resistant, ten moderately resistant and 16 germplasm were susceptible. Presence of Piz-t, Pita/Pita-2 and Pi9 gene ensured a resistant reaction in outdoor blast nursery whereas germplasm carrying Pib was susceptible when present alone. Presence of multiple genes, however, contributed to slow blasting resistance in the field. These results are useful in identification and incorporation of resistant genes from the germplasm into elite cultivars through marker assisted selection in rice breeding programs.     

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.     

水稻抗稻瘟病基因Pi35功能性分子标记的开发及其应用

稻瘟病是水稻生产上的严重病害,利用抗病基因培育抗病品种是控制稻瘟病最经济而有效的措施。在日本,稻瘟病部分抗性基因Pi35作为广谱持久抗性基因已广泛应用于水稻育种和稻瘟病防治实践。但是,Pi35基因在我国的资源和品种中的分布情况不清,制约了这一重要基因在我国育种实践中的应用,急需开发实用的分子标记,并系统研究该基因在我国的品种及其亲本中的分布情况,为稻瘟病抗性育种服务。本研究通过比对抗、感品种中Pi35等位基因序列,发现一个能检测抗、感病性差异的特异SNP(3780 T),并据此开发了Pi35基因的功能性分子标记Pi35-d CAPS。利用该标记检测了抗源藤系138的衍生品种10份、微核心种质204份和主栽品种67份,结合测序鉴定,确认5份藤系138衍生品种(垦鉴稻3号、垦鉴稻6号、垦稻8号、绥粳3号和龙粳34)及2份微核心种质(粳稻品种抚宁紫皮粳子和籼稻品种细麻线)携带Pi35基因。本研究结果为通过分子育种手段高效利用Pi35基因改良我国水稻(特别是籼稻)品种的稻瘟病抗性提供了手段。     

水稻抗稻瘟病基因Pi-ta的分子标记辅助选择

利用已建立的水稻抗稻瘟病基因Pi-ta显性分子标记对30个品系和157个来自不同国家的一些水稻品种进行分子鉴定,并采用稻瘟病菌菌株ZN57（IC-17）和ZN61（IB-49）人工接种试验进行致病性测试。结果表明,大部分品系和少数水稻品种含抗病基因Pi-ta,且对稻瘟病菌菌株ZN57和ZN61表现抗病反应。除此之外,利用两对显性分子标记YL1     

Molecular mapping of a novel blast resistance gene Pi38 in rice using SSLP and AFLP markers

Blast, caused by Magnaporthe grisea, is the most devastating disease of rice worldwide. In this study, the main objective was to identify and map a new gene for blast resistance, in an indica rice cultivar ‘Tadukan’ against blast fungal isolate B157, using molecular tools. F₂ segregating population was derived from ‘CO39’ (susceptible) and ‘Tadukan’ (resistant), and molecular mapping of the blast resistance gene was carried out using simple sequence length polymorphism (SSLP) and amplified fragment length polymorphism (AFLP) methods. Two SSLP markers, RM206 and RM21 and three AFLP markers (AF1: E‐aca/M‐ctt; AF2: E‐aca/M‐cat and AF3: E‐acc/M‐cac2) were identified to be linked to the resistance gene. The co‐segregation analysis using SSLP markers implied that the blast resistance gene designated Pi38 resides on rice chromosome 11.     

Analysis of rice blast resistance gene <Emphasis Type="Italic">Pi</Emphasis>-<Emphasis Type="Italic">z</Emphasis> in rice germplasm using pathogenicity assays and DNA markers

The Pi-z gene in rice confers resistance to a wide range of races of the rice blast fungus, Magnaporthe oryzae. The objective of this study was to characterize Pi-z in 111 rice germplasm accessions using DNA markers and pathogenicity assays. The existence of Pi-z in rice germplasm was detected by using four simple sequence repeat (SSR) markers (RM527, AP4791, AP5659-1, AP5659-5) closely linked to Pi-z, and was verified using pathogenicity assays with an avirulent strain (IE1k) and two virulent races (IB33 and IB49). Among 111 germplasm accessions evaluated, 73 were found to contain the Pi-z gene using both SSR markers and pathogenicity assays. The remaining 38 germplasm accessions were found to be inconsistent in their responses to the blast races IB33, IEIk and IB49 with expected SSR marker alleles, suggesting the presence of unexpected SSR alleles and additional R gene(s). These characterized germplasm can be used for genetic studies and marker-assisted breeding for improving blast resistance in rice.     

稻瘟病抗性基因Pi25特异性CAPS标记的开发与验证

为在水稻育种中快速与高效利用稻瘟病抗性基因Pi25, 本文利用该基因不同等位基因编码区序列差异开发了4套CAPS标记(CAP1/Hinc II、CAP3/Bgl II、CAP3/Nde I和CAP3/Hpy 99I), 并利用169份稻种资源、98个重组自交系(RIL)以及217个水稻转基因后代, 对4套标记的准确性和选择效果进行了验证。结果表明, 4套标记均能准确地检测Pi25/pi25座位。其中, 标记CAP1/Hinc II和CAP3/Hpy 99I特异性识别并酶切显性等位基因, 而标记CAP3/Bgl II和CAP3/Nde I特异性识别并酶切隐性等位基因。利用稻瘟病菌株JS001-20接种RIL与转基因材料, 抗性表现与标记检测的结果完全一致, 表明该CAPS标记准确可靠。分析稻种资源后发现, Pi25基因频率较低(1.2%), 说明该基因在我国水稻稻瘟病抗性育种中还没有被充分利用。本文的研究结果特别是开发的2对识别并酶切显性等位基因的CAPS标记可用于分子标记辅助选择, 改良我国早籼稻的稻瘟病抗性。     

Development of a SNLP marker from the Pi-ta blast resistance gene by tri-primer PCR

The Pi-ta gene from indica introgressed into japonica rice has been used to control the blast disease caused by the fungal pathogen Magnaporthe grisea (Herbert) Barr. (anamorph Pyricularia oryzae Cav.) worldwide. A single nucleotide length polymorphism (SNLP) was identified at the intron region of the Pi-ta gene to develop a codominant Pi-ta gene marker suitable for genotyping with an automated machine. The DNA primer specific to the resistant Pi-taallele was labeled with blue dye (FAM, 6-carboxyfluorescein) as a forward primer, the DNA primer specific to the susceptible pi-ta allele was labeled with green dye (HEX, 4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein) as another forward primer and the DNA primer identical to both Pi-ta/pi-ta alleles was unlabeled as the reverse primer for polymerase chain reaction (PCR). Using these three primers, a 181-bp blue peak in homozygous resistant and a green peak of 182–183 bp in homozygous susceptible, and both peaks in heterozygous plants were produced by PCR. The utility of marker was verified using a segregating F₂ population, inbred cultivated lines, dominant markers and pathogenicity testing. A codominant Pi-ta marker was thus developed for effective Pi-ta assisted selection for crop improvement. Using highly homologous competitive primers for allele detection by PCR can benefit the study of genome organization of the complex locus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic variation in resistance to blast disease (Pyricularia oryzae Cavara) in Japanese rice (Oryza sativa L.), as determined using a differential system

A total of 324 Japanese rice accessions, including landrace, improved, and weedy types were used to 1) investigate genetic variations in blast resistance to standard differential isolates, and 2) across the genome using polymorphism data on 64 SSR markers. From the polymorphism data, the accessions were classified into two clusters. Accessions from irrigated lowland areas were included mainly in cluster I, and upland and Indica types were mainly in cluster II. The accessions were classified into three resistance subgroups, A2, B1 and B2, based on the reaction patterns to blast isolates. The accessions in A2 were postulated to have at least two resistance genes Pish and Pik-s, whereas those in B1 had various combinations of the resistance genes Pish, Pia, Pii, Pi3, Pi5(t), and Pik alleles. The B2 accessions were resistant to almost all isolates, and many accessions of cluster II were included, and had Pish, Pia, Pii, Pi3, Pi5(t), certain Pik, Piz and Pita alleles, and unknown genes. The frequencies of accessions of B1 originating in Hokkaido, and those of B2 originating in the Kanto and Tohoku regions were remarkably higher than in the other regions.     

Role of the rice blast resistance gene Pi-cd in rice (Oryza sativa) breeding programmes

A series of DNA markers for various agronomic traits may accelerate the success of marker-assisted selection in practical plant breeding programmes. Here, we developed DNA markers for the blast resistance gene Pi-cd. In this study, we examined the effects of the Pi-cd locus on not only blast resistance but also agronomic traits in agriculture. We developed three pyramiding lines (PLs) coupling Pi-cd with three blast resistance genes, pi21, Pi35 and Pi39. The effect of Pi-cd on blast resistance was dependent on the coupled resistance genes. Then, we evaluated the effects of Pi-cd on 13 agronomic traits. Amylose content and 1,000-grain weight showed significant differences between the PLs and current commercial varieties, which had no negative effects on agronomic trait values. Furthermore, we investigated the distribution of genotype for the Pi-cd locus among varieties of upland rice. The KT genotype specific to rice blast resistance may be predominant in the varieties. The results suggested that Pi-cd has the potential to be useful for improving blast resistance in rice breeding programmes.     

Genomic variation and genetic relationships in Ipomoea spp.

Random amplified polymorphic DNA (RAPD) markers were used to develop genetic fingerprints and analyse genetic relationships among 29 Ipomoea accessions from different geographical locations around the world, including unique wild species, and reproducible profiles were obtained for all accessions using random decamer primers. The primers generated 46 polymorphic markers, one primer alone having 10 products, enabling the discrimination of all 29 accessions. A high level of genetic variability in sweet potato collections was suggested by the degree of polymorphism. Half of the Japanese land races were closely related while accessions from Papua New Guinea and The Philippines were distinct and exhibited the greatest genetic diversity. The wild species Ipomoea gracilis and Ipomoea tiliacea formed a group distinct from the cultivated sweet potato. The wild tetraploid accession K233 and the species Ipomoea trifida were progressively more related genetically to the cultivated sweet potato and are the probable progenitors of Ipomoea batatas, and may be suitable as germplasm for genetic enhancement. RAPDs proved to be useful for sweet potato systematics and should be valuable for germplasm management, gene tagging and efficient choice of parents in breeding programmes.     

Analysis of population structure and genetic diversity reveals gene flow and geographic patterns in cultivated rice (<Emphasis Type="Italic">O. sativa</Emphasis> and <Emphasis Type="Italic">O. glaberrima</Emphasis>) in West Africa

To fully exploit the diversity in African rice germplasm and to broaden the gene pool reliable information on the population genetic diversity and phenotypic characteristics is a prerequisite. In this paper, the population structure and genetic diversity of 42 cultivated African rice (Oryza spp.) accessions originating from West Africa (Benin, Mali and Nigeria, Liberia etc.) were investigated using 20 simple sequence repeats (SSR) and 77 amplified fragment length polymorphisms (AFLP). Additionally, field trials were set up to gain insight into phenotypic characteristics that differentiate the genetic populations among rice accessions. The analysis revealed considerably high polymorphisms for SSR markers (PIC mean?=?0.78) in the germplasm studied. A significant association was found between AFLP markers and geographic origin of rice accessions (R?=?0.72). Germplasm structure showed that Oryza sativa accessions were not totally isolated from Oryza glaberrima accessions. The results allowed identification of five O. glaberrima accessions which grouped together with O. sativa accessions, sharing common alleles of 18 loci out of the 20 SSR markers analyzed. Population structure analysis revealed existence of a gene flow between O. sativa and O. glaberrima rice accessions which can be used to combine several interesting traits in breeding programs. Further studies are needed to clarify the contributions of this gene flow to valuable traits such as abiotic and biotic stresses including disease resistance.     

Mapping of a blast field resistance gene Pi39(t) of elite rice strain Chubu 111

We investigated the mode of inheritance and map location of field resistance to rice blast in the elite rice strain Chubu 111, and yield under severe blast conditions. Chubu 111 carries the complete resistance gene Pii, although field testing showed this strain to be susceptible to infection. The level of field resistance of Chubu 111 was so high that chemicals used to control blast were not required, even in an epiphytotic area. Genetic analysis of field resistance to blast in 149 F₃ lines derived from a cross between Chubu 111 and the susceptible cultivar ‘Mineasahi’ suggested that field resistance is controlled by a dominant gene, designated Pi39(t), that cosegregates with the single sequence repeat marker loci RM3843 and RM5473 on chromosome 4. Comparative studies of polymorphism at RM3843 among Chubu 111 and six cultivars or lines in its pedigree suggested that the donor of the resistance gene was the Chinese cultivar ‘Haonaihuan’. Marker‐assisted selection of Pi39(t) should be useful in rice‐breeding programmes for field resistance to blast.     

Resistance spectrum assay and fine mapping of the blast resistance gene from a rice experimental line, IRBLta2-Re

In the present study, we performed the resistance assessment by rice blast inoculation on IRBLta2-Re and IRBLta-CP1, the experimental lines supposed to carry rice blast resistance genes Pita2 and Pita, respectively. The analysis by using 196 rice blast isolates derived from China indicated that the resistance spectrum of IRBLta2-Re was broader than that of IRBLta-CP1. Both IRBLta2-Re and IRBLta-CP1 have the Pita gene by analyzing the functional single amino acid difference of Pita/pita locus. To identify the additional gene in IRBLta2-Re, 1250 F₂ individuals from the cross between CO39 and IRBLta2-Re were used as the mapping population. The F₂ population was inoculated with the blast isolate 08-T4 which was incompatible to IRBLta2-Re, but compatible to CO39 and IRBLta-CP1. In the phenotypic data analysis, the F₂ population segregated in a 3:1 ratio for resistant and susceptible plants, respectively, suggesting that IRBLta2-Re has an additional resistance gene other than Pita, which was tentatively designated Pita3(t) (supposed to be Pita2). To identify the Pita3(t), a total of 50 microsatellite and 12 position specific microsatellite markers distributed by two sides of the Pita gene were selected in the parent polymorphism screening. The results showed that PT4 and PT5 were co-segregated with the target gene. A contig map corresponding to the resistance gene and Pita genes was constructed based on the fine mapping and bioinformatics assay. The resistance gene, Pita3(t), was, thus, assumed to be in an interval of approximately 178 kb which containing a total of 5 NBS–LRR genes, and was about 500 kb away from the Pita gene.     

Frequency and distribution of the brown rust resistance gene Bru1 and implications for the Louisiana sugarcane breeding programme

Brown rust, caused by the fungus Puccinia melanocephala, poses an increasing threat to sugarcane industries worldwide. Recently, markers R12H16 and 9020‐F4 were developed for a major resistance gene Bru1 that contributes to a significant proportion of brown rust resistance in multiple sugarcane industries. Marker‐assisted screening of Louisiana sugarcane germplasm showed a low frequency (4.3%, five out of 117 clones) of Bru1 among sugarcane cultivars and elite breeding clones. Likewise, among progeny of crosses involving wild/exotic germplasm, only 14 of 208 clones (6.7%) tested Bru1 positive. However, Bru1 frequency was higher (28.7%, 52 of 181 clones) in wild/exotic germplasm, which indicated that diverse genetic resources are available for Bru1 introgression. Commercial Bru1‐positive cultivar, ‘L 01‐299', was resistant to brown rust. However, Bru1‐positive cultivar, ‘L 10‐146’, was susceptible while Bru1‐negative cultivars, such as ‘L 99‐233’, showed resistance to brown rust. Bru1‐negative clones with brown rust resistance offer an opportunity to identify alternate sources of resistance, which can be pyramided with Bru1 for effective and durable resistance in sugarcane against the changing pathogen.     

Identification of cultivars and accessions of Lolium, Festuca and Festulolium hybrids through the detection of simple sequence repeat polymorphism

Molecular diversity and genetic affinity in the Lolium/Festuca grass complex have been assessed using simple sequence repeat (SSR) marker technology. The genotypic set was derived from three accessions of perennial ryegrass, two cultivars of Italian ryegrass, two cultivars of meadow fescue, two cultivars of tall fescue and 10 accessions from different intergeneric hybrid (Festulolium) combinations. The majority of the genomic DNA‐derived SSR primer pairs from perennial ryegrass (LPSSR) and Italian ryegrass (LMSSR) produced clear, simple and distinctive amplification products from the majority of the genotypes. The efficiency of cross‐specific amplification for LPSSR markers varied from 38% in meadow fescue to 93% in two cultivars of Festulolium and from 57% in meadow fescue to 87% in Italian ryegrass for LMSSR markers. Of 40 amplified markers, 14 (35%) produced species‐difference alleles in the relation to cultivars used in the present study. Thirty‐five LPSSR locus‐derived alleles were found to be specific to Lolium species, four to meadow fescue and six to tall fescue. For LMSSR alleles, eight were specific to Lolium species and five were only associated with Italian ryegrass, and null alleles were detected for meadow fescue in all instances. These species‐difference markers could clearly identify different accessions of Festulolium. Cluster analysis separated the individual taxa and showed grouping of intergeneric hybrids based on genomic composition. The data distinguished between the species and reflected the known pedigree of the cultivars and the differences between the species. The dendrogram also distinguished between the Festulolium accessions and clearly demonstrated the relations between Festulolium hybrids and their parent species.     

Genetic diversity of Cannabis sativa germplasm based on RAPD markers

Random amplified polymorphic DN A (RAPD) markers were generated from 13 cultivars and accessions of Cannabis sativa L. Approximately 200 fragments generated by 10 primers of arbitrary sequence were used to assess the level of DNA variation. Statistical analysis was performed using the Dice coefficient of similarity and principal coordinate analysis. The grouping of the accessions according to the cluster analysis was in good agreement with their origin and lines with common ancestors were grouped together. Principal coordinates 1 and 2 revealed a clear separation of Italian and Hungarian germplasm and a third group, including a mixture of genotypes coming from different places; the third coordinate separated the Korean group which is probably the most divergent germplasm. Variability within the two cultivars ‘Carmagnola’ and ‘Fibranova1’ was also shown, suggesting good possibilities for long–term selection work. RAPD markers provide a powerful tool for the investigation of genetic variation in cultivars/accessions of hemp.     

Exploring genetic diversity of rice cultivars for the presence of brown planthopper (BPH) resistance genes and development of SNP marker for Bph18

Brown planthopper (BPH) is the most devastating insect pest in rice‐growing areas. Information on availability of BPH resistance alleles and their sources enhances BPH‐resistant breeding programmes. In this study, 260 highly diversified rice cultivars or breeding lines were screened for the presence of five major BPH resistance genes (Bph10, Bph13, Bph18, Bph20 and Bph21) using gene‐specific markers. The analysis revealed that 137 of the 260 cultivars possess at least one BPH resistance gene. Bph10 was predominant while Bph20 was the least distributed. Moreover, two and three different resistance gene combinations were found in the cultivars. Molecular markers play an important role in molecular breeding programmes. A tightly linked PCR‐based co‐dominant Bph18 marker was developed, which is cost effective and time effective and simpler than available Bph18 CAPS marker (7312.T4A). We strongly believe that the identified BPH‐resistant cultivars can be used as alternative resistance gene sources and also as resource for novel BPH resistance genes. The developed Bph18 marker will be highly useful in molecular breeding applications of BPH‐resistant breeding programmes.     

Association mapping of straighthead disorder induced by arsenic in Oryza sativa

Straighthead is a physiological disorder in rice (Oryza sativa L.) resulting in sterile florets, poorly developed panicles and yield loss. Because of its sporadic nature and unidentified causes for the disorder, molecular marker assisted selection is essential for resistance improvement in breeding programmes. To take advantage of recent advances in gene‐mapping technology, we executed a genome‐wide association mapping to identify genetic regions associated with straighthead disorder using 547 accessions of germplasm from the USDA rice core collection. Straighthead was evaluated in arsenic treated soil and genotyping was conducted with 75 molecular markers covering the entire rice genome about every 25 cM. A mixed‐linear model approach combining the principal component assignments with kinship estimates proved to be particularly promising for association mapping. The extent of linkage disequilibrium was described among the markers. Six markers were found to be significantly associated with straighthead, explaining 35% of the total phenotypic variation. However, only two SSR markers, RM413 and RM277 on chromosome 5 and 12, respectively, have a significant association with low rating indicating straighthead resistance. Confirmation of the marker‐straighthead association using segregating populations is necessary before marker‐assisted selection can be applied.     

Analysis of a major rice blast resistance gene in the rice restorer line Hanghui 1179

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most devastating diseases of rice (Oryza sativa) worldwide. Identification and utilization of resistance genes in rice breeding is considered to be an effective and economical method to control this disease. Hanghui 1179 (HH1179) is a new native rice restorer line developed in South China. The hybrids derived from HH1179 show broad-spectrum resistance against rice blast in South China, and a further understanding of the genetic resistance in HH1179 will provide useful information for breeding resistant cultivars. In the present study, we used bulked segregant analysis combined with specific-length amplified fragment sequencing to identify a dominant gene from HH1179 that provides resistance against the rice blast isolate GD13-14. Association analysis indicated that the resistance gene is located on chromosome 6 and we mapped the target gene to a 100.8 kb region (between markers InDel-8 and RM19818) that contains the Pi2/Pi9/Piz/Piz-t/Pi50 gene cluster. Candidate gene prediction and cDNA sequencing indicated that the target resistance gene in HH1179 is Pi2. Our findings will be valuable for resistance breeding with restorer line HH1179.