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.     

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)...     

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.     

A novel aphid resistance locus in cowpea identified by combining SSR and SNP markers

The utility of combining simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) marker genotyping was determined for genetically mapping a novel aphid (Aphis craccivora) resistance locus in cowpea breeding line SARC 1‐57‐2 and for introgressing the resistance into elite cultivars by marker‐assisted backcrossing (MABC). The locus was tagged with codominant SSR marker CP 171F/172R with a recombination fraction of 5.91% in an F₂ population from ‘Apagbaala’ x SARC 1‐57‐2. A SNP‐genotyped biparental recombinant inbred line population was genotyped for CP 171F/172R, which was mapped to position 11.5 cM on linkage group (LG) 10 (physical position 30.514 Mb on chromosome Vu10). Using CP 171F/172R for foreground selection and a KASP‐SNP‐based marker panel for background selection in MABC, the resistance from SARC 1‐57‐2 was introduced into elite susceptible cultivar ‘Zaayura’. Five BC₄F₃ lines of improved ‘Zaayura’ that were isogenic except for the resistance locus region had phenotypes similar to SARC 1‐57‐2. This study identified a novel aphid resistance locus and demonstrated the effectiveness of integrating SSR and SNP markers for trait mapping and marker‐assisted breeding.     

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.     

Characterization of broad‐spectrum resistance to Soybean mosaic virus in soybean [Glycine max (L.) Merr.] cultivar ‘RN‐9’

Soybean mosaic virus (SMV) can cause serious yield losses in soybean. Soybean cultivar ‘RN‐9’ is resistant to 15 of 21 SMV strains. To well‐characterize this invaluable broad‐spectrum SMV‐resistance, populations (F₁, F₂ and F_2:3) derived from resistant (R) × susceptible (S) and R × R crosses were tested for SMV‐SC18 resistance. Genetic analysis revealed that SC18 resistance in ‘RN‐9’ plus two elite SMV‐resistant genotypes (‘Qihuang No.1’ and ‘Kefeng No.1’) are controlled by independently single dominant genes. Linkage analysis showed that the resistance of ‘RN‐9’ to SMV strains SC10, SC14, SC15 and SC18 is controlled by more than one gene(s). Moreover, Rsc10‐r and Rsc18‐r were both positioned between the two simple sequence repeats markers Satt286 and Satt277, while Rsc14‐r was fine‐mapped in 136.8‐kb genomic region containing sixteen genes, flanked by BARCSOYSSR_06_0786 and BARCSOYSSR_06_0790 at genetic distances of 3.79 and 4.14 cM, respectively. Allelic sequence comparison showed that Cytochrome P450‐encoding genes (Glyma.06g176000 and Glyma.06g176100) likely confer the resistance to SC14 in ‘RN‐9’. Our results would facilitate the breeding of broad‐spectrum and durable SMV resistance in soybeans.     

节水抗旱稻恢复系的抗褐飞虱分子标记辅助选育及抗性评价

褐飞虱是水稻的主要害虫之一,利用水稻抗褐飞虱基因培育抗虫品种是目前公认最经济有效、环境友好的策略。本研究利用水稻功能基因组已克隆的抗褐飞虱基因,通过分子标记辅助选择和常规回交育种相结合的方法,将抗褐飞虱基因Bph6、Bph9、Bph14和Bph15单独和聚合导入到节水抗旱稻恢复系旱恢3号,获得了一系列含有单基因、双基因、三基因和四基因的改良系。采用标准苗期集团筛选法进行褐飞虱抗性鉴定,评价这些基因在旱恢3号背景下的效应及相互作用。表明单基因改良系中, Bph9的抗性最强,且Bph9 Bph6 Bph15 Bph14;在聚合改良系中,抗性均优于单基因改良系,四基因聚合改良系的抗性最强,不同基因型组合的抗性效应是Bph6+Bph9+Bph14+Bph15Bph6+Bph9 Bph6+Bph9+Bph14 Bph6+Bph9+Bph15 Bph6+Bph14+Bph15 Bph9+Bph14+Bph15 Bph14+Bph15。在自然条件下,改良系与旱恢3号在株高、有效穗和千粒重等农艺性状上差异不显著,其他性状与旱恢3号相仿或略差。本试验表明单独和聚合导入Bph6、Bph9、Bph14和Bph15基因能显著提高节水抗旱稻恢复系的褐飞虱抗性,这4个基因的加性效应明显,可为今后节水抗旱稻抗褐飞虱育种提供理论依据和材料基础。     

Breeding cowpea for resistance to Striga gesnerioides in the Nigerian dry savannas using marker‐assisted selection

Historically, conventional breeding has been the primary strategy used to develop a number of Striga‐resistant varieties currently grown in the Sahel of Western Africa. In this study, we have successfully developed and applied a marker‐assisted selection strategy that employs a single backcross programme to introgress Striga resistance into farmer preferred varieties of cowpea for the Nigeria savannas. In this strategy, we have introduced the Striga resistance gene from the donor parent IT97K‐499‐35 into an elite farmer preferred cowpea cultivar ‘Borno Brown’. The selected 47 BC₁F₂ populations confirmed the recombinants with desirable progeny having Striga resistance gene(s). The 28 lines selected in the BC₁F_2:4 generation with large seed size, brown seed coat colour and carrying marker alleles were evaluated in the field for resistance to Striga resistance. This led to the selection of a number of desirable improved lines that were immune to Striga having local genetic background with higher yield than those of their parents and standard varieties.     

Identification and marker‐assisted introgression of QTL conferring resistance to bacterial leaf blight in cowpea (Vigna unguiculata (L.) Walp.)

Bacterial leaf blight (BLB), caused by Xanthomonas axonopodis pv. vignicola (Xav), is widespread in major cowpea [Vigna unguiculata (L.) Walp.] growing regions of the world. Considering the resource poor nature of cowpea farmers, development and introduction of cultivars resistant to the disease is the best option. Identification of DNA markers and marker‐assisted selection will increase precision of breeding for resistance to diseases like bacterial leaf blight. Hence, an attempt was made to detect QTL for resistance to BLB using 194 F_2 : 3 progeny derived from the cross ‘C‐152’ (susceptible parent) × ‘V‐16’ (resistant parent). These progeny were screened for resistance to bacterial blight by the leaf inoculation method. Platykurtic distribution of per cent disease index scores indicated quantitative inheritance of resistance to bacterial leaf blight. A genetic map with 96 markers (79 SSR and 17 CISP) constructed from the 194 F₂ individuals was used to perform QTL analysis. Out of three major QTL identified, one was on LG 8 (qtlblb‐1) and two on LG 11 (qtlblb‐2 and qtlblb‐3). The PCR product generated by the primer VuMt337 encoded for RIN2‐like mRNA that positively regulate RPM1‐ and RPS2‐dependent hypersensitive response. The QTL qtlblb‐1 explained 30.58% phenotypic variation followed by qtlblb‐2 and qtlblb‐3 with 10.77% and 10.63%, respectively. The major QTL region on LG 8 was introgressed from cultivar V‐16 into the bacterial leaf blight susceptible variety C‐152 through marker‐assisted backcrossing (MABC).     

Pyramiding three rust‐resistance genes confers a high level of resistance in soybean (Glycine max)

Pyramiding Asian soybean rust (ASR) resistance (Rpp) genes in a single genotype has been shown to increase ASR resistance in soybean. However, it remains unclear which combinations of Rpp genes are superior. Therefore, here, we developed six new Rpp‐pyramided lines carrying different combinations of Rpp genes and evaluated their resistance against 13 Bangladeshi rust (Phakopsora pachyrhizi) isolates (BdRPs) alongside seven previously developed Rpp‐pyramided lines. We found that lines carrying one, two and three Rpp genes had high ASR resistance without sporulation in 13.8%, 35.2% and 73.1% of the assays, respectively. Among the new lines that were developed, those with Rpp3 + Rpp4 and Rpp3 + Rpp4 + Rpp5 had high levels of ASR resistance, while the line containing Rpp2 + Rpp4 + Rpp5 showed immunity phenotype at two weeks after inoculation by the BdRP‐22 infection. Thus, pyramiding larger numbers of Rpp genes confers soybean with a higher level of resistance to ASR pathogens and can produce an immunity phenotype at two weeks after inoculation.     

Development of elite rice restorer lines in the genetic background of R022 possessing tolerance to brown planthopper, stem borer, leaf folder and herbicide through marker-assisted breeding

Rice leaf folder, stem borer and brown planthopper (BPH) are the most devastating rice insect pests. Developing and planting insect-resistant rice varieties is the most economical and effective measure for controlling these pests. BPH can be controlled with native BPH-resistance genes in rice, while at present rice leaf folder and stem borer can only be controlled through planting transgenic Bt rice. In this study, the breeding of a new restorer line KR022 possessing stacked BPH-resistance genes Bph14 and Bph15, Bt gene cry1C and glufosinate-resistance gene bar, is reported for the first time. A rice restorer line R022 with BPH-resistance genes Bph14 and Bph15 was used as a recurrent parent to cross with the transgenic rice T1C-19 of cry1C and bar genes during the breeding process. The restorer line KR022 was developed from the backcross populations of R022 and T1C-19 through molecular marker-assisted selection and glufosinate-resistance selection. The cry1C and bar genes were found to integrate on chromosome 11 of KR022, and the genome recovery of KR022 was up to 95.8 % of the R022 genome. The quantification of Cry1C protein expression showed that it was expressed at different levels in the leaf, stem, panicle, endosperm, and root of KR022 and its hybrid rice. The insect-resistance evaluation indicated that KR022 and its hybrid rice had good resistance to rice leaf folder and stem borer, both in laboratory settings and in the field. Furthermore, they exhibited increased resistance to BPH at both the seedling and mature stage. The field trial showed there was no significant difference in key agronomic traits between KR022 and its recurrent parent R022, and four hybrids from KR022 yield much higher than the control II-You 838. Moreover, KR022 and its hybrid rice were found to have resistance to the herbicide glufosinate. These results demonstrate that KR022 is effective as a rice restorer line for the breeding of “green super rice”, possessing multiple tolerances to rice BPH, stem borer, leaf folder and glufosinate.     

A new locus for resistance to brown planthopper identified in the indica rice variety DV85

The brown planthopper (BPH) is one of the most destructive insect pests of rice. Resistant varieties have proved to be one of the most economic and effective measures for BPH management. In this study, an indica rice ‘DV85’ showed resistance to biotype 2 of BPH by bulked seedling test, and a recombinant inbred line (RIL) population derived from a cross between a susceptible rice ‘Kinmaze’ and ‘DV85’ was phenotyped to map genetic factors conferring BPH resistance in ‘DV85′. Composite interval mapping revealed that one quantitative trait locus (QTL) with a LOD score of 10.1 was detected between XNpb202 and C1172 on chromosome 11. This QTL was designated as Qbph11. Qbph11 explained 68.4% of the phenotypic variance of BPH resistance in this population. The allele from the resistant parent ‘DV85’ at Qbph11 reduced the damage caused by BPH feeding and would be very useful in breeding resistant rice varieties via marker‐assisted selection.     

Diversity of five glutenin loci within durum wheat (Triticum turgidum L. ssp. durum (Desf.) Husn.) germplasm grown in Algeria

The HMW and B‐LMW glutenin subunits composition of 120 durum wheat germplasm grown in Algeria was examined using SDS‐PAGE. All together, 39 glutenin patterns were detected, including eight for HMW and 21 for B‐LMW glutenin subunits. Twenty‐six different alleles were identified for the five glutenin loci studied, that is, Glu‐A1 (3), Glu‐B1 (7), Glu‐A3 (5), Glu‐B3 (9) and Glu‐B2 (2). Two new alleles were found at Glu‐B3 locus: Glu‐B3new₁ encodes for five subunits (7 + 8 + 14 + 16 + 18) and Glu‐B3new₂ codes for five subunits (4 + 6* + 12 + 15 + 15*), of which subunit 15* with mobility between bands 15–16 was not described previously. At the Glu‐1 loci, the Glu‐A1c/Glu‐B1e allelic composition was predominant. For the B‐LMW glutenins, the most common allelic composition was Glu‐A3a/Glu‐B3a/Glu‐B2a. The collection analysed shows glutenin alleles and allele combinations related to high gluten strength. This information could be useful to select varieties with improved quality and also as a source of genes to develop new lines when breeding for quality.     

Targeted transfer of trait for Verticillium wilt resistance from Gossypium barbadense into G. hirsutum using SSR markers

Verticillium wilt (VW) is a soil‐borne disease of cotton that is destructive worldwide. Transferring desired traits from Gossypium barbadense is challenging through traditional interspecific introgression. We previously demonstrated that a molecular marker, BNL3255‐208, is associated with VW resistance in G. barbadense. This breakthrough opens the way for marker‐assisted selection (MAS) breeding. Here, the highly resistant G. barbadense cv. ‘Pima90‐53’ and the severe diseased Gossypium hirsutum cv. ‘CCRI8’ were used as donor parent and recipient parent, respectively. Our goal was to transfer the disease resistance from donor to recipient via MAS. Among 71 MAS obtained lines, as many as 19 lines had enhanced resistance. Among those lines, 11 lines showed high resistance and four lines displayed resistance to VW. Moreover, seven lines displayed improved fibre quality. After combining the markedly improved resistance and fibre properties, we identified two elite innovated introgression lines – ZY2 and ZY31 – that did not seem to differ in other agronomic traits from the recipient parent. This study first successfully transferred of G. barbadense resistance into G. hirsutum by MAS.     

Expression of a Triticum turgidum var. dicoccoides source of Fusarium head blight resistance transferred to synthetic hexaploid wheat

Yield and quality reductions caused by Fusarium head blight (FHB) have spurred spring wheat (Triticum aestivum L.) breeders to identify and develop new sources of host plant resistance. Four wheat synthetic hexaploids (×Aegilotriticum sp.) were developed, each having a quantitative trait locus (QTL), Qfhs.ndsu‐3AS, providing FHB resistance from Triticum turgidum L. var. dicoccoides chromosome 3A. Synthetics were produced by hybridizing a ‘Langdon’‐T. dicoccoides‐ recombinant chromosome 3A substitution line (2n = 4x = 28, AABB with two accessions of T. tauschii (2n= 2x = 14, DD). Synthetics were inoculated and evaluated for FHB resistance in two separate greenhouse seasons. One synthetic, 01NDSWG‐5, exhibited FHB severity ratings of 36% and 32% in the separate seasons, compared with ratings of 9% and 30% for ‘Alsen’, a FHB‐resistant spring cultivar, and ratings of 70% and 96% for ‘McNeal’, a susceptible spring cultivar, respectively. Synthetic × Alsen backcross‐derived lines were produced to initiate combining different sources of FHB resistance.     

Mapping quantitative trait loci conferring blast resistance in upland indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A genetic analysis of blast resistance in upland rice variety is very crucial. In this study, we performed a linkage mapping of quantitative trait loci (QTLs) for blast resistance using an advanced backcross population from a cross between Way Rarem (susceptible indica variety) and Oryzica Llanos 5 (durable resistant indica variety). A transgressive segregation was observed in the advanced backcross population of Way Rarem//Oryzica Llanos 5. A total of 16 QTLs have been identified along chromosomes 1, 3, 5, 6, 7, 8, 9, and 11 against eight blast pathogen isolates. Each QTL accounted from 11.31 to 45.11% of the variation in blast resistance. Most QTLs showed race specificity, demonstrating the small effect of such QTLs. Unexpectedly, several superior blast resistance alleles were contributed by Way Rarem, the susceptible-recurrent parent. Among eight candidate defense response genes detected in several loci, a single gene (oxalate oxidase) present on chromosome 3 was found to be associated with blast resistance in upland indica rice. Ultimately, these advanced backcross lines with resistance to blast tagged by markers might be useful for pyramiding blast resistance alleles in upland rice.     

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.     

聚合稻瘟病、白叶枯病和褐飞虱抗性基因的三系恢复系改良效果的评价

结合分子标记辅助轮回选择和田间鉴定的方法, 将三黄占2号的抗稻瘟病基因Pi-GD-1(t)和Pi-GD-2(t)(分别简称G1和G2)、CBB23中的抗白叶枯病基因Xa23 (简称X)和IR65482-7-216-1-2-B(简称IR65482)的抗褐飞虱基因Bph18(t) (简称B)导入温恢845、温恢117和温恢143等3个中籼恢复系,获得了8个兼抗稻瘟病和褐飞虱聚合系,温恢845-G1-G2-B-4、温恢845-G1-G2-B-5、温恢117-G1-G2-X-B-3、温恢143-G1-G2-B-3、温恢143-G2-X-B-9、温恢143-G2-X-B-10、温恢143-G1-G2-B-11和温恢143-G1-G2-B-37。这些聚合系及其与不育系五丰A的测交种,对稻瘟病和褐飞虱的抗性水平接近或略低于稻瘟病抗性亲本三黄占2号和稻飞虱抗性亲本IR65482。部分改良恢复系如温恢117-G1-G2-X-B-3、温恢143-G2-X-B-9和温恢143-G2-X-B-10及其测交种对白叶枯病表现为抗病或中抗。改良恢复系及其测交种在正常条件下的农艺性状与原始恢复系及其测交种相仿或更优,具有生产应用价值。研究结果表明,Xa23在不同恢复系背景下抗性表达完全,而Pi-GD-1(t)、Pi-GD-2(t)和Bph18(t)对稻瘟病和褐飞虱抗性的改良效果与恢复系的遗传背景有关。     

Genetic analysis and fine mapping of a dominant dwarfness gene from wild rice (Oryza barthii)

Plant architecture has been proposed as a means to enhance the potential yield of rice, especially by reducing height to provide lodging resistance. In this study, we developed a near‐isogenic line (NIL) using cultivar ‘Dianjingyou 1’ (DJY1) as a recipient parent and wild rice (Oryza barthii) as the donor parent. The NIL had semi‐dwarf stature and more tillers than DJY1. Cytological examination showed decreased numbers of cells in the stems of the NIL compared to DJY1. Genetic analysis indicated that this phenotype was controlled by a newly identified dominant dwarf gene, tentatively named as Dd7. A large population derived from the hybrid NIL‐Dd7/DJY1 was developed to fine‐map Dd7. Based on the physical location of molecular markers, the Dd7 locus was finally delimited to an 82‐kb region in chromosome 7. Gene prediction identified 14 open reading frames (ORFs) within this region. NIL‐Dd7 seems to confer no undesirable pleiotropic effects and therefore has potential value for rice breeding.