SSR analysis of cultivated groundnut (Arachis hypogaea L.) germplasm resistant to rust and late leaf spot diseases

Cultivated groundnut (Arachis hypogaea L.) is an agronomically and economically important oilseed crop grown extensively throughout the semi-arid tropics of Asia, Africa and Latin America. Rust (Puccinia arachidis) and late leaf spot (LLS, Phaseoisariopsis personata) are among the major diseases causing significant yield loss in groundnut. The development of varieties with high levels of resistance has been constrained by adaptation of disease isolates to resistance sources and incomplete resistance in resistant sources. Despite the wide range of morphological diversity observed in the cultivated groundnut gene pool, molecular marker analyses have thus far been unable to detect a parallel level of genetic diversity. However, the recent development of simple sequence repeat (SSR) markers presents new opportunities for molecular diversity analysis of cultivate groundnut. The current study was conducted to identify diverse disease resistant germplasm for the development of mapping populations and for their introduction into breeding programs. Twenty-three SSRs were screened across 22 groundnut genotypes with differing levels of resistance to rust and LLS. Overall, 135 alleles across 23 loci were observed in the 22 genotypes screened. Twelve of the 23 SSRs (52%) showed a high level of polymorphism, with PIC values ≥0.5. This is the first report detecting such high levels of genetic polymorphism in cultivated groundnut. Multi-dimensional scaling and cluster analyses revealed three well-separated groups of genotypes. Locus by locus AMOVA and Kruskal–Wallis one-way ANOVA identified candidate SSR loci that may be valuable for mapping rust and LLS resistance. The molecular diversity analysis presented here provides valuable information for groundnut breeders designing strategies for incorporating and pyramiding rust and late leaf spot resistances and for molecular biologists wishing to create recombinant inbred line populations to map these traits.E.S. Mace and D.T. Phong contributed equally to this work.     

Components of resistance to late leaf spot and rust among interspecific derivatives and their significance in a foliar disease resistance breeding in groundnut (Arachis hypogaea L.)

Late leaf spot (LLS) and rust cause substantial yield losses and reduce the fodder and seed quality in groundnut (Arachis hypogaea L.). Adoption of resistant cultivars by the semi-arid tropic farmers is the best option to overcome yield losses. Knowledge on components of resistance to these diseases should facilitate the development of groundnut cultivars with enhanced resistance to LLS and rust. The objectives of the experiments were to study the genetic variability and relationships among components of resistance to LLS and rust, and assess their significance in disease resistance breeding. Fifteen interspecific derivatives for LLS and 14 for rust and a susceptible control, TMV 2, were evaluated in a randomised complete block design with two or three replications under greenhouse conditions. The experiments were repeated twice. Genotypic differences were highly significant for all the traits studied. Resistance to LLS is due to longer incubation and latent periods, lesser lesions per leaf, smaller lesion diameter, lower sporulation index, and lesser leaf area damage and disease score. Selection based on components of resistance to LLS may not lead to plants with higher retained green leaf area. The remaining green leaf area on the plant should, therefore, be the major selection criteria for resistance to LLS in breeding programs. Resistance to rust is due to longer incubation and latent periods, fewer pustules per leaf, smaller pustule diameter, lower sporulation index, and lesser leaf area damage and disease score. Rust resistant components appear to work additively, therefore, selection based on resistance components together with green leaf area retained on the plant should be the basis of selecting for resistance to rust in breeding programs. ICGV 99005, 99003, 99012, and 99015 for rust and ICGV 99006, 99013, 99004, 99003, and 99001 for LLS are the better parents for use in resistance breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

SSR markers associated for late leaf spot disease resistance by bulked segregant analysis in groundnut (Arachis hypogaea L.)

Late leaf spot (LLS) caused by Phaeoisariopsis personata is the major foliar disease that reduces the pod yield and severely affects the fodder and seed quality in groundnut. Molecular markers linked with LLS can improve the process of identification of resistant genotypes. In the present study, a LLS susceptible genotype (TMV 2) and the LLS resistant genotype (COG 0437) were crossed and their F₂ population was used for marker analysis. The phenotypic mean data on F_2:3 progenies were used as phenotype. Parents were surveyed with 77 SSR (Simple Sequence Repeat) primers to identify polymorphic markers. Among SSR markers, nine primers were found polymorphic between the parents TMV 2 and COG 0437. These markers were utilized for bulked segregant analysis (BSA). Among the polymorphic SSR markers, three primers viz., PM 375₁₆₂, pPGPseq5D5₂₂₀ and PM 384₁₀₀ were able to distinguish the resistant and susceptible bulks and individuals for LLS. In single marker analysis, the markers PM 375, PM 384, pPGPseq5D5, PM 137, PM 3, PMc 588 and Ah 4-26 were linked with LLS severity score. The phenotypic variation explained by these markers ranged from 32 to 59?%. The markers identified through BSA were also confirmed with single marker analysis. While validating the three primers over a set of resistant and susceptible genotypes, the primer PM 384₁₀₀ allele had association with resistance. Hence PM 384 could be utilized in the marker assisted breeding programme over a wide range of genetic background.     

Foliar fungal disease‐resistant introgression lines of groundnut (Arachis hypogaea L.) record higher pod and haulm yield in multilocation testing

Introgression lines (ILs) of groundnut with enhanced resistance to rust and late leaf spot (LLS) recorded increased pod and haulm yield in multilocation testing. Marker‐assisted backcrossing (MABC) approach was used to introgress a genomic region containing a major QTL that explains >80% of phenotypic variance (PV) for rust resistance and 67.98% PV for LLS resistance. ILs in the genetic background of TAG 24, ICGV 91114 and JL 24 were evaluated for two seasons to select 20 best ILs based on resistance, productivity parameters and maturity duration. Multilocation evaluation of the selected ILs was conducted in three locations including disease hot spots. Background genotype, environment and genotype × environment interactions are important for expression of resistance governed by the QTL region. Six best ILs namely ICGV 13192, ICGV 13193, ICGV 13200, ICGV 13206, ICGV 13228 and ICGV 13229 were selected with 39–79% higher mean pod yield and 25–89% higher mean haulm yield over their respective recurrent parents. Pod yield increase was contributed by increase in seed mass and number of pods per plant.     

小麦品种小偃9323抗条锈基因的遗传分析和分子作图

小偃9323是小偃6号的同源材料,具有早熟、抗逆性强、适应性广、抗条锈性强等许多优良的生物学特性。为明确其抗条锈性及遗传规律,利用当前流行的中国条锈菌小种CYR32对抗病品种小偃9323与感病品种铭贤169及其杂交后代F₁、F₂、F₃和BC₁代进行苗期抗条锈性遗传分析,并对其抗条锈基因进行SSR分子标记。结果表明,小偃9323对CYR32小种具有良好的抗性,由1对隐性基因所控制。利用F₂代分离群体,筛选到6个与抗病基因连锁的SSR标记,分别是Xwmc807、Xbarc3、Xwmc684、Xwmc201、Xwmc553和Xwmc179;该抗病基因位于小麦6AL染色体上,其最近的标记为Xwmc201和Xwmc553,遗传距离分别是2.6 cM和3.7 cM。分析表明,该基因不同于已知抗条锈基因,暂被命名为YrXY9323。用YrXY9323两侧遗传距离最近的标记Xwmc201和Xwmc553对42个黄淮麦区主栽小麦品种进行分子检测,结果表明有19%的品种具有与YrXY9323相同的标记位点。本结果对YrXY9323在小麦抗条锈病育种中的应用提供了理论依据。     

Marker‐assisted backcrossing to develop foliar disease‐resistant genotypes in TMV 2 variety of peanut (Arachis hypogaea L.)

TMV 2 is a very popular peanut variety among the Indian farmers, but it is highly susceptible to fungal foliar diseases like late leaf spot (LLS) and rust. Marker‐assisted backcrossing (MABC) in TMV 2 using foliar disease‐resistant donor, GPBD 4 and the disease resistance‐linked markers (GM2009, GM2079, GM2301, GM1839 and IPAHM103) resulted in a large number of backcross populations and also straight cross populations. Foreground selection followed by field evaluation under disease epiphytotic conditions could identify a few superior genotypes. Two homozygous backcross lines TMG‐29 and TMG‐46 showed enhanced resistance to LLS and rust diseases (score of 3.00 for both) along with 71.0% and 62.7% increase in the pod yield per plot, respectively, over the check, TMV 2. These foliar disease‐resistant and productive lines can be released as commercial varieties or can be used as genetic resources in the peanut improvement.     

Genetic analysis of partial resistance to coffee leaf rust ( Hemileia vastatrix Berk & Br.) introgressed into the cultivated Coffea arabica L. from the diploid C. canephora species

The objective of this study was to identify polymorphic molecular markers associated with partial resistance to coffee leaf rust, Hemileia vastarix. A segregating F ₂ population derived from a cross between the susceptible Coffea arabica cv. Caturra and a C. canephora-introgressed Arabica line exhibiting high partial resistance was analyzed. Rust resistance measured as rust incidence (RI) and defoliation (DEF) was evaluated in field conditions in three consecutive years (2003–2005). During the 2003 season, which was characterized by favorable conditions for a rust epidemic, the F ₂ plants exhibited different levels of resistance ranging from very susceptible (50.1% for DEF and 49.5% for RI) to highly partial resistance (9.1% for DEF and 3.7% for RI). Molecular analysis enabled identification of seven polymorphic markers (5 AFLP and 2 SSR) exhibiting significant association with partial resistance. Coexistence of resistance homozygous alleles (RR) at codominant SSR loci was correlated with high resistance. This study is the first attempt to develop PCR-based sequence specific markers linked to partial rust resistance in coffee.     

Development of NILs from heterogeneous inbred families for validating the rust resistance QTL in peanut (Arachis hypogaea L.)

Heterogeneous inbred families segregating for rust resistance were identified from the two crosses involving susceptible (TAG 24 and TG 26) and resistant (GPBD 4) varieties of peanut. Rust‐resistant (less than score 5) and rust‐susceptible (more than score 5) plants were identified in each HIF and evaluated under rust epiphytotic conditions. The set of plants belonging to the same HIF, but differing significantly in rust resistance, not in other morphological and productivity traits, was regarded as near‐isogenic lines (NILs). Largely, rust‐resistant NILs had GPBD 4‐type allele, and susceptible NILs carried either TAG 24 or TG 26‐type allele at the three SSR loci (IPAHM103, GM1536 and GM2301) linked to a major genomic region governing rust resistance. Comparison of the remaining genomic regions between the NILs originating from each of the HIFs using transposon markers indicated a considerably high similarity of 86.4% and 83.1% in TAG 24 × GPPBD 4 and TG 26 × GPBD 4, respectively. These NILs are useful for fine mapping and expression analysis of rust resistance.     

小麦品种中梁22抗条锈病基因的遗传分析和分子作图

对中梁22/铭贤169杂交F₂群体苗期抗条锈病鉴定及中国春单体系抗病基因的染色体定位发现, 中梁22携带1个显性(暂命名YrZhong22)和1个隐性抗病基因, 前者位于5B染色体。由中梁22´铭贤169的F₂群体构建抗病、感病池, 用SSR标记结合集群分离分析法(BSA), 建立了与YrZhong22连锁的4个微卫星标记Xwmc289、Xwmc810、Xgdm116和Xbarc232, 并将YrZhong22定位于小麦5BL染色体。YrZhong22与相邻微卫星位点Xwmc810和Xgdm116的遗传距离分别是2.7 cM和4.4 cM。系谱分析及分子标记分析表明, YrZhong22可能是一个来自中间偃麦草的新抗条锈病基因。     

小麦抗叶锈病基因Lr45的SSR分子标记

用定位于2A染色体的59对SSR、EST-SSR引物,对小麦抗叶锈病基因Lr45进行分子标记,共筛选出11对揭示TcLr45多态性的引物。用157株F₂抗感群体对这11对引物进一步检测,得到4个与Lr45共分离的SSR标记(Xgwm95、Xgwm47、Xgwm372和Xgwm122)。将经PAGE检测的标记Xgwm95的抗感差异带及Xgwm47的抗性片段进行克隆测序发现其中含有微卫星序列,且均为二核苷酸重复。Xgwm372和Xgwm122经琼脂糖凝胶电泳发现与Lr45的供体黑麦有共同的标记片段,可直接用于分子标记辅助选择。     

Molecular tagging of Asiatic soybean rust resistance in exotic genotype EC 241780 reveals complementation of two genes

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi severely reduces seed yield in soybean. Molecular tagging of ASR resistance can help in the process of resistance breeding. In this study, an F₂ population of cross (susceptible cultivar ‘NRC 7’ × resistant exotic genotype EC 241780) was used for bulked segregant analysis (BSA) with 25 SSR (simple sequence repeat) primers linked with six Rpp genes. Among them, five polymorphic SSR markers, viz., Sct 187, SSR 1859, Satt 191 (Rpp1b like loci) and Satt 215, Sat_361 (Rpp2 loci) distinguished the ASR resistant and susceptible bulks and individuals. In combined marker analysis, the markers Satt 191 (Rpp1b like loci) and Satt 215 (Rpp2 loci) were linked with ASR severity score and were also confirmed in individual 110 F₂ segregants. Hence, these markers could be utilized in the marker assisted rust resistance breeding of Rpp1b like and Rpp2 genes. In silico candidate gene analysis for hypersensitive response revealed that Satt 191 linked region was rich in genes encoding apoptotic ATPase having leucine‐rich repeat (LRR) domain.     

Molecular mapping of a stripe rust resistance gene in wheat cultivar Wuhan 2

Stripe rust (or yellow rust), caused by Puccinia striiformis f. sp. tritici, is one of the most destructive diseases of wheat worldwide. Growing resistant cultivars is the best approach to control the disease. To identify and map genes for stripe rust resistance in wheat cultivar ‘Wuhan 2', an F₂ population was developed from a cross between the cultivar and susceptible cultivar Mingxian 169. The parents, 179 F₂ plants and their derived F_2:3 lines were evaluated for responses to Chinese races CYR30 and CYR31 of the pathogen in a greenhouse. A recessive gene for resistance was identified. DNA bulked segregant analysis was applied and resistance gene analog polymorphism (RGAP) and simple sequence repeat (SSR) techniques were used to identify molecular markers linked to the resistance gene. A genetic map consisting of five RGAP and six SSR markers was constructed. The recessive gene, designated Yrwh2, was located on the short arm of chromosome 3B and flanked by SSR markers Xwmc540 and Xgwm566 at 5.9 and 10.0 cM, respectively. The chromosomal location of the resistance gene and its close marker suggest that the locus is different from previously reported stripe rust resistance genes Yr30, QYr.ucw-3BS, Yrns-B1, YrRub and QYrex.wgp-3BL previously mapped to chromosome 3B. Yrwh2 and its closely linked markers are potentially useful for developing stripe rust resistance wheat cultivars if used in combination with other genes.     

Rapid identification of a major effect QTL conferring adult plant resistance to stripe rust in wheat cultivar Yaco“S”

Stripe rust is a devastating disease in common wheat (Triticum aestivum) worldwide. Growing cultivars with adult-plant resistance (APR) is an environmental friendly approach that provides long-term protection to wheat from this disease. Wheat cultivar Yaco“S” showed a high level of APR to stripe rust in the field from 2008 to 2014. The objective of this study was to detect the major quantitative trait loci (QTL) for APR to stripe rust in Yaco“S”. One hundred and eighty-four F_2:3 lines were developed from a cross between Yaco“S” and susceptible cultivar Mingxian169. Illumina 90K and 660K single nucleotide polymorphism (SNP) chips were implemented to bulked pools and their parents to identify SNPs associated with the major QTL. A high-density linkage map was constructed using simple sequence repeat (SSR) and SNP markers. Inclusive composite interval mapping detected a major effect QTL Qyryac.nwafu-2BS conferring stable resistance to stripe rust in all tested environments. Qyryac.nwafu-2BS were mapped to a 1.3 cm interval and explained 17.3–51.9% of the phenotypic variation. Compared with stripe rust resistance genes previously mapped to chromosome 2B, Qyryac.nwafu-2BS is likely a new APR gene to stripe rust. Combining SNP iSelect assay and kompetitive allele specific PCR technology, we found that the APR gene could be rapidly and accurately mapped and it is useful for improving stripe rust resistance in wheat breeding.     

甘蔗抗褐锈病新基因抗感病池构建及SSR多态性引物筛选

由黑顶柄锈菌(Puccinia melanocephala H.Sydow&P.Sydow)引起的甘蔗褐锈病是危害中国甘蔗生产的主要病害之一.为了鉴定和发掘抗褐锈病新基因,防止褐锈病爆发流行和保证甘蔗安全生产,本研究以杂交组合'粤糖03-393'x'ROC 24'抗感分离真实性F,代群体为材料,构建抗感基因池,合成44...     

小麦突变体D51抗秆锈性遗传分析及其抗性基因SSR标记

D51是优良品系龙6239经辐射诱变和组织培养相结合获得的高产优质抗秆锈突变体材料,对我国优势秆锈病21C3CPH、21C3CKH和21C3CTR小种均表现免疫。利用来自D51/龙6239、D51/中国春的2个F₂群体和来自D51/龙6239的1个F₂群体分别在苗期和成株期进行21C3CPH小种接种,抗病反应型鉴定表明,3个F₂群体中抗感分离比例均为3∶1,说明D51抗秆锈性受一对显性基因控制,全生育期表达,部分D51/龙6239 F₂植株的F₃株系的抗病鉴定进一步验证F₂鉴定的可靠性。利用675对小麦SSR引物和185株D51/龙6239 F₂分离群体对SrD51基因进行标记定位,将SrD51定位在5D染色体的短臂上。其中SSR标记Xgwm190和Xwmc150与SrD51基因的遗传距离分别为18.58和21.33 cM,并分别位于目的基因的两侧。由于已知的秆锈抗性基因仅有Sr30被定位在小麦5DL上,且Sr30不抗34C2MKK和34C2MFK,而突变体D51的原亲本龙6239不抗21C3CPH、21C3CKH和21C3CTR,却对34C2MKK和34C2MFK表现免疫抗性。因而推断此突变体的秆锈抗性基因可能是一个新基因,暂命名为SrD51。     

小麦新品种“山农20”抗病基因的分子检测

山农20是2011年和2012年分别通过国家黄淮南、北片审定的小麦高产多抗新品种,在国家区试抗病性鉴定和生产中都表现出良好的抗黄淮麦区主要病害的特性。本研究利用与小麦抗白粉病、条锈病、叶锈病、纹枯病基因和抗赤霉病主效QTL紧密连锁的SSR、SCAR、STS等标记对该品种进行了分子检测,发现山农20含有6个抗白粉病基因(Pm12、Pm24、Pm30、Pm31、Pm35和Pm36),6个抗条锈病基因(Yr5、Yr9、Yr15、Yr24、Yr26和YrTp1),2个抗叶锈病基因(Lr21和Lr26),1个抗纹枯病基因(Ses1),但未检测到抗赤霉病主效QTL。分子检测结果部分解释了山农20的优良抗病性,也为利用分子标记辅助选择培育抗病稳产小麦新品种提供参考。     

Detection of molecular markers linked to the durable adult plant stripe rust resistance gene Yr18 in bread wheat (Triticum aestivum L.)

Stripe rust of wheat caused by Puccinia striiformis West. f. sp. tritici presents a serious problem for wheat production worldwide, and identification and deployment of resistance sources to it are key objectives for many wheat breeders. Here we report the detection of simple sequence repeat (SSR) markers linked to the durable adult plant resistance of cv. ‘Otane’, which has conferred this resistance since its release in New Zealand in 1984. A double haploid population from a cross between ‘Otane’ and the susceptible cv. Tiritea’ was visually assessed for adult plant infection types (IT) in the glasshouse and field, and for final disease severity in the field against stripe rust pathotype 106E139A⁺. At least three resistance loci controlled adult plant resistance to stripe rust in this population. Quantitative trait loci (QTL) mapping results revealed that two of these, one on chromosome 7DS corresponds to the durable adult plant resistance gene Yr18 and other on chromosome 5DL were contributed from ‘Otane’; while the remaining one on chromosome 7BL, was contributed from the susceptible ‘Tiritea’. Interval mapping placed the ‘Otane’‐resistant segment near the centromere of chromosome 7DS at a distance of 7 cM from the SSR marker gwm44. The stability of QTL in the two environments is discussed. SSR gwm44 is potentially a candidate marker for identifying the durable resistance gene Yr18 in breeding programmes.     

CIMMYT小麦种质C615抗叶锈病QTL分析

由Puccinia triticina引起的叶锈病是小麦主要病害之一, 引进种质C615具有叶锈病成株期抗性, 但其抗病性遗传机制尚不清楚。本研究以抗病亲本C615与高感叶锈病亲本宁麦18构建的F_2:7代重组自交系群体为材料, 利用337对多态性SSR标记构建遗传连锁图谱, 结合2016、2017连续两年的叶锈病鉴定结果进行复合区间作图, 结果在1BL、2DS、3BS、4DL和6BS染色体上共发现了5个抗性QTL, 暂命名为QLr.njau-1BL、QLr.njau-2DS、QLr.njau-3BS、QLr.njau-4DL和QLr.njau-6BS。其中, QLr.njau-1BL、QLr.njau-3BS和QLr.njau-4DL在两年均被检测到, 分别解释10.1%~15.7%、10.9%~13.5%和8.2%~9.0%的表型变异; 另2个QTL只在一年被检测到, 解释6.2%和9.2%的表型变异。除QLr.njau-2DS外的4个抗性QTL均来源于抗病亲本C615。QLr.njau-1BL和QLr.njau-4DL分别与已报道的慢病性基因Lr46、Lr67在同一区域, QLr.njau-3B可能为一个新的抗叶锈病QTL。此外, 本研究在C615/扬麦13 (轮回亲本)BC₄F₅回交群体中选出了15个农艺性状优良且抗叶锈病的株系, 利用与C615所含抗性QTL紧密连锁的7个SSR标记对其进行基因型检测, 结果显示所有这15个株系均含有来自C615的抗性QTL, 且有3个株系聚合了全部抗性位点, 表明C615可作为抗源亲本用于高产、抗病育种。本研究结果将为分子标记选育抗叶锈品种提供材料和技术支撑。     

Phenotypic and genotypic screening for rust resistance in common bean germplasm in Uganda

Rust caused by Uromyces appendiculatus (Pers., Pers.) Unger is one of the major foliar diseases of common bean (Phaseolus vulgaris) in Uganda. The use of host resistance remains the best option in managing this disease. The objective of this study was to identify sources of broad-spectrum rust resistance in common bean germplasm including landraces, commercial cultivars and introduced genotypes using a combination of phenotypic and genotypic screening with 22 simple sequence repeat (SSR) markers located on chromosome Pv04. A total of 138 genotypes were field screened from 2014 and 2015 using an alpha lattice design. The variance and correlation of disease incidence, area under the disease progression curve (AUDPC) and total grain yield were computed using GenStat. The polymorphism information content of the genotypes was determined, and the association of the markers and the disease resistance traits were analyzed using PowerMarker and TASSEL respectively. Resistance of each genotype was compared to the presence and absence of amplified markers. There were highly significant differences (P < 0.001) among the genotypes for disease incidence, AUDPC and total grain yield and a strong correlation (P < 0.001) between disease incidence and AUDPC in both years. The SSR markers, BARC_PV_SSR04725, bean_ssr_0778 and bean_ssr_2892 were associated (P ≤ 0.05) with rust resistance. Fifteen 15 genotypes which included the landraces Nabufumbo, and Kapchorwa white, and the commercial cultivar NABE 2 were identified as new sources of rust resistance that would be useful in future bean breeding programmes in Uganda.     

Identification and molecular mapping of a leaf rust resistance gene in spelt wheat landrace Altgold

Leaf rust, caused by Puccinia triticina, is an important disease for wheat production, both in China and worldwide. In laboratory studies spelt wheat (Triticum aestivum ssp. spelta) landrace Altgold was resistant to P. triticina races THT and PHT and genetic analysis indicated that it possessed a dominant leaf rust resistance gene, temporarily designated LrAlt. F₆ recombinant inbred lines (RILs) derived from a cross with the susceptible common wheat cultivar Nongda 3338 were used to map LrAlt with SSR markers. The resistance gene was distal to SSR loci Xbarc212, Xwmc382, Xgwm636, and Xwmc407 on the short arm of chromosome 2A. The closest markers Xbarc212 and Xwmc382 which co-segregated were 1.8 cM away from LrAlt. The relationships of LrAlt and other wheat leaf rust resistance genes located on the short arm of chromosome 2A were discussed, suggesting that LrAlt might be a new leaf rust resistance gene.