Development and Validation of SCAR Markers Co-Segregating with an Agropyron Elongatum Derived Leaf Rust Resistance Gene Lr24 in Wheat

Summary An Agropyron elongatum-derived leaf rust resistance gene Lr24 located on chromosome 3DL of wheat was tagged with six random amplified polymorphic DNA (RAPD) markers which co-segregated with the gene. The markers were identified in homozygous resistant F₂ plants taken from a population segregating for leaf rust resistance generated from a cross between two near-isogenic lines (NILs) differing only for Lr24. Phenotyping was done by inoculating the plants with pathotype 77-5 of Puccinia triticina. To enable gene-specific selection, three RAPD markers (S1302₆₀₉, S1326₆₁₅ and OPAB-1₃₈₈) were successfully converted to polymorphic sequence characterized amplified region (SCAR) markers, amplifying only the critical DNA fragments co-segregating with Lr24. The SCAR markers were validated for specificity to the gene Lr24 in wheat NILs possessing Lr24 in 10 additional genetic backgrounds including the Thatcher NIL, but not to 43 Thatcher NILs possessing designated leaf rust resistance genes other than Lr24. This indicated the potential usefulness of these SCAR markers in marker assisted selection (MAS) and for pyramiding leaf rust resistance genes in wheat.     

SCAR marker tagged to the alien leaf rust resistance gene Lr19 uniquely marking the Agropyron elongatum-derived gene Lr24 in wheat: a revision

A sequence characterized amplified region (SCAR) marker tagged to an Agropyron elongatum‐derived leaf rust resistance (Lr) gene Lr19 was validated on 18 known alien Lr gener in near‐isogenic lines (NILs) in the variety ‘Thatcher’, along with three wheat cultivers carrying Lr24 and two carrying Lr19. The marker was expressed only in the Lr24 lines confirming that the marker tagged the geneLr24. The monomorphic expression of the SCAR marker in 10NIL pairs for Lr19 and Lr24 revealed that each NIL pair possessed the same gene, Lr24. The donor parents used in the NIL pairs for Lr19 (‘Sunstar*6/C80‐1′) and Lr24 (‘TR380‐14*7/3Ag#14′) amplified the same fragment. Nonsegregation for leaf rust in the F₂ population of the cross between the above donor parents confirmed the presence of the same gene in the two parents. Apparently, a genuine parent stock of ‘Sunstar*6/C80‐1’ was not involved in the development of the NIL pairs for Lr19 due to an improper maintence bredding protocol either at source or destination which went undetected in the absence of signs of virulence for either gene in the region.     

小麦抗白粉病近等基因系遗传背景的分子标记检测

利用AFLP分子标记技术,对近年来培育的几套近等基因系的遗传背景进行了检测,发现所检测到的遗传多样性主要集中于供体亲本之间,而近等基因系内部已达到很高的遗传一致性;包含Pm4b的近等基因系、包含Pm2/Pm6的近等基因系与轮回亲本遗传上比较接近,其内部选系之间也达到很高的一致性,是理想的近等基因系;发现Pm21中簇毛     

Yield and quality evaluations on a pair of processing tomato lines nearly isogenic for the Tm2a gene for resistance to the tobacco mosaic virus

A pair of processing tomato (Lycopersicon esculentum Mill.) lines, nearly isogenic for the Tm-2^a gene for resistance to tobacco mosaic virus, were grown in replicated trials under commercial production conditions in five locations worldwide. The lines were evaluated for 17 processing traits including fruit yield, size, soluble solids concentration, color, firmness, and viscosity. Eight of those traits differed significantly among the nearly-isogenic lines (NILs). Most notably, the NIL heterozygous for Tm-2^a yielded, on average, 16% more than the NIL homozygous for the susceptible allele and 33% more than the NIL homozygous for Tm-2^a. Viscosity was lower in the heterozygous NIL and the homozygous and heterozygous resistant NILs had softer fruit with larger stem scars compared to the homozygous susceptible NIL. These results indicate the presence of the Tm-2^a locus affects many traits of importance for processing tomatoes and may be used, in the heterozygous state, to significantly increase yield. Whether the observed effects are due to the Tm-2^a gene itself or genes associated via linkage drag could not be determined. This revised version was published online in July 2006 with corrections to the Cover Date.     

The influence of leaf rust resistance genes Lr29, Lr34, Lr35 and Lr37 on breadmaking quality in wheat

Leaf rust, caused by Puccinia triticina, is considered one of the most important diseases of wheat. In South Africa the genes Lr29, Lr34, Lr35 and Lr37 confer effective resistance to leaf rust, qualifying them for use in cultivar improvement. To study possible secondary effects of these genes, a collection of BC6 lines containing each of the genes singly, was evaluated for breadmaking quality. The recurrent parent Karee, and Thatcher NILs used as resistance donors in the primary crosses, as well as Thatcher, were included as checks. The presence of Lr29, Lr34, Lr35 and Lr37 caused a significant increase in flour protein and water absorption. For most of the other characteristics the NILs performed statistically similar to the recurrent parent. Some sib lines performed significantly better than others, emphasising the value of selecting for improved quality among backcross lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

The extent of similarity between near-isogenic lines of the Australian spring wheat variety Triple Dirk with their recurrent parent

Summary The near-isogenic lines of the wheat variety Triple Dirk supposed to differ only for Vrn genes have been investigated for their similarity to Triple Dirk and for purity. The NILs were found to differ from Triple Dirk. It was also discovered that Triple Dirk and the NILs each exist of more than one genotype. It is recommended that each NIL is tested for its Vrn-genotype and purified. New sets of Vrn NILs can only be made with these checked NILs and with one-plant progeny of the newly used recurrent parents.     

STMS markers for grain protein content and their validation using near-isogenic lines in bread wheat

The present study, undertaken as a continuation of an earlier study on quantitative trait loci (QTL) analysis of grain protein content (GPC) in bread wheat (Prasad et al. 1999), includes the following: (1) identification of an additional molecular marker associated with GPC; (2) development of near‐isogenic lines (NILs) for high GPC; and (3) the use of three sets of NILs (a total of 10 NILs) to validate the two available markers linked with QTL for GPC. A total of 114 sequence‐tagged microsatellite site (STMS) primer pairs (that were not used in the previous study) were used for detection of polymorphism between the two parents (PH132, with high GPC; WL711, with low GPC) of a mapping population of 100 recombinant inbred lines (RILs). A total of 95 primer pairs gave amplification products, of which only 30 detected reproducible polymorphism between the parental genotypes. Bulked segregant analysis was conducted using these 30 primers on two bulks (each comprising eight RILs) representing the two extremes of the normal distribution. A solitary primer pair (WMC415) showed association with GPC, which was further confirmed through selective genotyping. Subsequently, 100 RILs were genotyped. A single‐marker linear regression analysis showed significant association between the marker WMC415 and GPC, thus identifying a quantitative trait locus (designated as QGpcccsu‐5A1), which explained 6.21% of the variation for GPC among the RILs. The above STMS marker, together with the STMS marker (WMC41) identified earlier, explains approximately 25% of the variation for GPC. In order to conduct validation of the above two available markers, 10 NILs were developed for high GPC using two genotypes (WL711 and HD2329) with low GPC as recipient parents and another two genotypes (PH132 and PH133) with high GPC as donor parents. NIL 2233 (with 11.7% GPC), derived from HD2329, when tried with WMC41 gave a characteristic amplification profile similar to that of its donor parent PH132, and NIL 2215 (with 11.9% GPC) derived from WL711, when tried with WMC415 gave an amplification profile that resembled its donor parent PH133. The remaining eight NILs with high GPC gave patterns similar to those of their corresponding recipient parents with both the markers, suggesting that either the QTL, other than those associated with the above markers, were actually transferred from the donor parents and contributed to high GPC in these NILs or that recombination had occurred between the markers identified and the corresponding QTL. Thus, the marker validation conducted using NILs, while demonstrating the utility of these two microsatellite markers for use in marker‐assisted selection in plant breeding, also suggested that many more QTL exist that would need to be identified using closely linked molecular markers.     

Genetics of stem rust resistance in the spring wheat cultivar Thatcher and the enhancement of stem rust resistance by <Emphasis Type="Italic">Lr34</Emphasis>

Three recombinant inbred line populations from the crosses RL6071/Thatcher, RL6071/RL6058 (Thatcher Lr34), and Thatcher/RL6058, were used to study the genetics of stem rust resistance in Thatcher and TcLr34. Segregation of stem rust response in each population was used to determine the number of genes conferring resistance, as well as the effect of the leaf rust resistance gene Lr34 on stem rust resistance. The relationship between resistance in seedling and adult plants was also examined, and an attempt was made to identify microsatellite markers linked to genes that were effective in adult plants. In field plot tests at least three additive resistance genes segregated in the RL6071/RL6058 population, whereas two resistance genes segregated in the RL6071/Thatcher population. The presence of the gene Lr34 permitted the expression of additional stem rust resistance in Thatcher-derived lines both at the seedling and adult plant stages. Seedling resistance to races TPMK and RKQQ was significantly associated with resistance in adult plants, whereas seedling resistance to races QCCD and QCCB may have made a minor contribution. The seedling resistance genes Sr16 and Sr12 may have contributed to resistance in adult plants. A molecular marker linked to resistance in adult plants was identified on chromosome 2BL.     

小麦抗叶锈基因Lr24、Lr35的STS、SCAR标记在近等基因系(NILs)上的特异性验证

用一套分别含有不同抗叶锈基因的53个以Thatcher为遗传背景的近等基因系（near-isogeniclines,NILs）对已报道的分别与抗叶锈基因Lr24和Lr35连锁的STS、SCAR进行特异性验证。结果对于与Lr24连锁的STS标记,在53个NILs中只在TcLr24亲本中扩增出片段大小与报道相同的310bp的条带,在TcLr35中也扩增出了一条片段,但片段大小不同于310bp约为270bp。对于与Lr35连锁的SCAR标记,只在TcLr35亲本中扩增出片段大小为900bp的条带,与报道片段大小一致。验证结果表明与抗病基因Lr24和Lr35连锁的STS、SCAR分子标记在NILs中特异性都较好,进一步证明了这两个分子标记可方便地用于小麦抗叶锈基因Lr24、Lr35的分子标记辅助选择育种。     

Development of near-isogenic lines for a major QTL on 3BL conferring Fusarium crown rot resistance in hexaploid wheat

By essentially fixing the genetic background, near-isogenic lines (NILs) are ideal for studies of the function of specific loci. We report in this paper the development of NILs for a major QTL located on the long arm of chromosome 3B conferring Fusarium crown rot (FCR) resistance in hexaploid wheat. These NILs were generated based on the method of the heterogeneous inbred family analysis. 13 heterozygous lines were initially selected from three segregating populations using a single SSR marker linked with the major FCR QTL. The two isolines for each of the putative NILs obtained showed no obvious morphological differences, but differences among the NIL pairs were large. Significant differences in FCR resistance between the isolines were detected for nine of the 13 putative NIL pairs. The presence of the FCR allele from the resistant parent reduced FCR severity by 29.3–63.9% with an average of 45.2% across these NILs. These NILs will be invaluable in further characterising this major FCR locus, in studying the mechanism of FCR resistance and in investigating possible interactions between FCR resistance and other traits of agronomic importance.     

Climacteric and non-climacteric behavior in melon fruit: 2. Linking climacteric pattern and main postharvest disorders and decay in a set of near-isogenic lines

A set of near-isogenic lines (NILs) of melon (Cucumis melo L.) was used to test the relationship between the climacteric pattern and postharvest disorders at harvest and after 30 days at 8 °C. The NILs contained different chromosome introgressions in the linkage group III from the non-climacteric exotic Korean accession PI 161375 transferred into the genetic background of the non-climacteric Spanish cultivar ‘Piel de Sapo’ (PS). A quantitative trait locus (QTL) in this linkage group induced climacteric behavior in eight NILs accompanied by a peak of ethylene production and fruit dehiscence to different degrees. The cultivar ‘Nicolás’ and one NIL showed a non-climacteric pattern of respiration rate and ethylene production. The climacteric NILs were used to test the relationship between this pattern and postharvest disorders. The reference climacteric lines ‘Fado’ and ‘Védrantais’ were more sensitive to CI and associated Cladosporium rot than the NILs or PS. In general, a more intense climacteric behavior was accompanied by fruit dehiscence, and higher total losses and greater skin scald after storage, than in PS. A higher incidence of chilling injury (CI) in the climacteric NILs was found compared with the non-climacteric ones, although with exceptions (one NIL for CI in the form of scald; the same NIL and one more for pitting). The climacteric onset and netting scald were not related, and CI in the form of skin spots was only found in climacteric NILs and was positively correlated with the maximum peak of ethylene production. Some climacteric NILs did not follow the rule of a higher susceptibility to other disorders and decay after storage compared with PS, such as for example in fruit over-ripening (detected externally or internally), Cladosporium rot at the peduncle and Alternaria rot. Mealiness was independent of climacteric behavior. Three climacteric NILs obtained better flavor scores after storage than PS, although the maximum peak of ethylene production was positively correlated with off-flavor. Genotypic correlation between disorder data and the physiological data of climacteric fruit revealed positive (flavor index) or negative postharvest consequences (skin injuries, rots or off-flavors). At least one QTL can be assigned to most of the quality traits analyzed.     

The degree of similarity of backcross lines of Triticum aestivum cultivars manitou and neepawa with Aegilops speltoides accessions as donors

Summary The degree of similarity of a BC line with its recurrent parent is not related to the presence of expressions for morphological characters originating from the donor like purple coleoptile, purple anther and waxy leaf. BC lines derived from one donor do not resemble each other more than they do other BC lines. The absence of characters conditioned by dominant or co-dominant genes may be caused by the presence of inhibitor genes.     

The degree of phenotypic similarity of near-isogenic lines of the wheat cultivar Centana with their recurrent parent

Summary The degree of phenotypic similarity of Centana and its near-isogenic lines (NILs) has been investigated. Some NILs have a disease reaction, a gliadin pattern and a yield level which differs from Centana. This means that they still possess many donor genes. One of the NILs, Shortana, must be a derivative of a backcross line crossed with an unknown genotype resulting in a good yielding variety.     

Effect of three anthocyaninless genes on germination in tomato (Lycopersicon esculentum Mill.) I. Seed germination under optimal conditions

The effect of three mutant genes affecting anthocyanin biosynthesis – anthocyaninless of Hoffmann (ah), anthocyaninwithout (aw) and baby lea syndrome (bls) – on tomato germination capacities at 24 ° C was investigated. The study was performed on nine anthocyanin containing lines – Apedice, Apeca, Ailsa Craig, Monfavet 167, Monfavet 168, Por, Piernita, VF 36 and VFNT cherry and 13 isogenic/near isogenic lines (IL/NIL) from them: 4 differing for gene ah, 3 differing for gene aw and 6 differing for gene bls). In the majority of the anthocyaninless IL/NILs germination began earlier than that in the wild type lines. Significant differences were observed in the time to 50% germination between the anthocyanin-containing and the anthocyaninless IL/NILs except for the most rapidly germinating line VFNT cherry. Seed weight, water uptake, effect of testa removal and presence or absence of maternal effects in F1 germination responses were investigated in order to elucidate the causes of the more rapid germination in the anthocyaninless IL/NILs. The increased amount of water uptake by the anthocyaninless lines, the shorter treatment time necessary for their testa removal, the important maternal effects in the genetic variability of the time to 50% germination and the differences in germination capacities between the wild type and the mutant IL/NILs after testa removal, indicated that the seed coat and the endosperm had to be affected by the effects of the three genes in a way that caused enhanced germination. This revised version was published online in July 2006 with corrections to the Cover Date.     

AFLP and SCAR markers linked to the suppressor gene (Rf) of a dominant genetic male sterility in rapeseed (Brassica napus L.)

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene (Ms) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene (Rf). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus. In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual (MsMsrfrf) and a fertile individual (MsMsRfrf) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.     

Effect of row type,flowering type and several other spike characters on resistance to Fusarium head blight in barley

Fusarium head blight (FHB) is a destructive disease of barley. The genetics and expression of resistance to FHB in barley is complex, and various spike characters are thought to possibly influence resistance. Tests using spray-inoculation of Fusarium graminearum at anthesis in greenhouse environments showed that two-rowed and cleistogamous varieties from Japan belong to the highest resistance group, while six-rowed and chasmogamous varieties are mostly susceptible. In order to evaluate the effect of such spike characters, including row type and flowering type, on FHB resistance, near-isogenic lines (NILs) differing in these characters were tested for their resistance. Two testing methods were used: the pot-plant and cut-spike methods, in which spikes at anthesis were spray-inoculated in greenhouse environments. The chasmogamous NILs and some six-rowed NILs were significantly more diseased than cleistogamous and two-rowed parent lines, respectively, and the difference in FHB severity was greater and more stable between cleistogamous/chasmogamous NIL pairs than between two-/six-rowed pairs. Slight or no differences were observed in glaucous/non-glaucous, normal/dense spike, normal/uzu type and normal/deficiens NIL pairs. The results indicate that the contribution of cleistogamy and/or the genetic background toward FHB resistance is more than that of row type and the other tested spike characters. Further, it should be possible to develop six-rowed varieties with FHB resistance nearly as good as that of the two-rowed varieties.     

Effect of three anthocyaninless genes on germination in tomato (Lycopersicon esculentum Mill.) II. Seed germination under stress conditions

Seeds from four pairs of tomato isogenic/near-isogenic lines (IL/NIL) differing for anthocyaninless of Hoffmann (ah), three pairs of IL/NILs differing for anthocyaninwithout (aw) and six pairs of IL/NILs differing for baby lea syndrome (bls) were evaluated for their germination ability under stress conditions: low and high temperature, salt and osmotic stress. Mutant genes affecting anthocyanin biosynthesis enhanced tomato germination capacity under all above mentioned treatments, except for the aw gene under a PEG-6000 treatment. Testa removal accelerated germination under stress conditions, but differences in time to 50% germination between the wild type lines and their ah-, aw- and bls-IL/NILs although diminished, were not eliminated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization and identification of cold tolerant near-isogenic lines in rice

To exploit the genetic mechanism of cold tolerance in rice, cold tolerant near-isogenic lines (NILs) were developed by backcrossing Kunmingxiaobaigu (KMXBG), reported to be the most cold-tolerant variety at the booting stage, as donor, with the cold sensitive Japanese commercial japonica variety, Towada. Comparisons of cold tolerance-related traits between five BC₆F₅ NILs and recurrent parent Towada under cold treatment and normal temperatures at the booting stage showed that the differences between the NILs and Towada were significant only for spikelet fertility-related traits. Analyses of cold tolerance in the NILs at the budding (germination), seedling and booting stages indicated both correlated effects and differences. Lines 1913-4 and 1916-1 showed strong and stable tolerance at all three stages. Whole genome marker screening showed that the proportion of genetic background recovery was more than 98%. Seventeen markers from KMXBG were introgressed in two or more NILs, and cold tolerance genes were possibly present in these marker regions. The NILs should be excellent materials for both rice improvement and map-based cloning of cold tolerance QTLs.     

Characterization of segregation distortion on chromosome 3 induced in wide hybridization between indica and japonica type rice varieties

We previously surveyed chromosomal regions showing segregation distortion of RFLP markers in the F₂ population from the cross between a japonica type variety ‘Nipponbare’ and an indica type variety ‘Milyang23’, and showed that the most skewed segregation appeared on the short arm of chromosome 3. By comparison with the marker loci where distortion factors were previously identified, this region was assumed to be a gametophytic selection-2 (ga2) gene region. To evaluate this region, two near isogenic lines (NILs) were developed. One NIL had the ‘Nipponbare’ segment of this region on the genetic background of ‘Milyang23’ (NIL9-23), and the other NIL had the ‘Milyang23’ segment on the genetic background of ‘Nipponbare’ (NIL33-18). NIL9-23 and ‘Milyang23’, NIL33-18 and ‘Nipponbare’, and ‘Nipponbare’ and ‘Milyang23’ were respectively crossed to produce F₁ and F₂ populations. The F₁ plants of NIL9-23 × ‘Milyang23’ and NIL33-18 × ‘Nipponbare’ showed high seed fertility and the same pollen fertility as their parental cultivars, indicating that ga2 does not reduce seed and pollen fertility. Segregation ratio of a molecular marker on the ga2 region in the three F₂ populations was investigated to clarify whether segregation distortion occurred on the different genetic backgrounds. Segregation distortion of the ga2 region appeared in the both F₂ populations from the NIL9-23 and ‘Milyang23’ cross (background was ‘Milyang23’ homozygote) and the ‘Nipponbare’ and ‘Milyang23’ cross (background was heterozygote), but did notin the F₂ population from the NIL33-18 and ‘Nipponbare’ cross (background was ‘Nipponbare’ homozygote). This result indicates that ga2 interacts with a ‘Milyang23’ allele(s) on the different chromosomal region(s) to cause skewed segregation of the ga2 region. In addition, segregation ratio was the same between the F₂ populations from NIL9-23 × ‘Milyang23’ and ‘Nipponbare’ × ‘Milyang23’ crosses, suggesting that the both genotypes, ‘Milyang23’ homozygote and heterozygote, of gene(s) located on the different chromosomal region(s) have the same effect on the segregation distortion. This revised version was published online in July 2006 with corrections to the Cover Date.     

利用分子标记辅助选择优质蛋白玉米近等基因系

为了扩增优质蛋白玉米育种新种质,用普通玉米种质转育创制优质蛋白玉米(QPM)近等基因系;利用SSR标记phi057进行辅助选择,以优质蛋白玉米种质为o2基因供体,转导、构建来自不同遗传背景普通玉米种质为受体的QPM近等基因系。结果表明：利用共显性SSR标记引物phi057在o2基因供体自交系与普通玉米受体自交系间表现多态性,能区分O2O2、O2o2和o2o2等3种基因型,来自不同遗传背景种质所构建获得的QPM近等基因系赖氨酸含量有不同程度提高,赖氨酸含量达到0.36%~0.42%,不同遗传背景受体自交系赖氨酸增加幅度9.1%~64.0%。普通玉米种质导入供体o2基因后能够提高种质赖氨酸含量。