Inheritance and gene tagging of male fertility restoration of cytoplasmic-nuclear male-sterile line NJCMS1A in soybean

The F₁, F₂ and F_2:3 of the NJCMS1A × 'Zhongdou 5' cross were used to analyse the inheritance of the male fertility restoration of the cytoplasmic-nuclear male-sterile line NJCMS1A in soybean. The results of genetic analysis showed two pairs of dominant genes conferring the male fertility restoration of NJCMS1A, which further confirmed previous results. The F₂ population from the NJCMS1A × 'Zhongdou 5' cross was used for tagging the restorer genes for NJCMS1A with 664 pairs of simple sequence repeat primers selected randomly from the genetic linkage map of soybean published by Cregan et al. (1999) . Satt626 on linkage group M and Satt300 on linkage group A1 of the integrated linkage map by Song et al. (2004) were found to link to the two restorer genes of NJCMS1A. The maximum-likelihood estimates of the genetic distance between the two markers, Satt626 and Satt300, and the two restorer genes of 'Zhongdou 5' were 9.75 and 11.18 cM, respectively.     

大豆质核互作雄性不育系NJCMS3A双亲雄性育性基因的SSR标记

利用大豆质核互作雄性不育系NJMCS3A的质、核供体亲本N21566和N21249构建F₂和BC₁F₁育性分离群体进行雄性育性的遗传分析与基因定位。结果表明, F₁正反交可育,F₂和BC₁F₁的可育株与不育株分离比例经χ²测验分别符合3∶1和1∶1,表明NJCMS3A供体亲本雄性育性由一对基因控制,可育等位基因为显性。该基因可能是NJCMS3A的一个恢复基因。选用793对SSR引物对F₂和BC₁F₁群体分别进行育性基因定位,发现该育性基因位于O连锁群上,在Satt331和Satt477标记之间,与Satt331、CSSR133和Satt477标记距离的次序一致,分别为8.1~10.4 cM、11.4~16.4 cM、13.3~19.2 cM。     

QTL mapping of maize (Zea mays) stay-green traits and their relationship to yield

A maize genetic linkage map derived from 115 simple sequence repeat (SSR) markers was constructed from an F₂ population. The F₂ was generated from a cross between a stay-green inbred line (Q319) and a normal inbred line (Mo17). The map resolved 10 linkage groups and spanned 1431.0 cM in length with an average genetic distance of 12.44 cM between two neighbouring loci. A total of 14 quantitative trait loci (QTL) were detected for stay-green traits at different postflowering time intervals and identified by composite interval mapping. The respective QTL contribution to phenotypic variance ranged from 5.40% to 11.49%, with trait synergistic action from Q319. Moreover, maize stay-green traits were closely correlated to grain yield. Additional QTL analyses indicated that multiple intervals of stay-green QTL overlapped with yield QTL.     

Identification and molecular tagging of a stripe rust resistance gene in wheat line P81

Stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is one of the most devastating diseases in common wheat ( Triticum aestivum L.). With the objective of identifying and tagging a new gene for resistance to stripe rust in wheat line P81, F₁, F₂ and F_2:3 populations from the cross 'Chuanmai 28'/P81 were inoculated with Chinese PST race CYR32 in greenhouse and field trials. P81 carried a single dominant gene for resistance (designated YrP81 ) to CYR32. Tests of allelism showed that YrP81 was different from Yr5 , Yr10 , Yr15 and Yr26 . Simple sequence repeat (SSR) and resistance gene-analogue polymorphism (RGAP) between the parents were used for genotyping the F₂ populations. YrP81 was closely linked to four SSR loci on chromosome 2BS with genetic distances of 18.3 cM ( Xwmc25 ), 1.8 cM ( Xgwm429 ), 4.1 cM ( Xwmc770 ) and 5.3 cM ( Xgwm148 ). Two RGAP markers RGA1 (NLRR/XLRR) and RGA2 (Pto kin4/NLRR-INV2) were also closely linked to YrP81 with genetic distances of 4.7 and 6.3 cM, respectively. The linkage map of YrP81 and molecular markers was established in the order Xwmc25 - RGA2 - RGA1 - Xgwm429 - YrP81 - Xwmc770 - Xgwm148 . Pedigree analysis, response patterns with Chinese PST races and associations with markers suggested that YrP81 is a novel stripe rust resistance gene. The PCR-based microsatellite and RGAP markers identified here could be applied in selection of YrP81 in wheat breeding.     

The restorer gene for soybean M‐type cytoplasmic male sterility,Rf‐m,is located in a PPR gene‐rich region on chromosome 16

Over the past decade, M‐type cytoplasmic male sterility (CMS) line W931A and a variety of restorer lines have been exploited for the release of hybrid seeds in soybean (Glycine max). However, the identities of restorer genes in the nuclei of soybean restorer lines are still unclear. In this study, we analysed the inheritance pattern of restorer locus Rf‐m from restorer line WR016 and constructed a high‐resolution map of this locus. Results showed that Rf‐m in WR016 is a monogenic dominant gene located within a 162.4‐kb region on chromosome 16, which is flanked on each side by new developed simple sequence repeat (SSR) markers GmSSR1602 and GmSSR1610 at a distance of 0.11 and 0.25 cM, respectively. Nineteen open reading frames (ORFs) were predicted in this region. Of these, seven genes arranged in tandem on chromosome 16 encode pentatricopeptide repeat (PPR) proteins, which is similar to other reported restorer loci in plants. These results lay a solid foundation for map‐based cloning of the Rf‐m gene and will be helpful for marker‐assisted selection of elite CMS restorer lines.     

SSCP Marker Development and Analysis of Candidate Restorer Gene Rf1 for Cotton Cytoplasmic Male Sterility

[Objective] Locating the cotton cytoplasmic male sterility (CMS) restorer gene Rf₁ is important for investigating restorer gene mechanisms and improving restorer lines. In our previous study, a gene cluster, with nine Pentatricopeptide repeat(PPR) genes and nine other genes, was found within the 160-kb Rf₁ target region in Scaffold 333. The objective here was to improve the density of Rf1-linked markers in the target region and determine the expression profiles of candidate genes. [Method] Using the sequences of the 18 genes, we designed 155 single-strand conformation polymorphism (SSCP) primers covering all of the gene sequences to identify the polymorphic SSCP markers between the fertile and sterile pools. Additionally, real-time polymerase chain reaction(PCR) was performed to analyze the expression profiles of eight candidate genes in the four developmental stages of buds of sterile, maintainer and restoring lines, respectively. [Result] In total, 15 polymorphic primers were identified. A genotype analysis of the F₂ population was conducted using the 15 primers and 3 other polymorphic simple sequence repeats (SSR) markers. The markers were distributed in a 4.8 cM range. In addition, owing to the influence of sterile cytoplasm or restorer genes, most of the genes showed different expression patterns in the four developmental stages of the three lines' buds. [Conclusion] SSCP markers tightly linked to Rf₁ were identified and the expression profiles of candidate genes were determined. This study provides a basis for the further fine mapping of restorer genes and for candidate gene screening.     

Tagging of four fertility restorer loci for wild abortive—cytoplasmic male sterility system in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) using microsatellite markers

Tagging of restorer genes for wild abortive (WA) CMS source by studying a 222 individual plants from a F₂ population of a cross between IR58025A × IR42686R. The restorer line IR42686R that was used in this study had been previously derived through random mating composite population (RMCP) involving 12 parents facilitated by IR36 genetic male sterility. Four Rf genes were tagged to simple sequence repeats (SSR) markers on chromosomes 1, 7, 10, 12 by recessive class analysis. The recombination frequency between a positive marker and Rf locus was calculated using maximum likelihood estimator assuming that all the 46 extremely sterile individual plants were homozygous at the targeted Rf locus. The recombination frequency between the marker and the restorer trait were converted to genetic distances using Kosambi function. A new Rf locus designated as Rf7 on chromosome 12 was found to be linked to RM7003 at a genetic distance of 13.3 cM (LOD 6.12). We report here first, a new molecular marker (RM 6344) linked to Rf4 locus on chromosome 7 that was previously mapped by trisomic analysis. RM443 and RM315 were flanking the Rf3 gene at a genetic distance of 4.4 (LOD 10.29) and 20.7 cM (LOD 3.98) on chromosome 1, respectively. The Rf6 was flanked on both side with SSR markers RM258 and RM591 at a genetic distance of 4.4 (LOD 10.29) and 23.3 cM (LOD 3.39) located on chromosome 10. The random mating composite population is an excellent breeding approach to develop superior restorer lines and for pyramiding different Rf genes of different CMS systems. Rf genes tagged with closely linked SSR markers would be facilitating marker assisted selection (MAS) in hybrid rice breeding program by reducing time and workload for identifying potential restorers. L. Bazrkar and A. J. Ali equally contributed to this work.     

Molecular mapping of a new gene for resistance to frogeye leaf spot of soya bean in 'Peking'

Amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) techniques were used to map the _RGSp_eking gene, which is resistant to most isolates of Cercospora sojina in the soya bean cultivar ‘Peking’. The mapping was conducted using a defined F₂ population derived from the cross of ‘Peking’(resistant) בLee’(susceptible). Of 64 EcoRI and MseI primer combinations, 30 produced polymorphisms between the two parents. The F₂ population, consisting of 116 individuals, was screened with the 30 AFLP primer pairs and three mapped SSR markers to detect markers possibly linked to Rcs_Peking. One AFLP marker amplified by primer pair E‐AAC/M‐CTA and one SSR marker Satt244 were identified to be linked to Res_Peking. The gene was located within a 2.1‐cM interval between markers AACCTA178 and Satt244, 1.1 cM from Satt244 and 1.0 cM from AACCTA178. Since the SSR markers Satt244 and Satt431 have been mapped to molecular linkage group (LG) J of soya bean, the Res_Peking resistance gene was putatively located on the LG J. This will provide soya bean breeders an opportunity to use these markers for marker‐assisted selection for frogeye leaf spot resistance in soya bean.     

Genetic mapping for the Rf1 (fertility restoration) gene in sunflower (Helianthus annuus L.) by SSR and TRAP markers

The Rf1 gene in sunflower can effectively restore the pollen fertility of PET1 cytoplasm in male-sterile lines and has been widely used in commercial hybrid production. Identifying molecular markers tightly linked to this gene will be useful in marker-assisted selection to develop maintainer and restorer lines. Rf1 has been mapped to Linkage Group (LG) 13 of the public sunflower simple sequence repeat (SSR) map by aligning maps constructed from different populations and only one SSR marker was reported to be loosely linked to Rf1 . This paper reports the result of applying target region amplification polymorphism (TRAP) and SSR markers to map and develop a sequence-tagged site (STS) marker tightly linked to Rf1 using two populations derived from a cross between two U.S. public sunflower lines, RHA439 and cmsHA441. An SSR marker, ORS511, was 3.7 cM from the Rf1 gene and a TRAP marker, K11F05Sa12-160, was linked to Rf1 at a distance of 0.4 cM. This TRAP marker was converted to an STS marker for using in sunflower breeding.     

大豆品种成熟期基因型推测的研究

选择不同来源中国大豆品种23份和国外引进的成熟期近等基因系35份进行SSR分析,目的是鉴定与成熟期基因型紧密连锁的标记,进而推测中国大豆品种的成熟期基因型。结果表明,（1）210对SSR标记中125对在成熟期近等基因型中具有多态性,推测与成熟期有关的标记有8个;（2）在Clark近等基因系中,筛选出成熟期基因E3/e3特异性标记Satt229,E4/e4的特异SSR标记Sct_010、Satt294、Satt247、Satt452和Satt156;在Clark和Harosoy近等基因系中,筛选出E7/e7的特异性SSR标记为Satt071、Satt178;（3）根据8个与成熟期相关的标记的分子数据,构建了国外大豆近等基因系的UPGMA聚类图,共聚为4类,背景来源相同或相似的材料被聚为一类,明显分为Clark近等基因系和Harosoy近等基因系。（4）与近等基因系成熟期基因(E7)分子标记比对,推测出25份中国大豆品种的成熟期基因。     

Molecular mapping of the fertility-restoration gene Rf4 for WA-cytoplasmic male sterility in rice

The wild abortive cytoplasmic male sterility (CMS‐WA) system, an ideal type of sporophytic CMS in indica rice, is used for the large‐scale commercial production of hybrid rice. Searching for restorer genes is a good approach when phenotyping is very time‐consuming and requires the determination of spikelet sterility in testcross progeny. To establish more precisely the genetical and physical maps of the Rf4 gene, high‐resolution mapping of this locus was carried out using simple sequence repeat (SSR) markers and newly designed markers in a F₂ population. The genetic linkage analysis indicated that five SSR markers (RM6737, RM304, RM171, RM5841 and RM228) on the long arm of chromosome 10 were linked with the Rf4 gene. Rf4 was flanked by two SSR markers RM171 and RM6737 at distances of 3.2 and 1.6 cM, respectively. Also, within the region between Rf4 gene and RM171, a newly designed primer pair, AB443, produced two sterile‐specific markers, AB443‐400 and AB443‐500, 0.5 and 1.03 cM from the gene. The flanking markers identified give promise for their application in molecular marker‐assisted selection (MAS) and they are also suitable for starting chromosome walking to clone Rf4 gene in the near future.     

大豆灰斑病1号生理小种抗性基因的SSR标记

针对中国大豆灰斑病1号生理小种,以抗所有生理小种的品系东农40566为母本,以感所有生理小种的品种东农410为父本配制杂交组合,杂交得到F2代后连续自交3代得到F5代群体。该群体经人工接种灰斑病1号生理小种后,利用BSA法对500个SSR标记进行筛选,其中3个标记Satt565、SOYGPATR和Satt396在抗、感池间表现出稳定的多态性,并且在F2代个体中表现出抗性与多态性协同分离的趋势。3个标记与抗性基因的连锁顺序为Satt565—SOYGPATR—Hrcs1—Satt396,它们与抗性基因的连锁距离分别为12.7cM、6.5cM、14.7cM。推测抗大豆灰斑病1号生理小种的基因可能位于C1连锁群上。     

Molecular mapping of genic male-sterile genes ms15, ms5 and ms6 in tetraploid cotton

Two genic male sterile (GMS) lines, Lang-A conditioned by ms ₁₅ and Zhongkang-A conditioned by ms ₅ ms ₆ duplicate recessive genes in Gossypium hirsutum L., were chosen to map GMS genes. These two lines were crossed with Gossypium barbadense cv. 'Hai7124' to produce segregating populations. The ms ₁₅ gene was mapped on chromosome 12, and was flanked by two simple sequence repeat (SSR) markers, NAU2176 and NAU1278, with a genetic distance of 0.8 and 1.9 cM respectively. The ms ₅ and ms ₆ genes were mapped to one pair of homoeologous chromosomes, ms ₅ on chromosome 12 flanked by three SSR markers, NAU3561, NAU2176 and NAU2096, with genetic distances of 1.4, 1.8 and 1.8 cM, respectively, and ms ₆ on chromosome 26 flanked by two SSR markers, BNL1227 and NAU460, with a genetic distance of 1.4 and 1.7 cM respectively. These tightly linked markers with the ms ₁₅ , ms ₅ and ms ₆ genes can be used in the marker-assisted selection among segregating populations in a breeding programme, and provide the foundation for gene isolation by map-based cloning for these three genes.     

Association analysis using SSR markers to find QTL for seed protein content in soybean

Association analysis studies can be used to test for associations between molecular markers and quantitative trait loci (QTL). In this study, a genome-wide scan was performed using 150 simple sequence repeat (SSR) markers to identify QTL associated with seed protein content in soybean. The initial mapping population consisted of two subpopulations of 48 germplasm accessions each, with high or low protein levels based on data from the USDA’s Germplasm Resources Information Network website. Intrachromosomal LD extended up to 50 cM with r ² > 0.1 and 10 cM with r ² > 0.2 across the accessions. An association map consisting of 150 markers was constructed on the basis of differences in allele frequency distributions between the two subpopulations. Eleven putative QTL were identified on the basis of highly significant markers. Nine of these are in regions where protein QTL have been mapped, but the genomic regions containing Satt431 on LG J and Satt551 on LG M have not been reported in previous linkage mapping studies. Furthermore, these new putative protein QTL do not map near any QTL known to affect maturity. Since biased population structure was known to exist in the original association analysis population, association analyses were also conducted on two similar but independent confirmation populations. Satt431 and Satt551 were also significant in those analyses. These results suggest that our association analysis approach could be a useful alternative to linkage mapping for the identification of unreported regions of the soybean genome containing putative QTL.     

Development and primary genetic analysis of a fertility temperature-sensitive polima cytoplasmic male sterility restorer in Brassica napus

Over the past decade, the polima cytoplasmic male sterility ( pol CMS) three-line and two-line systems have been developed for the production of hybrid seed in Brassica napus oilseed rape in China. The discovery of the novel pol CMS restorer line FL-204 is described here. It restores male fertility of hybrid plants in the pol CMS system, but hybrid seed production can only be carried out under autumn sowing in Wuhan in south China under moderate temperatures at flowering. The restorer cannot be used as a male for hybrid seed production in northwestern China (Gansu) under spring sowing conditions, because there it is more or less male sterile due to high temperatures at flowering. Because of this behaviour, it is referred to as a fertility temperature-sensitive restorer (FTSR) in this paper. F₂, BC₁ as well as double haploid populations were constructed to determine the inheritance of fertility restoration of FL-204 in the autumn at Wuhan and under spring sowing conditions at Gansu, respectively. Deviations from Mendelian genetics were observed. It was hypothesized that the change of fertility was the result of the interaction between nuclear genes [restoring gene ( Rf ) and temperature-sensitive genes ( ts )] and the cytoplasm. The Rf gene in FL-204 was incapable of restoring male fertility of pol CMS lines under spring sowing conditions at Gansu where it is inactivated by the recessive ts gene present in FL-204. However, the ts gene(s) could be non-functional under moderate temperature conditions at flowering at Wuhan which allows full expression of male fertility in FL-204. The recessive ts gene(s) can only be expressed in plants containing the pol sterile cytoplasm. A method for the utilization of the FTSR pol CMS restorer FL-204 for the production of hybrid seed in B. napus oilseed rape is proposed.     

Cytogenetic analysis of F1, F2 and BC1 plants from intergeneric sexual hybridization between Sinapis alba and Brassica oleracea by genomic in situ hybridization

By intergeneric sexual hybridization between Sinapis alba and Brassica oleracea , F₁, F₂ and BC₁ progeny plants were produced. S. alba plants (genome SS, 2n = 24) were pollinated with B. oleracea (genome CC, 2n = 18), and the fertile F₁ plants were pollinated with B. oleracea to obtain BC₁ plants. GISH analysis showed that 10 out of 12 F₁ plants had 12 S. alba chromosomes (one full S chromosome set) and nine B. oleracea chromosomes (one C chromosome sets), representing the expected hybrids. However, two F₁ plants had 12 S chromosomes and 18 C chromosomes (two C chromosome sets), indicating unexpected hybrids. A maximum of three trivalents between C and S chromosomes were identified at metaphase I of semi-fertile F₁ pollen mother cells (PMCs), which indicates homology and chromosome pairing between these two genomes. The C genome had obviously been doubled in two F₂ plants from selfed semi-fertile F₁ plants. BC₁ plants consisted of 18 C chromosomes and different numbers of one, five and six additional S chromosomes, respectively. Monosomic alien addition lines developed in the present study can be used for B. oleracea breeding and Sinapis alba gene mapping.     

Development of co-dominant amplified polymorphic sequence markers for resistance of sunflower to downy mildew race 730

The inheritance of the reaction of sunflower to downy mildew was investigated using resistant and susceptible near isogenic lines (NILs) and their F₃ families. Resistance to race 730 was evaluated using the whole seedling inoculation technique. Seventy-three F₃ families were inoculated, among which 54 families were resistant and 19 susceptible, fitting a 3 : 1 segregation ratio. F₃ families were also studied using several PCR markers. Ten markers at the Pl6 locus, specific for the resistant line, also segregated in F₃ families with a 3 : 1 ratio. The same segregation ratio occurred for microsatellite haplotypes that resembled the resistant parent, and were amplified with ORS 166 and ORS 1043. The only common fragment that was observed between resistant and susceptible parental lines was one of the TIR-NBS-LRR resistance gene analogue markers, having a restriction site. Two co-dominant cleaved amplified polymorphic sequence (CAPS) markers were obtained. The mapping data indicate that several dominant markers and two CAPS markers, developed here, completely co-segregate with the Pl6 gene conferring resistance to race 730. CAPS markers will facilitate efficient marker-assisted selection for sunflower resistance to downy mildew race 730.     

Genetic linkage map of cassava (Manihot esculenta Crantz) based on AFLP and SSR markers

To generate a genetic linkage map of cassava ( Manihot esculenta Crantz), 58 F₁ progenies from a cross between Rayong 90 (female) and Rayong 5 (male) were examined in amplification fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses. A total of 469 polymorphic markers consisting of 378 AFLPs generated from 76 primer combinations and 91 SSRs were identified. These markers were analyzed using the joinmap ^® 3.0 package program to construct a genetic linkage map. A total of 33 linkage groups of a common map were constructed from 119 AFLPs and 18 SSRs, spanning 1095 cM with an average of 7.99 cM between markers. The genetic linkage map generated in this study will be useful for genetic studies in cassava particularly for the identification of genetic markers linked to traits of interest, although the complex cassava genome suggests that maybe a long term objective.     

Identification of a SCAR marker linked to a recessive male sterile gene (Tems) and its application in breeding of marigold (Tagetes erecta)

In marigold, an F₂ segregation population of 167 plants was constructed from a cross of a line (M525A) carrying the male sterility trait × an inbred line (f53f). In line M525A, the male sterility trait was controlled by the recessive gene, Tems . The intersimple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) techniques combined with bulked segregant analysis were used to develop markers linked to the trait. From a survey of the 38 ISSR primers and 170 SRAP primer combinations, only one SRAP marker that was closely linked to the target trait was identified and successfully converted into sequence characterized amplified region (SCAR) marker that was located within 2.4 cM from Tems locus. The marker was validated with five other two-type lines and in each case the male fertile plants were reliably identified. This SCAR marker therefore permits the efficient marker-assisted selection of male sterile individuals in breeding programmes of marigold and will greatly facilitate the breeding of F₁ cultivars.     

Molecular mapping of a new red mutant gene (Rs) in upland cotton

In recent years, there has been slow progress in improving cotton yield. It is known that the F₁ generation from the cross of the new red mutant and the normal green leaf plant has high photosynthetic efficiency. Therefore, cloning the new red mutant gene and further introducing it into other crops through transgenic techniques is a promising approach for achieving high photosynthetic efficiency through breeding. To map this new mutant gene, tentatively named R _s , the authors constructed an F₂ generation containing 1214 individual plants from mutant EH083 ( Gossypium hirsutum ) and Hai 7124 ( Gossypium barbadense ). Fifty-five pairs of simple sequence repeats and sequence-related amplified polymorphism (SRAP) primers on chromosome 7 were selected to screen the two parents. Finally, the R _s gene was mapped at the 0.3 cM interval flanked by markers NAU3735 and NAU1048.