Three AFLP markers tightly linked to the genic male sterility <Emphasis Type="Italic">ms</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript> gene in chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) and conversion to a CAPS marker

Genic male sterility (GMS) has long been used as a tool for hybrid seed production in chili pepper (Capsicum annuum L.). We developed DNA markers linked to the GMS ms ₃ gene in a segregating population using bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) techniques. The segregating population was subjected to BSA-AFLP with 512 primer combinations. Three AFLP markers (Eagg/Mccc₂₇₆, Eagc/Mctt₁₇₈, and Ecag/Mtgc₂₀₄) were identified as tightly linked to the ms ₃ locus. Among them, we converted the AFLP marker Ecag/Mtgc₂₀₄ to the cleavage amplified polymorphic sequence (CAPS) marker, named GMS3-CAPS, based on sequencing analysis of internal and flanking regions for the markers between male-fertile and sterile plants. This marker will be useful for pepper breeding using the GMS system.     

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.     

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.     

Development of SCAR and CAPS markers linked to a recessive male sterility gene in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.)

Genetic male sterility (GMS) has been a useful system for the production of hybrid varieties in self-pollinated plants. We obtained a GMS line developed from a spontaneous mutation in lettuce (Lactuca sativa L.). Genetic analysis in our previous study revealed that the sterility was controlled by a recessive gene which was named ms-S. For simple and quick screening of individuals showing male sterility, we attempted molecular mapping of the ms-S locus using an amplified fragment length polymorphism (AFLP) technique. From the examination of 4,096 AFLP primer combinations, 63 AFLP markers were found to be linked to the gene and nine of them were successfully converted into sequence characterized amplified region (SCAR) markers and cleaved amplified polymorphic sequence (CAPS) markers. Linkage analysis indicated that these nine markers were closely linked to the ms-S gene and all were located on the same side of the gene. The minimum genetic distance between the ms-S gene and a marker was 3.1 cM. These results provide additional information for map-based cloning of the ms-S gene and will be of great help for lettuce breeding using GMS to produce F₁ hybrids.     

Identification of AFLP markers linked to one recessive genic male sterility gene in oilseed rape, Brassica napus

The commercial utilization of heterosis in seed yield by means of hybrid varieties is of great importance for increasing oilseed rape production in China. This requires a functional system for the production of hybrid seed. The Brassica napus oilseed rape line 9012AB is a recessive epistatic genic male sterility (GMS) two‐type line, in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms1 and ms2) interacting with one pair of a recessive epistatic inhibitor gene (rf). Homozygosity at the rf locus (rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms1ms1ms2ms2 plants. This study was conducted to identify molecular markers for one of the male fertility/sterility loci in the B. napus male sterility line 9012AB. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from male sterile and male fertile plants of the homozygous two‐type line 9012AB were subjected to amplified fragment length polymorphic (AFLP) analysis. A total of 256 primer combinations were used and seven markers tightly linked to one recessive genic male sterile gene (ms) were identified. Among them, six fragments co‐segregated with the target gene in the tested population, and the other one had a genetic distance of 4.3 cM. The markers identified in this study will greatly enhance the utilization of recessive GMS for the production of hybrid seed in B. napus oilseed rape in China.     

Identification of AFLP and RAPD markers linked to anthracnose resistance in grapes and their conversion to SCAR markers

Resistance to anthracnose or black spot ( Elsinoe ampelina ), a serious fungal pathogen in viticulture and table grape production, was investigated on 25 grape cultivars. Bioassays performed with culture filtrates produced by the pathogen revealed 14 resistant genotypes. In most plants resistance originated from Vitis labrucsa but also genotypes with V. rupestris and V. riparia  ×  V. rupestris background showed resistance. Genetic analysis was conducted in F₁, S₁ and BC₁ plants developed from various cultivars. In total, 326 F₁ plants were evaluated, 172 genotypes proofed to be resistant, whereas 154 were susceptible to anthracnose. A Mendelian segregation ratio of 1 : 1 (χ² = 0.30–0.65) indicating that anthracnose resistance is controlled by a single dominant gene. To facilitate the use of marker-assisted selection in grape-breeding PCR-based markers were developed by random amplified polymorphic DNA and amplified fragment length polymorphism in bulk segregant analysis. Finally, OPB 15₁₂₄₇ was developed as a sequence characterized amplified region marker being diagnostic for the locus of resistance to anthracnose in all resistant genotypes tested. Within the 25 grape cultivars OPB 15₁₂₄₇ is diagnostic in the genetic background of both V. labrucsa and V. rupestris and V. riparia  ×  V. rupestris .     

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.     

Identification of AFLP markers linked to the epistatic suppressor gene of a recessive genic male sterility in rapeseed and conversion to SCAR markers

A recessive epistatic genic male sterility (REGMS) two‐type line, 9012AB, has been used for rapeseed hybrid seed production in China. The male sterility of 9012AB is controlled by two recessive duplicate sterile genes (ms1 and ms2) interacting with one recessive epistatic suppressor gene (esp). Homozygosity at the esp locus (espesp) suppresses the expression of the recessive male sterility trait in homozygous ms1ms1ms2 ms2 plants. In this study, we used a combination of bulked segregant analyses and amplified fragment length polymorphism (AFLP) to identify markers linked to the suppressor gene in a BC₁ population. From the survey of 1024 AFLP primer combinations, eight markers tightly linked to the target gene were identified. The two closest markers flanking both sides of Esp, P9M5₃₇₀ and S16M14₇₈₀, had a genetic distance of 1.4 cM and 2.1 cM, respectively. The AFLP fragment from P4M8₁₉₀, which co‐segregated with the target gene was converted into a sequence characterized amplified region marker. The availability of linked molecular markers will facilitate the utilization of REGMS in hybrid breeding in Brassica napus.     

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.     

Nitrogen Fixation by Hybrids of White Clover (Trifolium repens L.) and Trifolium nigrescens

Development of hybrids between white clover ( Trifolium repens L.) and Trifolium nigrescens provides a novel route for genetically improving the reproductive capacity of white clover, provided the hybrids are agronomically viable, particularly with respect to N₂ fixation. A comparative study of growth and rates of N₂ fixation over 21 days was conducted with the parental species, F ₁ hybrids and backcross hybrids, in flowing solution culture, without a supply of mineral N to the plants. T. nigrescens was unable to fix N₂ in association with the strains of Rhizobium leguminosarum biovar. trifolii selected for inoculation. Rates of N₂ fixation per plant increased in the order T. nigrescens < F ₁ hybrid < T. repens < backcross 1. Specific rates of N₂ fixation (days 0–21) increased in the order T. nigrescens < F ₁ hybrid < backcross 1 <  T. repens . Dry matter production and nodule biomass per plant increased at a higher rate in backcross 1 hybrids than in T. repens. The results suggest that the potential for N₂ fixation by backcross 1 hybrids is at least as great as that by T. repens .     

Identification of AFLP fragments linked to one recessive genic male sterility (RGMS) in rapeseed (Brassica napus L.) and conversion to SCAR markers for marker-aided selection

117AB is a recessive genic male sterility (RGMS) line in which the sterility is controlled by a duplicate recessive gene named ms, located at two separate loci. In the RGMS line, the genotype of the sterile plant (117A) is msmsmsms, and that of the fertile plant (117B) is Msmsmsms. The present study was aimed to identify DNA markers linked to the ms locus by amplified fragment length polymorphism (AFLP). From the survey of 512 AFLP primer combinations, 6 AFLP fragments (y1, k1, k2, k3, k4, k5) were identified as being tightly linked to the Ms locus. The genetic distances between the markers and the Ms locus were all less than 8 cM, among which two fragments, designated as k2 and k3, co-segregated with the target gene in the tested population. Fragment k2 was successfully converted into a sequence characterized amplified region (SCAR) marker. The markers detected could be valuable in marker-assisted breeding of RGMS in Brassica napus.     

Modified complementation test of male sterility mutants in pepper (Capsicum annuum L.): preliminary study to convert male sterility system from GMS to CMS

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS systems.     

A CAPS marker TAO1902 diagnostic for the I-2 gene conferring resistance to Fusarium oxysporum f. sp. lycopersici race 2 in tomato

Fusarium oxysporum f. sp. lycopersici inhabits most tomato-growing regions worldwide, causing tomato production yield losses. A molecular marker linked to resistance would be useful for tomato improvement programmes. Thus, a cleaved amplified polymorphic sequence (CAPS) marker TAO1₉₀₂ was developed to identify tomato genotypes possessing the I-2 gene, which confers resistance to F. o. lycopersici race 2. The Rsa I or Fok I restriction fragments corresponded to the presence or absence of the I-2 allele in a segregating 100 F₂ progeny, tomato cultivars, 16 resistant and 20 susceptible to Fusarium wilt, respectively, lines and F₁ hybrids, representing various tomato gene pools. TAO1₉₀₂ may be helpful for selection of F. o. lycopersici -resistant tomato germplasm.     

Conversion of the genic male sterility (GMS) system of bell pepper (Capsicum annuum L.) to cytoplasmic male sterility (CMS)

Non‐pungent bell pepper (Capsicum annuum L.) lacks the cytoplasmic male sterility (CMS) nuclear restorer allele, Rf, and CMS cannot be employed in its F₁ hybrid seed production. To demonstrate that the genic male sterility (GMS) system in non‐pungent bell pepper can be converted to the CMS male sterility system, the conversion of GMS to CMS for non‐pungent bell pepper line GC3 was conducted by introgression of S‐type cytoplasm and the Rf allele from tropical pungent donors. After morphological traits were evaluated, two lines from BC₁F₁ containing S‐type cytoplasm and four lines from BC₂F₂ containing Rf allele, phenotypically similar to GC3, were obtained and could be employed as CMS male sterile lines and restorer lines for non‐pungent bell pepper. Four molecular markers potentially linked to traits of interest were also evaluated in BC₁F₁ and BC₁F₂ populations. This is the first time that GMS has been successfully converted to CMS in bell pepper, a significant contribution for bell pepper hybrid seed production.     

Molecular mapping of a dominant genic male sterility gene Ms in rapeseed (Brassica napus)

Rs1046AB is a genic male sterile two‐type line in rapeseed that has great potential for hybrid seed production. The sterility of this line is conditioned by the interaction of two genes, i.e. the dominant genic male sterility gene (Ms) and the suppressor gene (Rf). The present study was undertaken to identify DNA markers for the Ms locus in a BC₁ population developed from a cross between a male‐sterile plant in Rs1046AB and the fertile canola‐type cultivar ‘Samourai’. Bulked segregant analysis was performed using the amplified fragment length polymorphism (AFLP) methodology. From the survey of 480 AFLP primer combinations, five AFLP markers (P10M13₃₅₀, P13M8₄₀₀, P6M6₄₁₀, E7M1₂₃₀ and E3M15₁₀₀) tightly linked to the target gene were identified. Two of them, E3M15₁₀₀ and P6M6₄₁₀, located the closest, at either side of Ms at a distance of 3.7 and 5.9 cM, respectively. The Ms locus was subsequently mapped on linkage group LG10 in the map developed in this laboratory, adding two additional markers weakly linked to it. This suite of markers will be valuable in designing a marker‐assisted genic male sterility three‐line breeding programme.     

Mapping of the <Emphasis Type="Italic">ms8</Emphasis> male sterility gene in sweet pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) on the chromosome P4 using PCR-based markers useful for breeding programmes

The nuclear male sterility gene ms8 is expected to facilitate the production of sweet pepper (Capsicum annuum L.) hybrids as it provides means for hybridization without the labor-intensive hand emasculation of female inbred lines. The development of molecular markers linked to ms8 locus will help the breeding practice for the selection of hybrid parental lines. In this study, F₂ population resulting from a cross between the sweet pepper male sterile line 320 and the male fertile variety Elf was used to identify DNA markers linked to the ms8 locus. With the use of RAPD–BSA technique, seven markers linked to the ms8 locus were found. Four of them were converted into SCAR markers. In addition, two COSII/CAPS markers linked to the ms8 locus were identified. Comparative mapping with reference pepper maps indicated that the ms8 locus is located on the lower arm of the pepper chromosome P4. Identified markers are useful for molecular breeding, however, at present markers tightly linked to ms8 locus are still lacking. Identification of molecular markers linked to the ms8 locus and determination of its chromosomal localization are useful for fine mapping and also provide the perspective for ms8 gene cloning.     

甘蓝型油菜隐性上位互作核不育基因(Ms1)精细定位

甘蓝型油菜细胞核雄性不育是杂种优势利用的重要途径.隐性上位互作核不育系9012A已经广泛用于杂交种子生产,其不育性受两对隐性重叠不育基因(ms1和ms2)与一对隐性上位抑制基因(rf互作控制.ms1和ms2同时纯合(ms1ms1ms2ms2)表现不育,但隐性纯合rf(rfrf)对ms1ms1ms2ms2的表达起抑制作用...     

Irrigation Water Management for Efficient Water Use in Mulched Onion

Caused by the necessarily imperfect seed placement accuracy of sowing machines and, additionally, caused by many other biotic and abiotic factors, the resulting plant stands exhibit nonregular spatial distributions of its plants. Based on several simplifying assumptions, a stochastic approach is developed which allows an estimation of the effects of nonregular spatial patterns on yield per area. In this approach, two random variables are attached to each plant: single plant yield E and individual space A . The latter is estimated by the area of Thiessen polygons. Yield per area, calculated by the expectation of the ratio E/A , can be approximately expressed dependent on the means ( Ē and Ā ) and coefficients of variation ( v _E and v _A ) of E and A and their correlation ( r _EA ). In relation to the commonly used estimate Ē/Ā for yield per area, one obtains yield decreases if v _A / v _E  <  r _EA . This inequality, however, will be usually valid in the field of applications. The theoretical approaches and results were applied to three experimental data sets for drilled seeds of winter oilseed rape ( Brassica napus L.) (plant density: 60 plants m⁻², row distance: 10 cm). These data sets are characterized by different accuracies of longitudinal distributions within rows (58 %, 101 %, 150 %): yield depression increases with an increasing variability of plant distances within rows.     

Identification of two SCAR markers co-segregated with the dominant Ms and recessive ms alleles in onion (Allium cepa L.)

Cytoplasmic genetic male-sterility is used to produce hybrid onion (Allium cepa L.) seeds worldwide. In this paper, we present the results of research aimed toward identifying PCR-based markers linked to the Ms locus through amplified fragment length polymorphism (AFLP). After screening 512 AFLP primer combinations, only one AFLP fragment was identified as being flanking linked to the dominant Ms allele. Subsequently, the AFLP marker was converted into a sequence-characterized amplified region (SCAR) marker, designated as DNF-566, co-segregated with the dominant Ms allele in first backcross (BC₁) segregated populations. Furthermore, we designed another molecular marker (RNS-357) co-segregated with the ms allele to identify different genotypes (i.e., MsMs, Msms, or msms). Both markers could be used for evaluating onion lines with different genetic backgrounds (including male-sterile lines, maintainer lines, male-fertile lines, and commercial based F₁ hybrid cultivars). The results of this study indicate that maintainer plants could be directly selected by using these 2 SCAR markers in the onion breeding process, and this may contribute significantly toward breeding onion F₁ hybrid cultivars.     

A new male sterile mutant LZ in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Genetic male sterility (GMS) genes in wheat (Triticum aestivum L.) can be used for commercial hybrid seed production. A new wheat GMS mutant, LZ, was successfully used in the 4E-ms system for producing hybrid wheat, a new approach of producing hybrid seed based on GMS. Our objective was to analyse the genetic mechanism of male sterility and locate the GMS gene in mutant LZ to a chromosome. We firstly crossed male sterile line 257A (2n = 42) derived from mutant LZ to Chinese Spring and several other cultivars for determining the self-fertility of the F₁ hybrids and the segregation ratios of male-sterile and fertile plants in the F₂ and BC₁ generations. Secondly, we conducted nullisomic analysis by crossing male sterile plants of line 257A to 21 self-fertile nullisomic lines as male to test the F₁ fertilities and to locate the GMS gene in mutant LZ to a chromosome. Thirdly, we conducted an allelism test with Cornerstone, which has ms1c located on chromosome 4BS. All F₁s were male fertile and the segregation ratio of male-sterile: fertile plants in all BC₁ and F₂ populations fitted 1:1 and 1:3 ratios, respectively. The male sterility was stably inherited, and was not affected by environmental factors in two different locations or by the cytoplasm of wheat cultivars in four reciprocal cross combinations. The results of nullisomic analysis indicated the gene was on chromosome 4B. The allelism test showed that the mutant LZ was allelic to ms1c. We concluded that the mutant LZ has common wheat cytoplasm and carries a stably inherited monogenic recessive gene named ms1g.