Inheritance and mapping of a yellow leaf mutant of carrot (Daucus carota)

A yellow leaf mutant at a locus named YEL was selected in a population of the cultivated carrot. Genetic analysis of segregating F₂ progenies and corresponding F₃ families, indicates that the phenotype expressed is controlled by a single recessive nuclear gene. The mutant is stably inherited and is associated with a reduced leaf‐biomass of approximately 30% compared with the wild‐type. Amplified fragment length polymorphism markers were developed and used in bulked segregant analysis. Seventeen marker candidates were detected by using 45 primer pairs. Ten of these could be linked with the YEL locus and mapped in a linkage group with a total length of 33.2 cM. Application of the yellow leaf mutant in carrot research is discussed.     

Development of a molecular CAPS marker for the self-incompatibility locus in Brassica napus and identification of different S alleles

Using primers annealing to S locus sequences the cleaved amplified polymorphic sequences (CAPS) method was applied to develop a marker and to characterize different alleles at the self‐incompatibility locus in Brassica napus. A segregating F₂ population from a cross of a self‐incompatible (SI) and a self‐compatible parent, as well as seven SI lines representing four different S alleles were used. Several primers specific to the S locus in B. oleracea and B. campestris, chosen from the literature, allow polymerase chain reaction (PCR) amplification of genomic DNA. However, only one primer pair amplified a single specific and reproducible PCR fragment of the expected length in B. napus. Digestion with restriction endonucleases revealed polymorphisms for two CAPS markers absolutely linked to the S locus. Using the codominant marker efMboI it was possible to discriminate all three F₂ genotypes. With this marker and an additional marker using another primer pair it was possible to distinguish between three of the four different S alleles and five of the seven SI lines, respectively.     

Inheritance and genetic linkage analysis of a firm-ripening tomato mutant

The tomato cv. ‘Santa Clara’ is widely cultivated among tomato producers in most of the South‐east of Brazil. Recently, some plants of this cultivar were identified with morphological alterations in both vegetative and reproductive organs. These plants showed firm (firme) ripe fruits, slow and delayed ripening. They also had yellow leaves associated with precocious senescence and flowers with pale stigmas. The objective of this work was to determine the genetic model of inheritance for this mutation and to evaluate its effects on shelf life and loss of firmness in mature fruits, as well as analyse the occurrence of genetic relationships between this putative mutant and other pleiotropic mutants. Mutated plants were crossed with the non‐mutant cv. ‘Santa Clara’ and some previously described pleiotropic mutants. Seeds of F₁ and F₂ generations and backcrosses were obtained for the segregation analysis. Morphological characteristics modified by this mutation are governed by a recessive gene with pleiotropic effects. In addition, the test of allelism showed a lack of genetic complementation between the ‘firme’ mutant and lutescent‐2 mapped on chromosome 10. Fruits of the ‘firme’ mutant had a slower rate of softening compared with the cv. ‘Santa Clara’ and its hybrids. The fruit shelf life of the mutant ‘firme’ was significantly superior to the other genotypes. No maternal effect was detected in either qualitative or quantitative characteristics. Based on the data, the mutation ‘firme’ in the cv. ‘Santa Clara’ is located in the region containing the l‐2 locus, which promoted alterations in ripening and post‐harvest physiology of fruits. The mutation ‘firme’ may represent a new allele of the gene lutescent‐2 or a gene linked to physiological events of fruit ripening.     

Distribution of S haplotypes in commercial cultivars of Brassica rapa

Self‐incompatibility in Brassicaceae plants is sporophytically controlled by a single multi‐allelic locus (S locus), which contains at least three highly polymorphic genes expressed in the stigma (SLG and SRK) and in the pollen (SCR/SP11). Using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis with SXG‐specific primer pairs, the S haplotypes of F₁ hybrid and open‐pollinated commercial cultivars of Brassica rapa were identified. The number of S haplotypes detected in the F₁ hybrid cultivars of Chinese cabbage, komatsuna, pak‐choi, turnip, open‐pollinated cultivars of Chinese cabbage and turnip were 9, 9, 4, 11, 13 and 12, respectively. Nine of them had different PCR‐RFLP profiles from those of the S‐tester lines that determined the SLG sequences. Four SLG sequences in the F₁ hybrid cultivars were determined and named S⁵³, S⁵⁴, S⁵⁵ and S⁵⁶, respectively. It is demonstrated that the PCR‐RFLP analysis using specific primer pairs of SLG and SRK is useful for identification of the S haplotypes, in both, S homozygous and S heterozygous plants of B. rapa. The possibility of using this method routinely in breeding programmes, and in the evaluation of F₁ hybrid seed purity, is discussed.     

AFLP-based STS markers closely linked to a fertility restoration locus (Rfm1) for cytoplasmic male sterility in barley

The Rfm1 gene restores the fertility of the msm1 and msm² male‐sterile cytoplasms in barley. Rfm1 is located on the short arm of chromosome 6H. To develop molecular markers tightly linked to Rfm1 for use in sophisticated marker‐assisted selection and map‐based cloning, an amplified fragment‐length polymorphism (AFLP) marker system with isogenic lines and a segregating BC₁F₁ population was used. Nine hundred primer combinations were screened and a linkage map was constructed around the Rfm1 locus by using 25 recombinant plants selected from 214 BC₁F₁ plants. Three AFLP markers were identified, e34m2, e46m19 and e48m17, linked to the locus. The most closely linked markers were e34m2, at 1.0 cM distally and e46m19, at 1.1 cM proximally. The two AFLP markers were converted to dominant STS markers. These markers should accelerate programmes for breeding restorer lines and will be useful for map‐based cloning.     

Allelic relationship between spontaneous and induced mutant genes for stem fasciation in chickpea

Stem fasciation is a morphological abnormality observed in plants where the stem is widened and leaves and flowers or pods are clustered at the apex. Several spontaneous mutants and one induced mutant for stem fasciation are found in chickpea (Cicer arietinum L.). This study was aimed at determining allelic relationship between spontaneous and induced mutant genes controlling stem fasciation and effects of stem fasciation on grain yield. Two spontaneous (ICC 2042 and ICC 5645) and one induced (JGM 2) stem fasciation mutants were crossed in all combinations, excluding reciprocals. The F₁ and F₂ plants from a cross between the two spontaneous mutants had fasciated stem. This indicated the presence of a common gene (designated fas1) for stem fasciation in the two spontaneous mutants. The F₁s of the crosses of the induced mutant JGM 2 with both spontaneous mutants had normal plants and segregated in a ratio of 9 normal : 7 fasciated plants in F₂. Thus, the gene for stem fasciation in the induced mutant JGM 2 (designated fas2) is not allelic to the common gene for stem fasciation in spontaneous mutants. The two genes in dominant condition produced normal non‐fasciated stem. The fasciated and the non‐fasciated F₂ plants did not differ significantly for number of pods per plant, number of seeds per plant, grain yield per plant and seed size, suggesting that it is possible to exploit the fasciated trait in chickpea breeding without compromising on yield.     

Inheritance of resistance to the peach root‐knot nematode (Meloidogyne floridensis) in interspecific crosses between peach (Prunus persica) and its wild relative (Prunus kansuensis)

The peach root‐knot nematode, Meloidogyne floridensis (MF), infects majority of available nematode‐resistant peach rootstocks which are mostly derived from peach (Prunus persica) and Chinese wild peach (P. davidiana). Interspecific hybridization of peach with its wild relative, Kansu peach (P. kansuensis), offers potential for broadening the resistance spectrum in standard peach rootstocks. We investigated the inheritance of resistance to MF in segregating populations of peach (‘Okinawa’ or ‘Flordaguard’) × P. kansuensis. A total of 379 individuals from 13 F₂ and BC₁F₁ families were challenged with a pathogenic MF isolate “MFGnv14” and were classified as resistant (R) or susceptible (S) based on root galling intensity. Segregation analyses in F₂ progeny revealed the involvement of a major locus with a dominant or recessive allele determining resistance in progeny segregating 3R:1S and 1R:3S, respectively. Testcrosses with a homozygous‐susceptible peach genotype (‘Flordaguard’ or ‘UFSharp’) confirmed P. kansuensis as a source of new resistance and the heterozygous allelic status of P. kansuensis at the locus conferring resistance to MF. We propose a single‐locus dominant/recessive model for the inheritance of resistance.     

Inheritance and allelic relationships of fertility restoration genes for seven new sources of male-sterile cytoplasm in sunflower

The inheritance of fertility restoration of six mitomycin C and streptomycin‐induced cytoplasmic male‐sterile (cms) mutants and one cms line derived from Native American cultivar PI 432513 in sunflower was evaluated. These seven new cms sources were also compared with the commercially used cms PET1 (Helianthus petiolaris Nutt.) cytoplasm, using USDA inbred lines with restoration genes (Rf₁) specific for cms PET1 and new restoration lines identified for cms PI 432513. Restoration genes for cms PI 432513 were found in ‘Armavir’, VNIIMK, P21 and male‐fertile (MF) plants of PI 432513. F₂ and F₃ segregation ratios of crosses between cms PI 432513 and these restoration sources indicated a single dominant gene controlled fertility restoration. Progenies of cms PI 432513 testcrossed with F₁’s of half‐diallel crosses among the respective four homozygous restoration lines and RHA 274 suggested that the restoration genes of RHA 274, VNIIMK, P21 and PI 432513 were at the same locus. Restoration genes from VNIIMK, P21 and PI 432513 satisfactorily restored pollen stainability in the heterozygous condition. A very weak expression of the Rf gene in ‘Armavir’ was observed in the heterozygous condition. Fertility restoration capability of these genes for the six mutant cms HA 89 and cms HA 89 (in PET1 cytoplasm) was observed. The mutant cms HA 89 lines were also restored completely by RHA 266, RHA 274, RHA 280 and RHA 296, and F₂’s segregation ratios indicated single dominant gene control, implying a common cytoplasmic male sterility in all lines. F₁’s of half‐diallel crosses among RHA 266, RHA 273, RHA 274, RHA 280 and RHA 296 were testcrossed onto the cms lines, and their all MF progenies among lines, except RHA 280, confirmed that fertility restoration was controlled by a single Rf₁ gene locus. The restoration gene in confection line RHA 280, namely Rf₃, was at a different locus than Rf₁ and was equally capable of restoring all the cms lines. Cms HA 89 mutants and cms PI 432513 are in H. annuus cytoplasm, and are agronomically equal in hybrid performance to the cms PET1 used in commercial sunflower hybrids. These new cms lines will provide immediate alternative cms sources for reducing the genetic vulnerability resulting from the exclusive use of the single cms source PET1 in sunflower hybrid production.     

SSR marker tightly linked to the Ti locus in Soybean [Glycine max (L.) Merr.]

Soybean is a major source of protein meal in the world. Soybean kunitz trypsin inhibitor (SKTI) protein is a responsible for the inferior nutritional quality of unheated or incompletely heated soybean meal. The primary objective of this research was to identify DNA markers linked to the Ti locus controlling presence and absence of kunitz trypsin inhibitor protein. Two mapping populations were developed. Population 1 was derived from a cross between cultivar Jinpumkong2 (TiTi) and C242 (titi). Population 2 was made from a mating between cultivar Clark (TiTi) and C242. The F₁ plants were grown in the greenhouse to produce F₂ seeds. Each F₂ seed from F₁ plants was analyzed electrophoretically to determine the presence of the SKTI protein band. One-thousand RAPD primers, 342 AFLP primer sets, and 35 SSR primers were used to map Ti locus in population 1 and 2. The presence of SKTI protein was dominant to the lack of a SKTI protein and kunitz trypsin inhibit protein band was controlled by a single locus. Twelve DNA markers (4 RAPD, 4 AFLP, and 3 SSR) and Ti locus were found to be genetically linked in population 1 consisted with 94 F₂ individual plants. Three SSR markers (Satt409, Satt228, and Satt429) were linked with Ti locus within 10 cM. Satt228 marker was tightly linked with Ti locus. Satt228 marker was tightly linked within 0–3.7 cM of the Ti locus and may be useful in a marker assisted selection program.     

Development of a kompetitive allele‐specific PCR marker for selection of the mutated Wx‐D1d allele in wheat breeding

Waxy (Wx) protein is a key enzyme for synthesis of amylose in endosperm. Amylose content in wheat grain influences the quality of end‐use products. Seven alleles have been described at the Wx‐D1 locus, but only two of them (Wx‐D1b, Wx‐D1e) were genotyped with codominant markers. The waxy wheat line K107Wx1 developed by treating ‘Kanto 107’ seeds with ethyl methanesulphonate carries the Wx‐D1d allele. However, no molecular basis supports this nomenclature. In the present study, DNA sequence analysis confirmed that a single nucleotide polymorphism in the sixth exon of Wx‐D1 changed tryptophan at position 301 into a termination codon. Based on this sequence variation, a PCR‐based KASP marker was developed to detect this point mutation using 68 BC₈F₁ plants and 297 BC₈F₂ lines derived from the cross ‘Ningmai 14’*9/K107Wx1. Combined with codominant markers for the Wx‐A1 and Wx‐B1 alleles, waxy and non‐waxy near‐isogenic lines were distinguished. The KASP marker was efficient in identifying the mutant allele and can be used to transfer waxiness to elite lines.     

Mapping of a gene for leaf scald resistance in barley line 'B87/14' and validation of microsatellite and RFLP markers for marker-assisted selection

Seedlings of the barley line ‘B87/14’ were resistant to 22 out of 23 Australian isolates of Rhynchosporium secalis, the causal agent of leaf scald.‘B87/14’‐based populations were developed to determine the location of the resistance locus. Scald resistance segregated as a single dominant trait in BC₁F₂ and BC₁F₃ populations. Bulked segregant analysis identified amplified fragment length polymorphisms (AFLPs) with close linkage to the resistance locus. Fully mapped populations not segregating for scald resistance located these AFLP markers on chromosome 3H, possibly within the complex Rrs1 scald locus. Microsatellite and restriction fragment length polymorphism markers adjacent to the AFLP markers were identified and validated for their linkage to scald resistance in a second segregating population, with the closest marker 2.2 cM from the resistance locus. These markers can be used for selection of the Rrs.B87 scald‐resistance locus, and other genes at the chromosome 3H Rrs1 locus.     

A PCR-based DNA marker for detection of mutant and normal alleles of the Wx-D1 gene of wheat

To assist waxy wheat breeding a DNA marker was developed to discriminate mutant and normal alleles at the Wx‐D1 locus. This polymerase chain reaction‐based marker distinguishes the mutant from the normal allele by targeting the previously reported deletion basis of the mutant. The marker codominantly identifies the normal allele of the Wx‐D1 gene from the mutant allele originated from the Chinese landrace ‘Baihoumai’. However, attempts with a number of primer combinations targeting this deletion failed to amplify the corresponding fragment from an unrelated wheat line (NP150) that has a mutant null allele at the same locus. This indicates that NP150 has a different mutant allele from that of ‘Baihoumai’. This marker is a useful tool to identify wheat cultivars with mutant and normal alleles of the Wx‐D1 gene, and is used in marker‐assisted selection of the Wx‐D1 gene in our waxy wheat breeding programme.     

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

In this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F₂‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F₂ population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.     

38–48 kDa subunits of buckwheat 13S globulins are controlled by a single locus

The inheritance of buckwheat (Fagopyrum esculentum Moench) seed storage proteins was investigated by control crosses between accessions containing different SDS‐PAGE protein patterns in the range of 38–48 kDa. Analysis of segregation of individual F₁ and F₂ seeds obtained by 17 crosses showed segregation of eight electrophoretic bands, from which 12‐banding variants were inferred. Two variants contained one band, nine contained two bands and one variant contained three bands and behaved as a single co‐dominant unit. The segregation of banding variants fits the expected ratios and thus supports the hypothesis of single Mendelian gene inheritance and that alleles are co‐dominant. The results therefore suggest that the genes controlling globulin subunits are tightly linked and inherited as a single locus. The locus was designated Glob‐1 and the twelve segregated protein variants as alleles a‐l. This is the first buckwheat globulin locus identified with multiple alleles, phenotypically expressed as groups of protein bands and thus applicable as a functional marker in buckwheat genomic studies.     

A gene producing one to nine flowers per flowering node in chickpea

Chickpea (Cicer arietinum L.) has a racemose type of inflorescence and at each axis of the raceme usually one or two and rarely three flowers are borne. Plants producing 3 to 9 flowers, arranged in acymose inflorescence, at many axis of the raceme, were identified in F₂ of an interspecific cross ICC 5783 (C. arietinum) × ICCW 9 (C. reticulatum)in which both the parents involved were single-flowered. A spontaneous mutation in one of the two parents or in the F₁was suspected. However, the possibility for establishment of a rare recombination of two interacting recessive genes could not be ruled out. The number of pods set varied from 0 to 5 in each cyme. Inheritance studies indicated that a single recessive gene, designated cym, is responsible for cymose inflorescence. The allelic relationship of cym with sfl, a gene for double-flowered trait, was studied from a cross involving multi flowered plants and the double-flowered line ICC 4929. Thecym gene was not allelic to sfl, suggesting that two loci control the number of flowers per peduncle in chickpea. The cym locus segregated independently of the locus sfl, ifc (inhibitor of flower color) and blv (bronze leave). This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular mapping of black rot resistance locus Xca1bo on chromosome 3 in Indian cauliflower (Brassica oleracea var. botrytis L.)

Black rot is the most devastating disease of cauliflower worldwide causing severe damage to crop. The identification of markers linked to loci that control resistance can facilitate selection of plants for breeding programmes. In the present investigation, F₂ population derived from a cross between ‘Pusa Himjyoti’, a susceptible genotype, and ‘BR‐161’, a resistant genotype, was phenotyped by artificial inoculation using Xcc race 1. Segregation analysis of F₂ progeny indicated that a single dominant locus governed resistance to Xcc race 1 in ‘BR‐161’. Bulk segregant analysis in resistant and susceptible bulks of F₂ progeny revealed seven differentiating polymorphic markers (three RAPD, two ISSR and two SSR) of 102 markers screened. Subsequently, these markers were used to genotype the entire F₂ population, and a genetic linkage map covering 74.7 cM distance was developed. The major locus Xca1bo was mapped in 1.6‐cM interval flanked by the markers RAPD 04₈₃₃ and ISSR 11₆₃₅. The Xca1bo locus was located on chromosome 3. The linked markers will be useful for marker‐assisted resistance breeding in cauliflower.     

Broad-few-leaflets and outwardly curved wings: two new mutants of chickpea

This study was aimed at the induction of morphological mutations for increasing genetic variability and making available additional genetic markers for linkage studies in chickpea (Cicer arietinum L.). A wilt‐resistant, well‐adapted chickpea cultivar of central India,‘JG 315’(Jawahar gram 315), was used for the induction of mutations. Seeds presoaked in distilled water for 2 h were treated with ethyl methane sulphonate (EMS) using six different concentrations (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6%) and two different durations (6 and 8 h). Several morphological mutants were identified in M₂. One of the mutants, isolated from a treatment of 0.3% EMS for 8 h, had five to nine large leaflets per leaf in comparison with 11‐17 normal‐sized leaflets per leaf observed in the parental cultivar ‘JG 315′. The mutant was designated broad‐few‐leaflets. Many leaves of this mutant showed a cluster of three to five overlapping leaflets at the terminal end. The other mutant, designated outwardly curved wings, was isolated from the 0.5% EMS treatment for 6 h. In this mutant, the wings were curved outwards, exposing the keel petal, while the wings in typical chickpea flowers are incurved and enclose the keel. The lines developed from the broad‐few‐leaflets and outwardly curved wings mutants were named JGM 4 (Jawahar gram mutant 4) and JGM 5, respectively. Inheritance studies indicated that each of these mutant traits is governed by a single recessive gene. The gene for broad‐few‐leaflets was designated bfl and the gene for outwardly curved wings was designated ocw. The locus bfl was found to be linked with the locus lg (light green foliage) with a map distance of 18.7 ± 6.3 cM.     

The use of AFLP markers for the identification of carrot breeding lines and F1 hybrids

Four inbred lines of carrot (cytoplasmic male‐steriles and corresponding maintainers) and eight of their F₁ hybrids were studied with the amplified fragment length polymorphism (AFLP) technique to examine their genetic relationship and produce markers useful for testing hybrid seed purity. Eighty‐six polymorphic amplicons were identified in bulked DNA samples using eight primer pair combinations. Genetic distance was estimated on the basis of the presence or absence of polymorphic bands. The dendrogram plotted on the basis of the AFLP data closely represented the pedigree relationships of the lines and their hybrids. From one to six amplicons specific for a breeding line were identified. Most of them were also present in the DNA bulks of respective F₁ hybrids. However, screening performed on individual plants of two parental lines and the corresponding hybrid indicated insufficient uniformity of parental lines, limiting the applicability of AFLP markers for testing hybrid seed purity.     

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 effects of the soft starch (h) and background loci on volume of starch granules in five inbreds of maize

Larger particle volume is beneficial for many aspects of maize starch processing, and may improve the performance of some starch attributes. This study focused on the soft starch (h) locus to identify its potentially influential role in starch particle volume distribution. The objectives were to study the genetic expression of starch particle volume of the h locus in different genetic backgrounds and the gene action conditioning starch particle volume of other loci in both normal‐starch and h‐starch backgrounds. Forty‐five populations (five intra‐inbred F_1s, 10 hybrid F_1s 10 F_2s, 10 BC₁F_1s to h/h parent, and 10 BC₁ to h:h conversion of normal parent) were planted in 1993 at two locations and in 1995 at one location. Selfed heterozygotes (±/h) in all generations provided intra‐ear comparisons of normal and h starch, and F₃ and BC₁F₂ generations provided inter‐ear comparisons. Significant differences were found between normal and h:h genotypes in all intra‐ear and inter‐ear comparisons. In all cases, general combining ability effects were highly significant, suggesting the presence of additive gene effects. Generation mean analysis of normal and h:h starch materials yielded similar results, indicating the predominance of additive and some dominance effects for other loci on starch particle volume. These results indicate the usefulness of the soft starch gene and additional genetic variation among inbreds in the improvement of starch particle volume for increased starch recovery in wet milling.