Segregation Distortion of Marker Nuclear Genes in Alloplasmic and Isoplasmic Lines of Barley

Inheritance of barley nuclear genes responsible for various morphological marker traits was studied in hybrid populations F₂ and F_a. Nine marker genes showed deviation from Mendelian monogenic inheritance depending on the cross direction and maternal cytoplasm. Segregation biases to both recessive mutant and dominant normal phenotypes were observed. Mechanisms of the segregation bias related to cytoplasm substitution in iso- and alloplasmic lines are discussed.     

Induced mutations in foxtail millet (Setaria italica Beauv.)

Summary Chlorophyll mutations induced by gamma rays, EMS and DES were studied in foxtail millet (Setaria italica), using two cultures, MU-1 (bristled) and MU-2 (non-bristled). No major differences in the mutagenic response of the two cultures were observed. The treatments included four doses of gamma rays (10Kr, 20Kr, 30Kr, 40Kr) and four durations (6 hrs, 12 hrs, 18 hrs, 24 hrs) each of EMS (0.1%) and DES (0.1%). The combined treatments of gamma rays + EMS and gamma rays + DES were also given.Frequencies of chlorophyll mutations were recorded by three different methods, viz. (a) mutations per cent M₁ plants, (b) mutations per cent M₁ spikes and (c) mutants per cent M₂ plants. No significant differences in the results obtained by these three methods were observed. The frequencies and spectrum of mutations are discussed.Chlorina type were most frequent andviridoalbina least frequent.Striata and virescens were also quite common.Albinos, reported frequently in other crops, were found to be less frequent in foxtail millet during the present study. Number of sectors per spike were also determined from segregation ratios and only one sector per spike was found at all doses. Efficiency and effectiveness of mutagens were also determined and discussed. The results are also discussed with respect to mutagen specificity.     

Investigation of partial sterility in advanced generation,sodium azide-induced lines of spring barley

Summary The partial sterility found in several advanced generation, sodium azide-induced lines of spring barley (Hordeum vulgare L.) was investigated. Plants of mutant lines were reciprocally crossed with plants of their untreated mother lines. Spike sterility was measured in the selfed offspring of the plants crossed and in F₁ and F₂ progeny. Pollen sterility and endosperm development were analyzed in the selfed offspring of the plants crossed. Results indicated that the sterility was inherited in the mutant lines and was not caused by translocations, inversions, endosperm lethals, embryo-endosperm lethals, or major gene mutations. Furthermore, the sterility was not cytoplasmically inherited, and was essentially eliminated in the F₁ and F₂ of crosses between partially sterile lines and their fertile parents. Results suggest that the sterility may be caused by an environmental interaction with deleterious, homozygous recessive, minor gene mutations that were in the heterozygous condition when the mutant lines were originally selected.Scientific paper No. 7441, College of Agriculture Research Center, Washington State University, Pullman, Wash., USA, Project No. 1006     

Induced mutations and barley improvement

Summary Seven barley varieties, originating from three X-ray induced mutations, have been officially approved in Sweden since 1958. Some have gained a wide area of cultivation. The list is as follows: Pallas, isolated 1947, approved 1958, mutant ert-k ³² of Bonus barley. — Mari, isolated 1950, approved 1960, mutant mat-a ⁸ of Bonus. — Hellas, approved 1967, mutant cross of Pallas × Herta. — Kristina, approved 1969, mutant cross of Domen × Mari. — Visir, approved 1970, Pallas × Long Glumes back-crossed to Pallas. — Mona, approved 1970, mutant cross of Mari × Monte Cristo back-crossed to Mari. — Gunilla, approved 1970, hybrid cross of the mutant 44/3 arisen from Gull barley in 1939; evolved in a series of steps, using one six-row and four two-row varieties, with mutant characters prevailing and Gull genes reiterated. — After the first approval of Pallas in 1958, 12 more years have led to the approval of a second mutant case and five mutant crosses. In addition, chromosome translocations, induced by irradiation in Bonus, have been instrumental in the production of hybrid barley in USA and are used in the barley improvement program of Sweden, as well as for theoretical analysis in numerous countries.     

Chimeras for mutations induced in dormant tomato seeds

Genetic chimeras of the VFNT cherry tomato line (Lycopersicon esculentum) were produced by mutagenizing seeds with ethyl methane sulfonate (EMS). The chimeras thereby produced were evaluated by progeny-testing the fruits of the genetically mosaic tissue. A total of 2011 M₁ plants was grown from treated seeds and evaluated by screening their 95175 (M₂) progeny for mutations affecting seedling phenotype. Three vigorous and fertile M₁ plants bearing mutant progeny with definitive phenotypes were selected for systematic harvesting and analysis. The specific location of each fruit was noted at harvest time, enabling the mutated sporogenous tissue of the mosaic M₁ plants to be traced. Sectoring appeared in both branch and floral tissues. In several cases, mutant progenies were restricted to individual branches or parts thereof. True-breeding recessive mutants whose monogenic mode of inheritance was later established occasionally segregated within M₁ fruit progenies at frequencies that indicate a non-homogeneous floral meristem origin. The data emphasize the necessity of making a well-distributed harvest of mosaic plants in order to detect as many variants as possible, as mosaic sectors may or may not recur late in ontogeny and may not contribute to sporogenous tissue early in development.     

Linkage mapping of two mutations that reduce phytic acid content of barley grain

 This study describes the inheritance and linkage map positions of two low phytic acid barley (Hordeum vulgare) mutations, lpa1-1 and lpa2-1, that dramatically reduce grain phytic acid content and increase inorganic seed phosphorus (P). Wide-cross, F₂ mapping populations were constructed by mating six-rowed varieties, ‘Steptoe’ and/or ‘Morex’, with two-rowed ‘Harrington’lpa donor lines homozygous for either lpa1-1 or lpa2-1. The barley lpa1-1 mutation showed normal inheritance patterns, whereas a deficiency of homozygous lpa2-1/lpa2-1 F₂ plants was observed. We identified a codominant, STS-PCR marker (aMSU21) that cosegregated with lpa1-1 in a population of 41 F₂ plants. The aMSU21 marker was then mapped to a locus on barley chromosome 2H, using a North American Barley Genome Mapping Project (NABGMP) doubled haploid population (‘Harrington’בMorex’). We determined that lpa2-1 is located within a recombination interval of approximately 30 cM between two AFLP markers that were subsequently mapped to barley chromosome 7H by integration with the same NABGMP population. Recent comparative mapping studies indicate conserved genetic map orders of several homologous molecular marker loci in maize and the Triticeae species that also show corresponding linkage to the biochemically similar lpa2 mutations of maize and barley. This observation suggests that barley and maize lpa2 mutations may affect orthologous genes. No such evidence for correspondence of the phenotypically similar lpa1 mutations of barley and maize has been revealed. Received: 22 September 1997 / Accepted: 2 December 1997     

Identification and Fine Mapping of a White Husk Gene in Barley (Hordeum vulgare L.)

Barley is the only crop in the Poaceae family with adhering husks at maturity. The color of husk at barely development stage could influence the agronomic traits and malting qualities of grains. A barley mutant with a white husk was discovered from the malting barley cultivar Supi 3 and designated wh (white husk). Morphological changes and the genetics of white husk barley were investigated. Husks of the mutant were white at the heading and flowering stages but yellowed at maturity. The diastatic power and α-amino nitrogen contents also significantly increased in wh mutant. Transmission electron microscopy examination showed abnormal chloroplast development in the mutant. Genetic analysis of F₂ and BC₁F₁ populations developed from a cross of wh and Yangnongpi 5 (green husk) showed that the white husk was controlled by a single recessive gene (wh). The wh gene was initially mapped between 49.64 and 51.77 cM on chromosome 3H, which is syntenic with rice chromosome 1 where a white husk gene wlp1 has been isolated. The barley orthologous gene of wlp1 was sequenced from both parents and a 688 bp deletion identified in the wh mutant. We further fine-mapped the wh gene between SSR markers Bmac0067 and Bmag0508a with distances of 0.36 cM and 0.27 cM in an F₂ population with 1115 individuals of white husk. However, the wlp1 orthologous gene was mapped outside the interval. New candidate genes were identified based on the barley genome sequence.     

Analysis of dominant spring mutations in rye

The dominant mutant genes responsible for the spring habit were studied in seven rye plants according to the developed scheme of two-step crosses and analysis of the F₂ progeny. The genotypes with a particular genetic formula (heterozygote) were obtained by crossing the studied plants with the winter rye Korotkostebel’naya 69 carrying the recessive genes that control the winter habit of plants. Heterozygotes yielded by different combinations were crossed with each other. The F₁ hybrids were either self-pollinated to obtain F₂ progeny or crossed with the winter rye. Analysis of the progeny suggests that all seven plants carry the same gene.     

Putative homozygous mutations in regenerated plants of rice

Summary Both normal and putative homozygous mutant (dwarf mutant) rice plants were regenerated from diploid seed callus, cultured in the presence of 1% NaCl. This trait was transmitted at least through the eighth genration (D₈) of regenerated plants (D₁) by self-pollination, as a homozygous mutation. However, the trait disappeared in the F₁, F₂, F₃ and F₄ obtained by reciprocal crosses of mutant plants with either control plants or with progeny of normal regenerated plants. Chimeric reversion of the homozygous mutant trait was observed and the revertant phenotype was transmitted stably to at least three successive generations. Similar dwarf types of homzygous mutation were observed independently in the two varieties, Norin 8 and Nipponbare, in an experimental series of ca. 3000 D₁ plants. The frequency of mutations among regenerated plants was calculated to be 1.8×10^-2. The mechanism responsible for these phenomena may be heritable gene inactivation induced by in vitro culture.     

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 by in length. The nucleotide coding sequence could be aligned with the 3′ end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3′ untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1. F₁ progeny derived from the cross between the cultivar Golden Promise and the homozygous nir1 mutant STA3999 were heterozygous for these bands as anticipated. Co-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatment/loss of detectable nitrite reductase cross-reacting material at M_r 63000) was scored in an F₂ population of 312 plants derived from the cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showed strict co-segregation (in approximately one quarter (84) of the 312 F₂ plants examined). Only those F₂ individuals heterozygous for the RFLP pattern gave rise to F₃ progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase apoprotein gene Nii are very tightly linked.     

Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants

To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T₈ plant, having uidA (or gus) driven by the barley endosperm-specific B₁-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T₄ plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F₁ progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F₂ seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F₂ plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F₃ and F₄ generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.     

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 by in length. The nucleotide coding sequence could be aligned with the 3 end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3 untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1. F₁ progeny derived from the cross between the cultivar Golden Promise and the homozygous nir1 mutant STA3999 were heterozygous for these bands as anticipated. Co-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatment/loss of detectable nitrite reductase cross-reacting material at M_r 63000) was scored in an F₂ population of 312 plants derived from the cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showed strict co-segregation (in approximately one quarter (84) of the 312 F₂ plants examined). Only those F₂ individuals heterozygous for the RFLP pattern gave rise to F₃ progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase apoprotein gene Nii are very tightly linked.     

Influence of the Russian wheat aphid <Emphasis Type="Italic">Diuraphis noxia</Emphasis> (Kurdjumov) (Homoptera,Aphididae) on productive qualities of spring bread wheat and barley grown from the seeds from aphid-infested spikes

The aftereffects of the Russian wheat aphid (RWA) Diuraphis noxia on sowing and productive qualities of barley and spring bread wheat grain were assessed. Seeds of 4 cultivars of barley (Volgar, Povolzhsky 65, Kazak, and Povolzhsky 16) and 4 cultivars of spring wheat (Kinelskaya 59, Kinelskaya Otrada, Kinelskaya Niva, and Kinelskaya 2010) from spikes infested and uninfested with RWA in 2007 and in 2014 were sown in the subsequent years, using 0.5 m² experimental plots in four replications, at a seeding rate of 300 grains/m². The least significant difference (LSD_0.5) was used to compare the mean ± standard deviation (SD) values. The field germination rate of seeds from spring wheat spikes damaged by RWA was reduced by 15%. Of the components of grain yield, barley and spring wheat grown from seeds from the infested spikes showed a 23-31% smaller number of productive tillers before harvesting, a 16% smaller number of grains per spike, a 13-16% lower grain weight per spike, and a total yield loss of 33-42%. In hulless bread wheat RWA fed on the developing kernels inflicting greater damage, whereas the hulled barley seeds were practically not damaged. The mean yield loss of the barley and spring wheat spikes infested with RWA was 24-32% and 50-66%, respectively. Due to the greater tillering capacity and formation of secondary productive tillers in barley, about 52% of the productive barley tillers and 37-39% of spring wheat ones were infested with RWA, which resulted in a comparable yield loss (20-25% in barley and 19-23% in spring wheat). Resistance to RWA was higher in spring wheat and barley cultivars with a shorter vegetation period, looser spikes, and thinner culm walls. The length of productive tillers damaged by RWA was reduced by 21-28%, which determined a lower incidence of leaf diseases.     

Effects of sodium acetate and sodium chloride on EMS-induced chlorophyll mutations in barley

Sodium acetate solutions to which sodium chloride was added, and acetate or chloride alone have been used as pre-, simultaneous, and post-treatment of dry and pre-soaked seeds of barley to study their effect on the types and frequencies of ethyl methanesulphonate (EMS)-induced chlorophyll mutations in spring barley, variety Elsa, and winter barley, varieties $\text{436}\text{35}$ and Ager. Application of acetate/chloride on dry seeds before or simultaneously with EMS both resulted in the frequency of chimeral plants with chlorophyll-deficient sectors in M₁ and chlorophyll mutants in M₂ approximately being halved as compared with the controls (EMS treatment alone).An opposite effect was observed after simultaneous treatment with acetate/ chloride and EMS (pH 4.5 and pH 7.0) and application of acetate/chloride after EMS treatment of pre-soaked seeds. In this case the mutagen sensitivity, i.e. the frequency of chimeral plants with induced chlorophyll-deficient sectors in M₁ and of chlorophyll mutants in M₂, was approximately doubled as compared with the control.Separate application of both acetate or chloride as a simultaneous treatment with EMS resulted also in an increase in the chlorophyll mutation frequency as compared with EMS treatment alone.Based on these results some aspects of the acetate/chloride effect are briefly discussed.     

The carboxyl-terminal helical domain of the ATP synthase γ subunit is involved in ε subunit conformation and energy coupling

The γ subunit located at the center of ATP synthase (F_OF₁) plays critical roles in catalysis. Escherichia coli mutant with Pro substitution of the γ subunit residue γLeu218, which are located the rotor shaft near the c subunit ring, decreased NADH-driven ATP synthesis activity and ATP hydrolysis-dependent H⁺ transport of membranes to ~60% and ~40% of the wild type, respectively, without affecting F_OF₁ assembly. Consistently, the mutant was defective in growth by oxidative phosphorylation, indicating that energy coupling is impaired by the mutation. The ε subunit conformations in the γLeu218Pro mutant enzyme were investigated by cross-linking between cysteine residues introduced into both the ε subunit (εCys118 and εCys134, in the second helix and the hook segment, respectively) and the γ subunit (γCys99 and γCys260, located in the globular domain and the carboxyl-terminal helix, respectively). In the presence of ADP, the two γ260 and ε134 cysteine residues formed a disulfide bond in both the γLeu218Pro mutant and the wild type, indicating that the hook segment of ε subunit penetrates into the α₃β₃-ring along with the γ subunits in both enzymes. However, γ260/ε134 cross-linking in the γLeu218Pro mutant decreased significantly in the presence of ATP, whereas this effect was small in the wild type. These results suggested that the γ subunit carboxyl-terminal helix containing γLeu218 is involved in the conformation of the ε subunit hook region during ATP hydrolysis and, therefore, is required for energy coupling in F_OF₁.     

Efficient localization of mutations by interval haplotype analysis

We have developed a mathematical algorithm to implement a method for localizing mutations using haplotype analysis. Our strategy infers haplotypes based on the determination of genotypes of a proximal and a distal marker for 21 chromosomal intervals distributed across the mouse genome (corresponding to two intervals for Chromosomes (Chrs) 1 and 2 and one for the remaining 17 autosomes). To simulate the analysis of mice homozygous for recessive mutations, we tested the efficacy of our method on over 200 data sets generated from two independent mapping panel data sets containing the genotypes of 46 F₂ progeny of an intercross and 94 F₂ progeny of a backcross. In all cases we were able to identify the chromosomal interval carrying the recessive mutation despite the fact that some of the data sets consisted of as few as 10 meioses. Our strategy proved sensitive and expedient, since the simulated genome-wide screen could be executed by genotype analysis of 40 microsatellite markers in small numbers of intercross or backcross progeny. Received: 2 June 1997 / Accepted: 22 October 1997     

Mutagenesis at the cinnabar locus in Drosophila melanogaster

A study was undertaken to isolate mutations affecting the temporal appearance of kynurenine hydroxylase in Drosophila melanogaster. Such mutations, lacking or having reduced enzyme activity at the larval or pupal stage only, could represent changes in regulatory functions. Mutagenesis was carried out using EMS. Potential mutations were isolated from mass F₁ cultures. The screening of large numbers of individuals was made possible by the use of the mutant red, which allowed visual classification for the presence or absence of the enzyme at both stages. From a series of six mutagenesis experiments 111,561 chromosomes were tested, and 122 phenotypically mutant F₁ individuals were found. From these, 38 inheritable mutations were isolated which, by phenotypic observation, lacked or had reduced enzyme activity at the larval and pupal stages. Assay of enzyme activity levels in several of the mutants confirmed the phenotypic data. All of the 27 mutations that could be tested further are recessive and behave as cinnabar alleles. Complementation tests were performed between these 27 mutant stocks, and no complementation in the production of eye color has been seen between the mutants examined. When extended collection periods were used, a significantly higher percentage of inheritable mutations was isolated from the first 3 days of the screen. Over 80% of the F₁ phenotypic mutants could be classified as mosaics, which indicates that cinnabar can be autonomous under certain conditions. The failure to isolate mutations in possible regulatory function is discussed.     

Segregation of the isozymes of flax genotrophs

The segregation of isozymes of peroxidase and acid phosphatase in progenies of crosses between large (L) and small (S and L₆) flax genotrophs has been determined. The peroxidase isozymes segregated as expected on a simple Mendelian model with a dominant and a recessive allele and with the L genotroph being a homozygous dominant. All the peroxidase isozymes which differed segregated together, so the isozymes are controlled by either a single locus or closely linked loci. The acid phosphatase isozymes in the F₁ were all L type, but the segregations observed in the F₂ were not always consistent with a simple Mendelian model.     

Pleiotropic morphological and abiotic stress resistance phenotypes of the hyper-abscisic acid producing Abo™ mutant in the periwinkleCatharanthus roseus

The pleiotropic properties of aabo abo (Abo^™) γ-ray induced mutant ofCatharanthus roseuscv. Nirmal, selected among the M₂ generation seeds for ability to germinate at 45°C, are described. The mutant produced seeds possessing tricotyledonous embryos, unlike the typically dicotyledonous embryos present in the wild type Abo⁺ seeds. In comparison to Abo⁺ adults, the mutant plants had short stature and lanceolate leaves. The vascular bundles in the leaves and stem were poorly developed. Leaf surfaces were highly trichomatous, epidermal, cortex and mesophyll cells were small sized and a large majority of stomata were closed. Besides high temperature, the mutant was salinity and water-stress tolerant. The abscisic acid (ABA) content in the leaves was about 500-fold higher. The genetic lesionabo responsible for the above pleiotropy was recessive and inherited in Mendelian fashion. The seedlings and adult plants of the mutant accumulated higher proline than Abo⁺ plants. The phenotypes ofabo abo mutants permitted the conclusions that (i) the mutant synthesizes ABA constitutively, (ii) both ABA-dependent and ABA independent pathways for proline and betaine accumulation are functional in the mutant, and (iii) cell division, elongation and differentiation processes in embryo and adult plant stages are affected in the mutant.     

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M₂) family derived from a heterozygous (M₁) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M₂) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M₃) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M₂) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.