Irradiated Pollen Research Articles

The effects of genotypes and irradiation doses on haploid embryo induction and plant production in bottle gourd [Lagenaria siceraria(Malign) Stanley

Cultivated Legenaria siceraria (Malign) Stanley is commonly known as the white-flowered gourd. It has been grown annually and it is a monocious, vigorous climber species. The mature fruit is often scooped out and the skin is used as containers, music instrument, decorative purposes or in some cases, fishing floats. Shoots, tendrils and leaves are also cooked and the seeds are removed for oil extraction or for use in cooking. Furthermore, L. siceraria is used as rootstocks conferring the watermelon grafted plant resistance to soil-borne diseases and adverse soil conditions. The objective of this study was to optimize bottle gourd haploid plants. Haploid plants are an invaluable source to rapidly produce new homozygous inbred lines. We analyzed the effect of various pollen irradiation doses (50, 75, 100, 125, 150, 175 and 200 Gray gamma rays of 60Co) on the induction of parthenogenetic haploid embryos after pollination with the irradiated pollen in four bottle gourd landraces (Adana, Birecik, Spain and Cyprus). Our results indicate that pollination with irradiated pollens at 50 and 75 Gy had a positive effect on haploid embryo induction. After pollination with irradiated pollens, embryos with different shape were rescued, cultured and converted to intact plants under tissue culture conditions. The genotypes had important effect on induction of haploid embryo by irradiated pollen technique. Seventy five plants from Adana genotype, 10 plants from Birecik genotype, 121 plants from Spain genotype, 141 plants from Cyprus genotype and totally 347 plants were produced from this research.

Induction of haploid plants in citrus through gamma-irradiated pollen and ascertainment of ovule age for maximum recovery of haploid plantlets

The present investigation was carried out for the induction of haploid plants in Citrus grandis through in situ parthenogenesis by pollination with gamma-irradiated pollen of C. limetta and C. sinensis, treated with 50, 100, 200, 300, and 400 Gy gamma ray doses followed by in vitro ovule culture. Ovule culture at 50 days after pollination (DAP) was found optimum for the maximum recovery of in vitro-raised plantlets as compared to 35 and 20 DAP. At 50 DAP, irrespective of the pollen parent, plantlet regeneration capacity decreased at 300 and 400 Gy irradiation treatment (0.62% and 0.60%, respectively) with maximum recovery in the nonirradiated control (3.07%). Chromosome counting in actively growing root tips of all the in vitro-raised ovule cultured plantlets revealed that two haploid plants with chromosome number of nine were induced in C. grandis from the ovule culture that was cultured at 50 DAP following pollination with irradiated pollen of C. sinensis at 400 Gy and C. limetta pollen at 300 Gy. However, irradiation doses below 300 Gy were incapable of inducing any haploids in either cross combination. Similarly, all the plantlets regenerated from in vitro embryo culture of mature seeds were found to be diploid in nature, irrespective of the pollen parent and irradiation dose. Molecular analysis of the in vitro-raised haploid and diploid plants using SSR markers confirmed the maternal origins of the haploid plants; however, the diploid plants were found zygotic in nature with one allele from the seed parent and the other one from the pollen parent.

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The technique of pollen irradiation for mutation induction technique has been successfully demonstrated in many crop species. Therefore, the mutagenic effects of UV irradiated pollen on cowpea accessions were investigated. Pollen grains of eight cowpea accessions were irradiated with 30,000µWs/cm2 UV for 60, 120, 180, 240, 300 and 360 minutes. Emasculated flowers of each accession were self-pollinated with irradiated pollen to evaluate the effects of pollen mutagenesis on seed setting in the M1 generation. Harvested seeds from M1 plants were advanced to M2 to evaluate the effects of UV radiation on seed germination, plant survival and for mutant selection in the M3 generation. Data were analyzed using descriptive statistics. Pollen irradiation with UV for a short period (60 min) increased seed setting in all the cowpea accessions at M1 except IB-Y-1 where it reduced seed setting by 28.6%. Observed LD50 of UV rays among the cowpea lines ranged from 142.6 to 210.1 minutes. No significant difference was observed in seed germination for all treatments at M2 except irradiation for 120 minutes in IT90K-284-2. A trend similar to seed germination was observed in seedling survival at M2. The three-primary leaf and four-primary leaf mutants selected at M3 generation reverted back to two-primary leaf seedlings at M4. Low mutation frequencies recorded in this study shows that cowpea is considered less amenable to the application of UV irradiation as a practical breeding method. Key words: Cowpea mutagenesis, pollen irradiation, ultra-violet induced mutant, seed germination, seed setting.

Evaluation of haploidization efficiency in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) through anther culture

The production of large numbers of haploids is the crucial point of the dihaploidisation process. Although in vitro haploid plants were successfully produced by irradiated pollen technique in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.), the frequency is still insufficient for using in a large-scale breeding programme. Thus, the present study was conducted to determine the efficacy of the anther culture technique on the production of in vitro haploids in the aforementioned species for which there have been no successful reports concerning by androgenesis. The anthers at uninucleate microspore stage were collected at different florescence times and then cultured on a solid MS medium supplemented by different combinations of 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), KN (kinetin) with the constant addition of NAA (naphthalene acetic acid) to induce callogenesis, embryogenesis and plantlet initiation. The combination of PGR, genotype and anther collection time played an important role in the androgenic response. The highest response was obtained from 57SI21 and G9 lines with the combination of 2.0 or 4.0 mg/l BAP + 0.05 mg/l NAA (E6 medium) at the first anther collection time. Plantlets were regenerated and rooted on MS medium supplemented by 0.01 mg/l IAA. In total, 74 plants were recovered and propagated with micro-cuttings. The ploidy analyses revealed that 35 plants (47.3 %) were haploid (n = 20), and the others (52.7 %) were diploid (2n = 40).

Androgenesis, gynogenesis, and parthenogenesis haploids in cucurbit species

Haploids and doubled haploids are critical components of plant breeding. This review is focused on studies on haploids and double haploids inducted in cucurbits through in vitro pollination with irradiated pollen, unfertilized ovule/ovary culture, and anther/microspore culture during the last 30years, as well as comprehensive analysis of the main factors of each process and comparison between chromosome doubling and ploidy identification methods, with special focus on the application of double haploids in plant breeding and genetics. This review identifies existing problems affecting the efficiency of androgenesis, gynogenesis, and parthenogenesis in cucurbit species. Donor plant genotypes and surrounding environments, developmental stages of explants, culture media, stress factors, and chromosome doubling and ploidy identification are compared at length and discussed as methodologies and protocols for androgenesis, gynogenesis, and parthenogenesis in haploid and double haploid production technologies.

English

English

Induction of parthenogenetic haploid plants using gamma irradiated pollens in ‘Hirado Buntan’ pummelo (Citrus grandis [L.] Osbeck)

Homozygous genotypes have great potential in breeding and genomic researches in higher plants. Induction of parthenogenesis via pollination with gamma-irradiated pollens is an efficient method to obtain haploid plants that can overcome the biological limitations of fruit trees. In this study, we investigated the induction of haploid plants in ‘Hirado Buntan’ pummlo (Citrus grandis) through in-situ gynogenesis by pollination with irradiated pollens of trifoliate orange (255, 325 and 500Gy) and ‘Tongshui 72-1 Jincheng’ sweet orange (255, 395 and 500Gy). The pollen stainability, pollen tube behavior after pollination were evaluated. The seeds extracted from the fruits were rescued by in vitro embryo culture, and the ploidy level of the obtained plantlets was determined by flow cytometry. It showed that two haploid plants and one triploid plant, were obtained from the cross-pollination of ‘Hirado Buntan’ pummelo with irradiated pollens of trifoliate orange; two haploid plants were obtained from the cross-pollination of ‘Hirado Buntan’ pummlo with irradiated pollens of ‘Tongshui 72-1 Jincheng’ sweet orange. The parthenogenic origin of the haploid plants was further confirmed by chromosomal cytogenetic and SSR analysis. The ‘Hirado Buntan’ haploid plants obtained here hold great potential in citrus genetic and genomic studies in the future.

First Report of Obtaining Haploid Plants Using Tissue Culture Techniques in Spinach

Monoic, dioic, and hermaphrodite flower types complicate spinach breeding and cultivar development. The availability of haploid plants will definitely accelerate spinach breeding; however, there are currently no reports in the literature about the use of tissue culture techniques to obtain spinach haploids. Therefore, in this study, pollination with irradiated pollen and anther culture methods were used to obtain haploid plants in spinach. For anther culture, three spinach varieties (Koto F1, Favorit F1, and Greenstar F1) and four different nutrient media were tested to obtain haploid embryos. Significant outcomes were not achieved from anther culture, and only two plants were obtained from all the experiments. Gynogenesis studies using irradiated pollen were performed with the same three spinach varieties and six gamma ray doses (100, 150, 200, 300, 400, and 500 Gy) from (cobalt60) Co60. Murashige and Skoog (MS) nutrient medium containing 1 mg·L−1 indoleacetic acid (IAA) was used for embryo germination. Current findings revealed that the varieties produced responded differently to the various doses of radiation. A total of 3414 embryos and 1710 plants were obtained from experiments carried out for 2 years. Considering the numbers of embryos and plants per 100 seeds, the Favorit F1 variety provided better results than the other two varieties. However, significantly different outcomes were not achieved with regard to irradiation doses. Embryos were observed at all doses tested. Flow cytometry analyses that were carried out on regenerated plants revealed whether the plants were diploid or doubled haploid, and molecular analyses revealed that diploids resulted from spontaneous chromosome doubling. The current findings offer significant results for spinach breeding using haploids.

Artificial feeding of honeybees based on an understanding of nutritional principles

Artificial pollen substitutes were developed to improve productivity from honeybees during periods of nutrient scarcity. The history of pollen-substitute development is outlined. Although many attempts have been made, no substitute has the same nutritional value as bee-collected pollen. Following a review of honeybee nutrition, Black (2006) described the ingredient and nutrient specifications for a pollen substitute, including the need for attractiveness to honeybees. Protein isolates were recommended to avoid toxicity from carbohydrates found in many ingredients used in previous studies. Twenty-seven plant- and animal-derived oils and a rum supplement, mixed at 2% with a low-lipid irradiated pollen, were evaluated for attractiveness by measuring consumption and bee congregation when placed in dishes within beehives. Coconut, linseed oil and rum were preferred significantly (P < 0.05) to the pollen, whereas pollen was preferred to lavender and sage oils. Almond and evening primrose oils were also highly, but not significantly, preferred compared with pollen and were used in combinations with coconut and linseed oils in subsequent experiments. Eleven predominantly pure protein sources, either singly or in combination, were mixed with 2% or 5% attractive oils and evaluated for attractiveness. Soybean protein isolate was selected, because attractiveness was not significantly different from bee-collected pollen. It was then used with oils in an experiment to evaluate either powdered cellulose or milled oat hulls as a fibre source. There were no significant differences in attractiveness of substitutes with the two fibre sources or pollen, and powdered cellulose was selected for further use on the basis of availability. An experiment with ~1000 newly hatched bees with a fertile queen in cages was undertaken to evaluate the diets when given as the sole nutrient source. Consumption, bee longevity and estimated hypopharyngeal gland development using head weight were measured One artificial diet (PI-5) contained 30% soybean protein isolate, 10% cellulose, 42% icing sugar, 12.5% water, 4.5% mixed oils and 1.3% minerals and vitamin plus cholesterol. Diet (PI-10) was similar, but contained twice as much oil replacing icing sugar. Other treatments were redgum pollen (P), a commercial pollen substitute, Feedbee® and defatted soybean meal. Diet consumption, lifespan and head weights were significantly less for the PI diets than for P, while Feedbee® and defatted soybean-meal diets were generally intermediate. The PI diets were discovered to contain excess sodium, due to the manufacturing process. Bees consuming the PI diets had lower concentrations of magnesium, copper, iron, manganese and zinc in their bodies than did those offered P. The fatty acid content of bee bodies also varied with diet. A revised formulation is recommended with reduced sodium and modified mineral and fatty acid composition.

In Silico and Fluorescence In Situ Hybridization Mapping Reveals Collinearity between the Pennisetum squamulatum Apomixis Carrier-Chromosome and Chromosome 2 of Sorghum and Foxtail Millet.

Apomixis, or clonal propagation through seed, is a trait identified within multiple species of the grass family (Poaceae). The genetic locus controlling apomixis in Pennisetum squamulatum (syn Cenchrus squamulatus) and Cenchrus ciliaris (syn Pennisetum ciliare, buffelgrass) is the apospory-specific genomic region (ASGR). Previously, the ASGR was shown to be highly conserved but inverted in marker order between P. squamulatum and C. ciliaris based on fluorescence in situ hybridization (FISH) and varied in both karyotype and position of the ASGR on the ASGR-carrier chromosome among other apomictic Cenchrus/Pennisetum species. Using in silico transcript mapping and verification of physical positions of some of the transcripts via FISH, we discovered that the ASGR-carrier chromosome from P. squamulatum is collinear with chromosome 2 of foxtail millet and sorghum outside of the ASGR. The in silico ordering of the ASGR-carrier chromosome markers, previously unmapped in P. squamulatum, allowed for the identification of a backcross line with structural changes to the P. squamulatum ASGR-carrier chromosome derived from gamma irradiated pollen.

Dark Period Following UV-C Treatment Enhances Killing of Botrytis cinerea Conidia and Controls Gray Mold of Strawberries.

Strawberries are available throughout the year either from production in the field or from high and low tunnel culture. Diversity of production conditions results in new challenges in controlling diseases before and after harvest. Fungicides have traditionally been used to control these diseases; however, their limitations necessitate a search for new approaches. We found that UV-C irradiation of Botrytis cinerea, a major pathogen of strawberry, can effectively kill this fungus if a dark period follows the treatment. The inclusion of a 4-h dark period resulted in almost complete kill of B. cinerea conidia on agar media at a dose of 12.36 J/m2. The UV-C dose did not cause a reduction in photosynthesis in strawberry leaves or discoloration of sepals, even after exposing plants repeatedly (twice a week) for 7 weeks. Although irradiation of dry conidia of B. cinerea with this dose resulted in some survival, the conidia were not infective and not able to cause decay even when inoculated onto a highly susceptible mature apple fruit. Irradiation of strawberry pollen at 12.36 J/m2 did not affect pollen germination, tube growth and length in vitro, or germination and tube growth in the style of hand-pollinated emasculated strawberry flowers. No negative effect of the UV-C treatment was observed on fruit yield and quality in high tunnel culture. In the fruit and flower petal inoculation tests, the UV-C treatment was highly effective in reducing fruit decay and petal infection. This UV-C treatment with an exposure time of 60 s may be useful in controlling gray mold in tunnel production of strawberries and may also have the potential for use in intensive field and indoor production of other fruits and vegetables providing that a 4-h dark period follows the irradiation.

Hygienic food to reduce pathogen risk to bumblebees

Bumblebees are ecologically and economically important pollinators, and the value of bumblebees for crop pollination has led to the commercial production and exportation/importation of colonies on a global scale. Commercially produced bumblebee colonies can carry with them infectious parasites, which can both reduce the health of the colonies and spillover to wild bees, with potentially serious consequences. The presence of parasites in commercially produced bumblebee colonies is in part because colonies are reared on pollen collected from honey bees, which often contains a diversity of microbial parasites. In response to this threat, part of the industry has started to irradiate pollen used for bumblebee rearing. However, to date there is limited data published on the efficacy of this treatment. Here we examine the effect of gamma irradiation and an experimental ozone treatment on the presence and viability of parasites in honey bee pollen. While untreated pollen contained numerous viable parasites, we find that gamma irradiation reduced the viability of parasites in pollen, but did not eliminate parasites entirely. Ozone treatment appeared to be less effective than gamma irradiation, while an artificial pollen substitute was, as expected, entirely free of parasites. The results suggest that the irradiation of pollen before using it to rear bumblebee colonies is a sensible method which will help reduce the incidence of parasite infections in commercially produced bumblebee colonies, but that further optimisation, or the use of a nutritionally equivalent artificial pollen substitute, may be needed to fully eliminate this route of disease entry into factories.

Creation and Genomic Analysis of Irradiation Hybrids in Populus

Establishing efficient functional genomic systems for creating and characterizing genetic variation in forest trees is challenging. Here we describe protocols for creating novel gene-dosage variation in Populus through gamma-irradiation of pollen, followed by genomic analysis to identify chromosomal regions that have been deleted or inserted in each F1 individual. Irradiated pollen is used in a controlled, interspecific cross to create F1 progeny that carry deletions and insertions of chromosomal regions. These insertions and deletions result in novel changes in gene dosage that in turn affect both qualitative and quantitative phenotypic traits. The protocols described here outline the processes involved in optimizing irradiation levels and performing controlled crosses, sowing seed and propagating seedlings, and genome-wide sequence-based analysis of deletions and insertions in the F1 progeny. The same approach could be applied to other vegetatively propagated species. © 2016 by John Wiley & Sons, Inc.

Induced parthenogenesis by gamma-irradiated pollen in loquat for haploid production.

Successful haploid induction in loquat (Eriobotrya japonica (Thunb.) Lindl.) through in situ-induced parthenogenesis with gamma-ray irradiated pollen has been achieved. Female flowers of cultivar ‘Algerie’ were pollinated using pollen of cultivars ‘Changhong-3’, ‘Cox’ and ‘Saval Brasil’ irradiated with two doses of gamma rays, 150 and 300 Gy. The fruits were harvested 90, 105 and 120 days after pollination (dap). Four haploid plants were obtained from ‘Algerie’ pollinated with 300-Gy-treated pollen of ‘Saval Brasil’ from fruits harvested 105 dap. Haploidy was confirmed by flow cytometry and chromosome count. The haploids showed a very weak development compared to the diploid plants. This result suggests that irradiated pollen can be used to obtain parthenogenetic haploids.

The creation of new aneuploid lines of the cotton (Gossypium hirsutum L.) with identification of chromosomes by translocation and SSR-markers

The use of monosomic lines significantly increases the effectiveness of molecular-genetic analysis and the development of superior quality breeding lines via substitutions by alien chromosomes. A complete set of aneuploid series for each cotton chromosome was required. Simple sequence repeats (SSR) have been applied as useful markers for understanding cotton genetics. Several DNA markers have already been assigned to the individual chromosomes of Gossypium hirsutum. The primary objective of this paper is to report the creation of new monosomic lines and the use of chromosome specific simple sequence repeat (SSR) markers and translocation lines for chromosome identification to confirm chromosome specificity of monosomic lines in the Uzbek collection. Here, we summarize data on the development of a monosomic stock collection of cotton (G. hirsutum L.) from Uzbekistan, including the origin of 95 primary monosomics, their cytogenetic characteristics, morphological features and their identification by translocation tests and chromosome SSR-markers. Our results indicated several different monosomic lines identified by chromosomes 2, 4, 6 and telosome 11 of the At subgenome and chromosomes 18 and 22 of the Dt subgenome, 22 monosomes were identified as duplicates of three monosomes (chromosomes 2, 4 and 6). Chromosome 4 of the At subgenome was recovered more frequently (18 times) than chromosome 2 of the At subgenome (4 times) and chromosome 6 of the At subgenome (3 times) during pollen irradiation at different doses and in the progeny of the desynaptic plants. These lines will be useful for molecular mapping and enhancement of Upland cotton.

Effect of different irradiation sources and doses on haploid embryoinduction in Altinbas (Cucumis melo L. var. inodorus) melons

In vivo induction of parthenogenesis using an irradiated pollen technique provides successful results in melon and accelerates breeding by shortening the time for obtaining 100% homozygous lines. Gamma rays (cobalt-60) are usually used for the pollen irradiation, although it is difficult to find a suitable source for irradiation. Haploid technology is widely used in melon breeding programs, so there is a need to find alternative sources for pollen irradiation. This study was performed to compare the efficiency of different irradiation sources to induce haploid embryos. One dose of cobalt-60 (300 Gy), two different doses (200 Gy and 300 Gy) of cesium-137, and two different doses (200 Gy and 300 Gy) of X-ray were used for the irradiation of melon pollen. All irradiation sources were efficient for fruit set, while the highest fruit set (60.38%) was obtained from 200 Gy cesium-137 and the lowest (20.01%) was obtained from 300 Gy X-ray. A total of 2294 haploid embryos were rescued, of which 2052 regenerated into plants. The number of rescued haploid embryos per fruit ranged between 8.77 (300 Gy cobalt-60) and 4.23 (200 Gy X-ray). The highest regeneration rate of haploid embryos into plants was 92.56% (300 Gy cobalt-60) and the lowest was 61.86 % (200 Gy cesium-137).

English

Cassava is a highly heterozygous species; hence, current methods used in classical cassava breeding cannot match the urgent need to high yielding varieties. Recently, progress was made through androgenesis and gynogenesis as pathways for raising doubled cassava haploid lines to overcome problems associated with cassava’s inherent reproductive biology, but these efforts were limited (no candidate cassava plantlets were regenerated). For the first time, this study shows that pollen irradiation coupled with self-pollination and embryo rescue regenerated 62 candidate cassava plantlets. Plants of an elite cassava variety, Nase14, served as a mother plant and as the pollen donor for the irradiation. Irradiation dosages of 50 to 250 Gray studied across five pollination events and 300 or 500 Gray in one pollination event caused a reduction in pollen germination up to 67.0%. By 15 days after pollination (DAP) with irradiated pollen, up to 89.7% of the pollinated flowers had aborted. By embryo rescue time (42 DAP), significant differences were observed in number of fruits, seeds and embryos generated, with the non-irradiated pollen treatments having significantly higher numbers. Sixteen (16) heterozygous SSR markers in the parent and ploidy analysis showed that none of the regenerated plants was haploid or homozygous. However, the plantlets resulting from pollination with non-irradiated pollen had 56.2% homozygous loci, while progeny derived from irradiated treatments had frequencies of homozygous loci between 28.1 and 55.0%. This is the first time to use irradiated pollen in cassava as a pathway to generate candidate plantlets as an initial step in double haploid production. Key words: Cassava, doubled haploids, embryo rescue, plant regeneration, pollen germination, pollen irradiation.

EFFICIENT HAPLOID PRODUCTION ON 'WILKING' MANDARIN BY INDUCED GYNOGENESIS

Seedlessness is a major criterion for the citrus fresh fruit market. Therefore, triploid breeding appears very promising for the selection of new citrus cultivars. CIRAD has developed a method to produce triploid hybrids by somatic hybridization between diploid and haploid lines, and demonstrates the potential for haploid citrus production by induced gynogenesis in clementine. In this work we analysed the efficiency of induced gynogenesis by irradiated pollen in 'Wilking' mandarin (Citrus reticulata). Five doses of gamma irradiation of 'Meyer' lemon pollen were tested (0, 150, 200, 250, 300 Grays). Embryos were extracted from small seeds, obtained in immature fruits, and cultivated in vitro. The ploidy level of plantlets was determined by flow cytometry analysis. Irradiated pollen appeared to be efficient for gynogenesis induction in 'Wilking'. A relatively high number of haploids (7 plantlets) were obtained, essentially after hybridisation with 'Meyer' lemon pollen irradiated at 250 Grays. The ploidy level of all haploid plantlets was confirmed by chromosome counts, and molecular markers demonstrated their gynogenetic origin. These haploid lines should be useful for triploid breeding by somatic hybridisation, and also constitute interesting resources for genomic studies. (Resume d'auteur)

Induction of haploid plants in iris (Iris pseudacorus) by pollen irradiation

This is the first report of successful regeneration of haploid lines in iris (Iris pseudacorus) developed by in situ parthenogenesis followed by embryo rescue. Native genotypes of I. pseudacorus (2n = 38) were prepared as female parents and I. spuria was set as the males. Anthers collected from the pollen donor were irradiated by X-ray with doses of 0, 100, 200, 300, and 400 Gy in 2012. A second round of experiments was conducted in 2013 with 300 and 400 Gy. The ovaries from each pollinated flower were collected and cultured and the produced plantlets were then acclimatized. Chromosome counting and flow cytometry were used to determine the ploidy levels. The viability of irradiated pollens was also evaluated. Pollination with irradiated pollen at doses ranging from 100 to 400 Gy can significantly reduce fruit set, particularly at higher doses (300 and 400 Gy). The embryo production was reduced in I. pseudacorus as doses increased; the highest doses led to reduction in the number of regenerants with the formation of just 6 and 4 embryos at 300 and 400 Gy, respectively. Haploid plantlets were produced when the pollen grains were exposed to 300 and 400 Gy of X-ray. Irradiation at 100 and 200 Gy was found to be insufficient to cause pollen sterility. The best dose for haploid production in I. pseudacorus is hereby found to be 300 or 400 Gy of X-ray.

Efficient marker-assisted screening of structural changes involving Haynaldia villosa chromosome 6V using a double-distal-marker strategy

Wild relatives of wheat possess many agronomic traits important to wheat improvement. Wheat–alien translocation and deletion lines are important genetic stocks in wheat breeding or physical mapping of important alien genes. However, screening for chromosomal structural changes by conventional cytogenetic analyses is time-consuming work. It is necessary to develop an efficient method of finding alien chromosomal structural changes. In this research, two amplicon markers, 6VS- 381 and 6VL- 358 , specific to the distal regions of the short and long arms, respectively, of Haynaldia villosa chromosome 6V, were developed based on the expressed sequence tags located on the distal regions of wheat group 6. Marker-assisted screening work was then carried out in two individual radiation-induced populations. The first contained 365 plants involving 43 M1:2 families derived from a pollen irradiation treatment of a wheat–H. villosa monosomic 6V addition line. The other was a M1 population containing 100 plants derived from an irradiation treatment of a wheat–H. villosa disomic 6V(6A) substitution line. The female parent in both treatments was a common wheat cultivar Chinese Spring. Those plants marked by a single positive distal marker were considered to be putative structural changes of 6V. After cytogenetic analysis, a total of 20 structure-changed chromosomes were identified, comprising 12 whole-arm translocations, four terminal translocations, one 6VL terminal deletion, one translocation deletion, and two intercalary translocations. The double-distal-marker strategy proposed in this study gives an efficient model for finding structural aberrations involving a specific alien chromosome.