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Virginia, united states.
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- Vavilovskii Zhurnal Genet Selektsii
- v.26(4); 2022 Jul
Language: English | Russian
Investigation of genetic polymorphism of Russian rape and turnip rape varieties using SSR and SRAP markers
Изучение генетического полиморфизма российских сортов рапса и сурепицы с использованием ssr- и srap-маркеров, i.a. klimenko.
Federal Williams Research Center of Forage Production and Agroecology, Lobnya, Moscow region, Russia
V.T. Volovik
A.a. antonov, v.a. dushkin, a.o. shamustakimova, yu.m. yu.m. mavlyutov.
Rapeseed (Brassica napus L.) and turnip rape (B. rapa L. subsp. campestris (L.)) are important agricultural plants widely used for food, fodder and technical purposes and as green manure. Over the past decades, a large number of perspective varieties that are being currently cultivated in every region of Russia have been developed. To increase the breeding eff iciency and facilitate the seed production, modern molecular-genetic techniques should be introduced as means to estimate species and varietal diversity. The objective of the presented research study was to investigate DNA polymorphism of the rapeseed and turnip rape varieties developed at Federal Williams Research Center of Forage Production and Agroecology and detect informative markers for varietal identif ication and genetic certif ication. To genotype 18 gDNA samples, 42 and 25 combinations of respective SSR and SRAP primers were used. The results obtained demonstrate that SRAP markers were more effective for polymorphism analysis: 36 % of the tested markers revealed genetic polymorphism compared with only 16.7 % of microsatellite loci. Molecular markers to detect differences at interspecif ic and intervarietal levels have also been found. For the investigated set, such microsatellite loci as Na12A02, Ni2C12, Ni02-D08a, Ra02-E01, Ni03H07а and SRAP-marker combinations as F13-R9, Me4- R7, F11-Em2, F10-R7, F9-Em2 and F9-R8 proved to be informative. Application of the two marker techniques made it possible to detect a higher level of DNA polymorphism in plants of different types (spring and winter varieties) if compared against the intervarietal differences within a species or a group. According to Nei’s genetic diversity index, in the cluster of winter rapeseed, VIK 2 and Gorizont varieties had the longest genetic distance, and in the spring cluster, these were Novosel and Veles. A high level of similarity was found between Vikros and Bizon winter rapeseed varieties. The results obtained have a high practical value for varietal specif ication of seed material and genetic certif ication of rapeseed and turnip rape varieties.
Рапс (Brassica napus L.) и сурепица (B. rapa L. subsp. campestris (L.)) – важные сельскохозяйственные культуры, широко используются для продовольственных, кормовых и технических целей, а также в качестве сидератов. За последние десятилетия создано большое количество перспективных сортов, культивируемых практически во всех регионах России. Для повышения эффективности селекционного процесса и успешного развития семеноводства необходимо внедрять современные молекулярно-генетические методы оценки видового и сортового разнообразия. Цель настоящей работы заключалась в изучении ДНК-полиморфизма сортов рапса и сурепицы селекции Федерального научного центра кормопроизводства и агроэкологии им. В.Р. Вильямса и выявлении информативных маркеров для сортовой идентификации и генетической паспортизации. Для генотипирования 18 образцов геномной ДНК использовали 42 и 25 комбинаций SSR- и SRAP-праймеров соответственно. Результаты показали, что маркеры SRAP более эффективны для анализа полиморфизма изучаемого материала: 36 % от общего числа испытанных маркеров демонстрировали генетический полиморфизм, тогда как для микросателлитных локусов этот показатель равнялся 16.7 %. Определены молекулярные маркеры для выявления различий на межвидовом и межсортовом уровнях. Информативными для исследуемой выборки сортов оказались микросателлитные локусы Na12A02, Ni2C12, Ni02-D08a, Ra02-E01, Ni03H07а и комбинации SRAP-маркеров F13-R9, Me4-R7, F11-Em2, F10-R7, F9-Em2 и F9-R8. Анализ сортового материала по двум системам маркирования показал более высокий уровень ДНК-полиморфизма у образцов растений разного типа развития (яровой/озимый) в сравнении с различиями между сортами в пределах вида или группы. Согласно индексам генетического разнообразия Нея, в кластере сортов озимого рапса наибольшей генетической удаленностью выделялись ВИК 2 и Горизонт, среди яровых – Новосёл и Велес. Высокий уровень сходства обнаружен между яровыми сортами рапса Викрос и Бизон. Полученная информация имеет практическое значение для контроля сортовой принадлежности и генетической паспортизации семенного материала сортов рапса и сурепицы.
Introduction
Cabbage oilseed crops such as rapeseed (Brassica napus L.) and turnip rape (B. rapa L. subsp. campestris (L.)) are cultivated in almost every region of Russia, and, for the foreseeable future, are regarded as the main reserve for increasing the production of vegetable oil and fodder protein. These plants are widely used in food, fodder, technical purposes and as green manure that increases soil fertility thanks to the plants’ root remains containing up to 6 tons of organic maters, 80 kg of nitrogen, 60 kg of phosphorus and 90 kg of potassium per hectare. As for their food and fodder properties, rapeseed and turnip rape exceed many other cultivated crops since their seeds are 40–48 % fat and 21–33 % protein and contain a high amount of essential amino acids (Volovik, 2015). Rapeseed can provide livestock with green forage from early spring to late fall thanks to their cold hardiness and fast regrowth after mowing. They are also an excellent silage material, and their seeds and seed by-pass products are processed to produce seed cake and coarse meal. In the recent years the varieties of rapeseed and turnip rape with low or no erucic-acid content became available and seed production has increased more than 7 times to reach the world’s third place after soybeans and cotton. Russia’s short-term plans are to increase rapeseed planting acreage to 2.5 mln he.
As for Russian research institutions working intensely to select cabbage oilseed crops, the leading ones are All-Russian Research Institute of Rapeseed, All-Russian Research Institute of Oilseed Crops and All-Russian Williams Fodder Research Institute. For the two last decades, they have produced the perspective varieties of rapeseed, turnip rape, white mustard and oil radish that have been recommended for oil production, livestock and poultry green forage, combination fodder, seed cake and coarse meal production. In 2021, “State Register” of the Russian Federation included 13 varieties of rapeseed and 3 varieties of turnip rape selected by Federal Williams Research Center of Forage Production and Agroecology (Kosolapov et al., 2019; State Register…, 2021).
For preservation and rational use of newly available varieties, intensification of the selection process and protection of intellectual property, modern and effective methods to estimate species and varietal diversity at a genetic level are to be introduced. One of such techniques that has been successfully applied in the recent years is molecular DNA markers, which, if compared against the traditional morphological indicators, possess a number of advantages. These include a high level of polymorphism; even genome distribution; reliability; a possibility to automate the assay procedure that does not depend on environmental conditions or a plant development phase (Agarwal et al., 2008; Khlestkina, 2011; Chesnokov, 2018). If the most informative and convenient DNA markers are selected, their capabilities to estimate the genetic variability of selection material are regarded as unlimited.
Laboratory for Molecular and Genetic Studies in Federal Williams Research Center of Forage Production and Agroecology has been developing a system for DNA identification and genetic certification of Russian fodder crops. For the time being, the varietal identification techniques have been adapted for perennial legume grasses such as red clover and different species of alfalfa (Klimenko et al., 2020a, b). The assay uses samples of the summary total DNA obtained through a modified method from an arbitrary selected sample of every variety’s germinants. Two types of molecular markers were used: SSR (simple sequence repeats), which detect the variability of microsatellite genome sequences, and SRAP (sequence related amplified polymorphism), which is based on PCR with a pair of primers for amplification of intron/exon regions (open reading frames). The techniques have been tested on different species of fodder crops to optimize the amplification conditions, detection and analysis of results.
A problem of reliable varietal identification is particularly topical for rapeseed due to its limited genetic variability conditioned by the intensive selection aimed at higher content and quality of oil. Currently, a significant number of published studies have been devoted to using different DNA markers for estimation of the genetic diversity of rapeseed varieties and hybrids (Plieske, Struss, 2001; Snowdon, Friedt, 2004; Klyachenko et al., 2018; Mozgova et al., 2019); to genetic mapping (Piquemal et al., 2005; Gao et al., 2007; Geng, 2012) and marking the genes of economically valuable traits (Chen et al., 2010; Ananga et al., 2012). However, only a few such studies have investigated Russian varieties. Four varieties of winter and spring rapeseed (Podmoskovniy, Vikros, VIK 2 and Severyanin) were studied by Byelorussian researchers to identify the gene alleles determining the concentration of oleic and linolic acids in rapeseed oil (Lemesh et al., 2015). The same varieties were investigated to detect the DNA markers of the genes responsible for erucic-acid synthesis (Amosova et al., 2014). Microsatellite markers were used to study the genetic polymorphism of Russian varieties Ratnik and SNK- 198 (Satina, 2010) as well as the genetic homogeneity of spring rapeseed varieties Bulat and Forward (Rogozhina et al., 2015). Such winter varieties as Stolychniy, Laureat, Gorizont, Nord and Severyanin were investigated to detect the quantitative trait loci (QTLs) associated with high winter hardiness (Mozgova et al., 2019).
The objective of the presented study was to investigate DNA polymorphism of rapeseed and turnip rape varieties developed by breeders of Federal Williams Research Center of Forage Production and Agroecology and to identify the informative markers for varietal differentiation and genetic certification.
Materials and methods
Plant material. The study investigated 15 varieties of winter (Severyanin, Stolychniy, VIK 2, Nord, Laureat, Gorizont, Garant) and spring (Vikros, Novik, Novosel, Veles, Grant, Podmoskovniy, Lugovskoy, Bizon) rapeseed and 3 varieties of winter (Zarya) and spring (Nadezhda, Svetlana) turnip rape.
DNA extraction and PCR analysis. The gDNA was extracted from 30 germinants of each abovementioned variety (bulk samples) using the basic SDS method (Kirby, Cook, 1967; Dellaporta et al., 1983) with some modifications (Klimenko et al., 2020b). The quality and concentration of the obtained DNA fractions were verified with agarose gel (1.5 %) electrophoresis and using a Nabi spectrophotometer (MicroDigital, South Korea).
To carry out SSR analysis, 42 markers from the database Brassica info (https://www.brassica.info) and available publications were applied. The efficiency of the primers devised for these markers had been demonstrated in the studies devoted to development of the technology of rapeseed genotyping (Satina, 2010) and selection of the samples with low erucic-acid and glucosinolate content (Hasan et al., 2008). A part of the markers included in the analysis was used for hybridization control and detection of Alternaria blight resistant genotypes in Indian mustard (B. juncea L.) (Chandra et al., 2013; Sharma et al., 2018).
The PCR-mixture of 20 μl contained 3 μl 10 × PCR buffer (Taq Turbo Buffer), 0.5 μl 50 × dNTPs mix, 0.4 μl Taq polymerase (5U), forward and reverse primers (0.1 μl each, 100 μm) and 0.1 μl of DNA sample (20 ng/μl). The amplification was performed in a T-1000 thermal cycler (Bio-Rad, USA) at two different temperature regimes. The first amplification program was an initial 3-min denaturation at 95 °C followed by 30 cycles of 30 s at 94 °C, 30 s at 55–57 °C, 30 s at 72 °C and a final 5-min elongation at 72 °C (Satina, 2010). The second program included an initial 5-min denaturation at 95 °C followed by 39 cycles of 1 min at 94 °C, 2 min at 46–51 °C (depending on the primer pair in use), 2 min at 72 °C and a final 10-min elongation at 72 °C (Chandra et al., 2013). The reproducibility of obtained results was attested in three-fold replication.
SRAP analysis was carried out using 25 primer combinations comprised from 10 single oligonucleotides: F9, F13, Me4, F10, F11, R9, R7, Em2, R14, R8 (Li, Quiros, 2001; Rhouma et al., 2017). The amplification program was an initial 4-min denaturation at 94 °C followed by 10 cycles with changing temperature and duration parameters (1 min at 94 °C, 1 min at 35 °C, 1 min at 72 °C); followed by 30 cycles (1 min at 94 °C, 1 min at 50 °C, 1 min at 72 °C) and a final 5-min elongation step run at 72 °C. The PCR-mixture composition was similar to that used for the microsatellite analysis.
PCR-products were separated using 90-min 50-V agarosegel electrophoresis (4 % MetaPhorR Agarose, Rockland or 1.6 % LE, Lonza, USA). As the reference markers, 20 bp DNA Ruler (Bio-Rad), 100 kb DNA Ladder (Thermo Fisher Scientific, USA) and 100 bp + 1.5 kb (SibEnzyme, Russia) were applied.
Analysis of the obtained results. PCR-product detection and size measurement was performed using a GelDoc XR+ imaging system (Bio-Rad) and the ImageLab software (Bio- Rad Lab., Inc.) for molecular-mass markers. The obtained results were transformed into a binary matrix, and PopGene v. 1.32 (Yeh et al., 2000) was applied to determine such genetic diversity indices as the effective number of alleles per locus; Shannon’s index; expected heterozygosity; Nei’s genetic distance (Nei, Li, 1979). Polymorphism information content (PIC) for every pair of primers was calculated by the formula presented in the study (Chesnokov, Artemyeva, 2015). To build the genetic similarity dendrogram, the unweighted pair group method with arithmetic averages was applied in NTSYSpc v 2.10 (Rohlf, 2000).
To obtain gDNA from the rapeseed and turnip rape germinants, a modified SDS method was used. The applied protocol proved more effective and less costly compared to other known protocols and commercial reagents kits. The results of electrophoresis and spectrophotometry attested to the DNA’s high concentration and purification degree from protein compounds and polysaccharides for all experimental samples (Fig. 1, 2).
Lanes 1–15 (rape varieties): Severyanin, Stolychniy, VIK 2, Nord, Laureat, Gorizont, Garant, Vikros, Novik, Novosel, Veles, Grant, Podmoskovniy, Lugovskoy, Bizon; 16–18 (turnip rape varieties): Zarya, Nadezhda, Svetlana.
SSR-analysis
For genotyping the full variety collection, out of 42 SSR primers, 7 primers providing stable and reproducible amplification were selected (Table 1).
Analysis of the amplification fragments obtained using the listed primers detected 42 alleles. Their number per locus was 6 on average, varying from 3 (Ni2C12 and Bna.M.010) to 10 (Ra02-E01a). The fragment size varied from 110 bps (Ni2C12) to 1200 bps (Ni02-D08a). The maximum allele frequency was registered for Bna.M.010 (0.83), and the minimum – for Ni03H07a (0.27); the mean value was 0.42. The primers developed for Ni03H07a, Ni02-D08a and Ra02-E01a markers made it possible to detect 8–10 alleles per locus and had the highest PIC (0.82).
SRAP-analysis
Based on the results of preliminary testing, the initial 25 combinations of SRAP primers were reduced to 10 pairs, amplifying stable polymorphic DNA fragments (Table 2). In total, 53 PCR fragments of 132–1674 nucleotide pairs in size were obtained. One combination contained from 4 (F9-R9) to 7 (F10-R8, F11-Em2, F10-R7) amplicons. A part of the markers proved to be informative to detect the amplification fragments for differentiating the type of plants (winter/ spring). Using 6 combinations made it possible to obtain the amplicons specific for varieties identification (marked with a star in the Table 2).
Fig. 3 demonstrates the electrophoregram of PCR results with the F9-R8 primer combination. Significant DNA profile differences were found between winter (I) and spring (II) rapeseed varieties (joined in curly brackets). The arrows mark the variety-specific PCR products characteristic for Stolychniy winter rapeseed (508 bps) and Nadezhda spring turnip rape (700 bps) as well as the absence of an amplicon in size of 460 bps in spring rapeseed Podmoskovniy though it was a specific characteristic for other varieties in this group. The performed analysis demonstrated that it is possible to identify rapeseed varieties Grant and Novosel with 3 marker combinations (F11-Em2, F10-R7 and Me4-R7), and Gorizont and Lugovskoy – with 2 (F13-R9 and Me4-R7). Variety VIK 2 was identified with SRAP primers F9-Em2, and spring ones Veles – with F10-R7. Specific DNA spectra for rapeseed varieties Stolychniy, Podmoskovniy and turnip rape Nadezhda were obtained with F9-R8 combination.
Winter rapeseed varieties: Severyanin (1), Stolychniy (2), VIK 2 (3), Nord (4), Laureat (5), Gorizont (6), Garant (7); spring rapeseed varieties: Vikros (8), Novik (9), Novosel (10), Veles (11), Grant (12), Podmoskovniy (13), Lugovskoy (14), Bizon (15). Winter turnip rape: Zarya (16); spring turnip rape: Nadezhda (17), Svetlana (18). H2O control (19). M – molecular weight marker (100 кb DNA Ladder).
The performed analysis demonstrated that it is possible to identify rapeseed varieties Grant and Novosel with 3 marker combinations (F11-Em2, F10-R7 and Me4-R7), and Gorizont and Lugovskoy – with 2 (F13-R9 and Me4-R7). Variety VIK 2 was identified with SRAP primers F9-Em2, and spring ones Veles – with F10-R7. Specific DNA spectra for rapeseed varieties Stolychniy, Podmoskovniy and turnip rape Nadezhda were obtained with F9-R8 combination
The obtained data were transformed into a binary matrix to calculate Nei’s genetic distances (Table 3). The lowest genetic similarity coefficient (0.7069) was found between rapeseed varieties Gorizont, Novosel and Grant, the highest – between spring varieties Vikros and Bizon (1.0) as well as Veles and Bizon (0.9655). A similarly high genetic distance (0.3228) indicated significant differences between pairs: Grant and VIK 2, and Lugovskoy and Stolychniy. Low distance values and high genetic similarity were demonstrated by spring varieties Bizon and Vikros (zero distance) and winter varieties Garant, Severyanin, Stolychniy, Nord, Laureat (0.0174).
Notе. According to the data of 1 (Rhouma et al., 2017); 2 (Сатина, 2010); 3 (Chandra et al., 2013).
The results of PCR analysis for SSR and SRAP markers were used to determine the genetic variability indices and build an UPGMA dendrogram depicting the varieties’ phylogenetic relationships. The variety material had a low degree of genetic heterogeneity, while higher values of expected heterozygosity (He) and the number of effective alleles (ne) were determined with SSR markers: 0.25 on average against 0.14 and 1.47 per locus if compared to 1.24, respectively. However, the SRAP method has enabled obtaining more PCR products applicable for varietal differentiation (Table 4).
Notе. No. 1–15 – rapeseed varieties Severyanin, Stolychniy, VIK 2, Nord, Laureat, Gorizont, Garant, Vikros, Novik, Novosel, Veles, Grant, Podmoskovniy, Lugovskoy, Bizon.
Analysis of the UPGMA dendrogram demonstrated that the winter/spring rapeseed varieties were divided into two distinguishable clusters (Fig. 4). The first one united such winter cultivars as Severyanin, Garant, Stolychniy, Nord, Laureat, Gorizont, VIK 2; the second – all the spring ones. In the winter cluster VIK 2 and Gorizont were the most distant from the other varieties. The distances between Stolychniy, Nord, Laureat as well as between Garant and Severyanin were much shorter, which was confirmed by their high genetic similarity indices being 0.9655 and 0.9828, respectively (see Table 3). The most distant among spring rapeseed were twozero varieties Novosel, Grant and Lugovskoy, which had the longest genetic distances in the cluster (0.3469 and 0.3228). Bizon and Vikros belonged to one subgroup, sharing a common branch of the dendrogram.
The bulk strategy of DNA sampling from 30 germinants per variety has significantly reduced the labor efforts and cost of the research if compared to the traditional method of individual sample genotyping. The method has proved its efficiency for different cultures especially in large-scale studies of vast populations (Liu et al., 2018). However, this approach is only justified if the analyzed set of samples is representative. For cross-pollinating species with a high level of intrapopulation variations, it should include at least 30–50 plants per variety, which significantly increases the likelihood of registering a rare alleles, the occurrence of which in the population does not exceed 10 % (Crossa, 1989; Semerikov et al., 2002). The plants of winter rapeseed are known for their high self-pollination capacity (up to 70 % of flowers) (Shpaar, 2012), many varieties are linear; while in spring rapeseed this capacity reaches 40 % (Osipova, 1998). That’s why in our study we used budk samples that combined 30 seedlings from each variety.
A significant part of SSR primers tested in our study generated monomorphic amplification fragments. They did not allow us to properly estimate the genetic variability and had low reproducibility in replicated experiments. A proportion of the markers proven effective for intervarietal DNA polymorphism detection comprised 16.7 %, being much lower than in other studies (Plieske, Struss, 2001; Hasan et al., 2008; Tian et al., 2017). It was probably due to the composition of the tested collection that had a narrow genetic basis considering the varieties’ pedigree. At the same time, such parameters of genetic variability as the number of allelic variants, singleallele frequency, PIC and He were comparable to those found in published data (Satina, 2010; Klyachenko et al., 2018).
In general, the used markers made it possible to detect DNA polymorphism between rapeseed and turnip rape as well as between the winter and spring varieties within each species. However, Na12A02 marker turned out to be variety-specific for Bizon winter rapeseed and Zarya spring turnip rape, and Ra02-E01а – for VIK 2 winter rapeseed and Svetlana spring turnip rape. The unique alleles of Podmoskovniy and Lugovskoy rapeseed were detected using Ni02-D08a loci. The indicated markers can be used for varietal DNA identification and genetic certification.
SSR primers for the markers of Indian mustard’s Alternaria blight resistance genes (Chandra et al., 2013), such as Ni02- D08a, Ni03H07a and RA02-E01a, proved to be the most effective. Their application enabled us to detect the specific amplification fragments for linear winter rapeseed variety VIK 2. They also proved effective for Gorizont, which had been obtained on the base of VIK 2 by seed freezing followed by their selection at low-temperature stress. These two varieties share high winter hardiness and are resistant to Alternaria blight. Thereby the results of our study can be useful for further selection of perspective breeding material and QTL analysis on disease resistance.
Among the spring rapeseed, Veles variety turned out to be substantially different while Lugovskoy and Garant had many similarities in the studied microsatellite parts of regions of the genome. Veles is a new perspective variety that has been approved for use since 2021 and was selected based on Vikros using the method of chemical mutagenesis, producing a high frequency of nucleotide changes. This is possibly the reason for Veles having unique alleles in three loci: Ni2C12, Ra02- E01a, Na12A02. For Vikros variety, a specific DNA profile was also obtained with Ni2C12 marker.
Rapeseed Grant was selected using the method of interspecies and intervarietal hybridization of early-maturing foreign breeding samples and the high-yielding varieties Lugovskoy and Vikros, developed at Federal Williams Research Center of Forage Production and Agroecology. Their common origin is probably the reason for the genetic similarity found between Grant and Lugovskoy varieties.
In general, SSR analysis failed to achieve optimum effect in identification of the investigated varieties: from the total set, including 42 primers for microsatellite genome loci, only four were attested as variety-specific for rapeseed, and only one (Ni03H07а) – for Nadezhda spring turnip rape.
For further investigation of DNA polymorphism, SRAP analysis was applied. SRAP is the third generation of molecular markers that were initially designed for the genes of B. oleracea L. (Li, Quiros, 2001) and are successfully used these days for genetic variability estimation and genetic mapping in different plants (Aneja et al., 2012; Rhouma et al., 2017; Liu et al., 2018). This is a cheap, effective and highly reproducible technique
2017; Liu et al., 2018). This is a cheap, effective and highly reproducible technique
The final dendrogram of phylogenetic relations made it possible to visually estimate the degrees of genetic similarities and differences of the studied material. For instance, close placing of such rapeseed varieties as Stolychniy, Nord and Laureat was probably determined by the features of their origin: they were selected for winter hardiness from a combination, in which one of the parental forms was Promin’, a well-known winter rapeseed variety
Garant, selected for winter hardiness, and Severyanin, which was obtained by seed freezing in a climatic chamber and the following individual-family selection, turned out to be in the common subgroup and at a short genetic distance (0.0174) from each other. In addition to high winter hardiness, these varieties are resistant to lodging and to damage by pathogenic fungi
A two-zero spring variety Novosel takes a special position in his group (Nei’s distance is 0.3469). Novosel was developed based on the foreign breeding samples and Russian varieties Lugovskoy and Vikros, characterized by early maturing and high yield. Specific properties of the new breeding achievement are shorter maturation period in comparison to standard varieties and high resistance to Alternaria blight.
Spring rapeseed Bizon and Vikros take the common branch of the dendrogram. The varieties were developed using the method of interspecies hybridization but from different parental forms; characterized by high yield productivity, early maturation and low glucosinolate content.
The presented study has proved the efficiency of SSR and SRAP markers for estimation of DNA polymorphism in rapeseed and turnip rape varieties developed in Federal Williams Research Center of Forage Production and Agroecology. During the study, SRAP technique has demonstrated a higher level of informativity: 36 % of the tested markers were polymorphic, while for the microsatellite loci this rate did not exceed 16.7 %.
Both techniques of molecular analysis enabled detecting the DNA markers for identification of 10 out of 15 rapeseed varieties tested and for 2 turnip rape samples. Microsatellite loci Na12A02, Ni2C12, Ra02-E01 and Ni02-D08a allowed obtaining unique PCR products for Bizon, Veles, Vikros, VIK 2, Podmoskovniy and Lugovskoy rapeseed varieties. Marker Ni03H07а proved effective for identifying Nadezhda turnip rape. In the used SRAP test kit, such primers as F13-R9, Me4- R7, F11-Em2, F10-R7, F9-Em2 and F9-R8 proved effective for detecting variety-specific amplicons or obtaining unique DNA profiles for different types of plants (winter/spring) in rapeseed varieties Grant, Novosel, Gorizont, Stolychniy, Lugovskoy, Podmoskovniy and in spring turnip rape Svetlana.
The results of the study can be used for development of the perspective breeding samples and hybrids, for genetic certification and seed material purity control.
Conflict of interest
The authors declare no conflict of interest.
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Acknowledgments
The presented investigation was supported by the means of the federal budget, directed for performing the government assignment (project No. 0442-2019-0001АААА-А19-119122590053-0).
Contributor Information
I.A. Klimenko, Federal Williams Research Center of Forage Production and Agroecology, Lobnya, Moscow region, Russia .
V.T. Volovik, Federal Williams Research Center of Forage Production and Agroecology, Lobnya, Moscow region, Russia .
A.A. Antonov, Federal Williams Research Center of Forage Production and Agroecology, Lobnya, Moscow region, Russia .
V.A. Dushkin, Federal Williams Research Center of Forage Production and Agroecology, Lobnya, Moscow region, Russia .
A.O. Shamustakimova, Federal Williams Research Center of Forage Production and Agroecology, Lobnya, Moscow region, Russia .
Yu.M. Yu.M. Mavlyutov, Federal Williams Research Center of Forage Production and Agroecology, Lobnya, Moscow region, Russia .
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Peer-reviewed
Research Article
Phenotypic, biochemical and genomic variability in generations of the rapeseed ( Brassica napus L.) mutant lines obtained via chemical mutagenesis
Contributed equally to this work with: Alexandra V. Amosova, Svyatoslav A. Zoshchuk
Roles Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing
* E-mail: [email protected]
Affiliation Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation
Roles Formal analysis, Investigation, Resources, Visualization, Writing – original draft
Affiliation Federal Williams Research Center of Forage Production and Agroecology, Lobnya, Moscow region, Russian Federation
Affiliation Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russian Federation
Roles Formal analysis, Investigation, Visualization, Writing – original draft
Affiliation Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Minsk, Belarus
Roles Formal analysis, Investigation, Visualization
Roles Conceptualization, Validation, Writing – original draft, Writing – review & editing
Roles Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review & editing
Roles Conceptualization, Investigation, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing
- Alexandra V. Amosova,
- Svyatoslav A. Zoshchuk,
- Valentina T. Volovik,
- Anna V. Shirokova,
- Nickolai E. Horuzhiy,
- Galina V. Mozgova,
- Olga Yu. Yurkevich,
- Margarita A. Artyukhova,
- Valentina A. Lemesh,
- Published: August 28, 2019
- https://doi.org/10.1371/journal.pone.0221699
- Reader Comments
The phenotypic, biochemical and genetic variability was studied in M2-M5 generations of ethyl methansulfonat (EMS, 0.2%) mutagenized rapeseed lines generated from canola, ‘00’, B . napus cv. Vikros. EMS mutagenesis induced extensive diversity in morphological and agronomic traits among mutant progeny resulted in selection of EMS populations of B . napus - and B . rapa- morphotypes. The seeds of the obtained mutant lines were high-protein, low in oil and stabilized in contents of main fatty acids which make them useful for feed production. Despite the increased level of various meiotic abnormalities revealed in EMS populations, comparative karyotype analysis and FISH-based visualization of 45S and 5S rDNA indicated a high level of karyotypic stability in M2-M5 plants, and therefore, the obtained mutant lines could be useful in further rapeseed improvement. The revealed structural chromosomal reorganizations in karyotypes of several plants of B . rapa- type indicate that rapeseed breeding by chemical mutagenesis can result in cytogenetic instability in the mutant progeny, and therefore, it should include the karyotype examination. Our findings demonstrate that EMS at low concentrations has great potential in rapeseed improvement.
Citation: Amosova AV, Zoshchuk SA, Volovik VT, Shirokova AV, Horuzhiy NE, Mozgova GV, et al. (2019) Phenotypic, biochemical and genomic variability in generations of the rapeseed ( Brassica napus L.) mutant lines obtained via chemical mutagenesis. PLoS ONE 14(8): e0221699. https://doi.org/10.1371/journal.pone.0221699
Editor: Xiu-Qing Li, Agriculture and Agri-Food Canada, CANADA
Received: February 21, 2019; Accepted: August 13, 2019; Published: August 28, 2019
Copyright: © 2019 Amosova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript.
Funding: This work was supported by the Russian Foundation for Basic Research, No. 17-29-08034ofi_m, to OVM, and the Program of Fundamental Research for State Academies, No. 0120136 3824, to OVM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Rapeseed ( Brassica napus L.) is one of the most economically important crops widely used in different industries as an important source of edible vegetable oil, animal fodder and biodiesel [ 1 , 2 ]. B . napus is considered to be a natural amphidiploid (genome AACC, 2n = 38) originated from spontaneous hybridization between the ancestors of B . rapa L. (AA; 2n = 20) and B . oleracea L. (CC; 2n = 18) followed by diploidization [ 3 – 5 ]. The polyphyletic origin of B . napus has also been confirmed by results of organelle and nuclear RFLP analyses [ 6 ]. Although both B . oleracea and B . rapa have a great diversity of morphotypes with various origins, B . napus is characterized by a relatively narrow genetic diversity [ 7 , 8 ]. Moreover, breeding selection resulted in a decrease of genetic basis of current rapeseed cultivars. Therefore, new genetic sources and approaches are needed to diversify the genetic basis of rapeseed germplasm, which will make the current breeding programs more effective [ 9 , 10 ]. Examples of such approaches may include intraspecific hybridization and a recombinant DNA technology [ 11 , 12 ], creation of synthetic rapeseed lines via artificial crosses between various Brassica species containing A and C genomes [ 13 – 15 ] and also chemical and physical mutagenesis [ 16 – 17 ]. Chemical mutagenesis is an effective and simple method for obtaining valuable starting material that can further be used in crop improvement programs [ 17 , 18 ]. Chemical mutagens (e.g., azide, diethyl sulphate, dimethyl sulphate, ethyl methanesulphonate and N-nitroso compounds) are known to induce non-lethal point DNA mutations at a high rate and create novel genetic diversity in various crops [ 16 , 19 – 22 ]. Particularly, this approach is widely used in rapeseed breeding to produce new cultivars with the desired morpho-agronomic traits and/or biochemical profile which are difficult to obtain though crossbreeding and selection [ 11 , 12 , 23 ].
The fatty acid biosynthesis pathway is a primary metabolic pathway in oil-bearing plants [ 24 ]. Acetyl-CoA is the basic component of the fatty acid chain, involved in the synthesis of 16- or 18-carbon products, which are the major (up to 90%) fatty acids in plants. Various desaturases located in the plastids and the endoplasmic reticulum are responsible for catalyzing these fatty acids to become monounsaturated (palmitoleic acid, C 16:1 , and C 18:1 ) or polyunsaturated ones (C 18:2 and C 18:3 ). The fatty acid composition of the rapeseed oil is the main trait determined its utilization mode and range [ 25 ]. Seeds of the double-low varieties (canola, ‘00’, with very low glucosinolates and erucic acid content) produce oil containing approximately 7% of saturated fatty acids (including palmitic (C 16:0 ) and stearic (C 18:0 )), 61% of the monounsaturated oleic acid (C 18:1 ) and polyunsaturated fatty acids (linoleic (C 18:2 , 20%), linolenic (C 18:3 , 10%) and eicosenoic (C 20:1 , 1%)). This fatty acid composition is considered optimal for nutritional purposes [ 26 ]. However, due to the food- and non-food use of the oil, the demand for rapeseed oils with other fatty acid compositions exists in the market [ 11 , 27 – 29 ].
The investigation of mutant rapeseed genomes is mostly related to the allele polymorphism analysis and mapping of the mutant genes associated to agronomic traits. The content of erucic acid in B . napus is found to be under additive control of alleles of FAE1.1 and FAE1.2 genes encoding the enzyme of erucic acid synthesis, 3-ketoacyl-CoA synthase, from the oleoyl-CoA [ 30 , 31 ]. It was shown that loss of functions of FAE1.2 (C subgenome) and one base pair substitution in FAE1.1 gene (A subgenome) led to formation of canola, ‘00’, plants [ 32 , 33 ]. The content of oleic acid is controlled by the fatty acid desaturase 2 (FAD2) gene that encodes endoplasmic delta-12 fatty acid desaturase 2 (112-FAD2) which converts the precursors of oleic acid to the precursors of linoleic acid in the lipid biosynthetic pathway [ 24 , 34 – 36 ]. Four homologous FAD2 genes (BnFAD2-1, BnFAD2-2, BnFAD2-3, and BnFAD2-4) located separately on rapeseed chromosomes of A and C subgenomes, and their possible role in the rapeseed genome is oleic acid regulation [ 37 , 38 ]. The linolenic acid content in B . napus is controlled by two fatty acid desaturase 3 (FAD3) genes (BnaA.FAD3 and BnaC.FAD3), encoding delta-15 linoleate desaturase which is responsible for dehydration of linoleic acid to linolenic acid [ 39 ]. These genes were detected in the A and C subgenomes of B . napus [ 40 ] and mapped in the N4 (A4) and N14 (C4) linkage groups, correspondingly [ 39 ].
More variability of rapeseed germplasms can be created via mutagenesis [ 23 , 36 , 39 , 41 – 42 ]. At the same time, experimental mutagenesis in allopolyploid B . napus might result in various genetic, chromosomal and genomic reorganizations promoting genetic instability in the progeny. However, in karyotypes of rapeseed mutants, the structure of chromosomes and possible intra- and intergenomic structural rearrangements and substitutions are poorly investigated. Due to small rapeseed chromosomes (1.53–3.30 μm) [ 43 ], the detailed chromosomal analysis is still problematic and needs special approaches, e.g., chromosome elongation with the use of DNA intercalators, application of chromosomal markers allowing identification of individual rapeseed chromosomes and their subgenomic affiliation [ 44 – 47 ]. Comprehensive study of the genotypic variability in mutant rapeseed lines in combination with the karyotype structure analysis (chromosomal complements in A and C subgenomes, the presence of chromosome rearrangements, chromosome substitutions and additions), description of phenotypic and biochemical variability was not performed. Integration of mutation techniques with the molecular, cytogenetic and biochemical analyses provides exciting opportunities for rapeseed breeding. Such approach could be useful in developing reliable tools for improving selection methods and also for introducing novel traits into rapeseed cultivars.
The objectives of the present study were to analyze phenotypic, biochemical and cytogenomic variability in M1-M5 generations of the ethyl methanesulfonate (EMS) mutagenized progeny of the spring canola B . napus cv. Vikros in order to reveal agronomically valuable and genetically stable rapeseed mutant genotypes. The current approach based on the analysis of morphological and agronomic traits, the biochemical profile, SNaPshot detection of mutant and wild-type FAD3 genes, meiosis and FISH localization of 5S and 5S rDNA has been applied.
Materials and methods
Ethics statement.
This study including plant sample collection and experimental research conducted on these materials was according to the federal law on environmental protection approved by the Council of the Russian Federation.
Plant material
Seeds of the spring canola, ‘00’, B . napus cv. Vikros (3480, Russian Federation) were obtained from the germplasm collections of Federal Williams Research Center of Forage Production and Agroecology, Lobnya, Moscow, Russian Federation. Before the mutagenesis assays, the progeny of three succeeding generations (I1-I3) of this B . napus cv. Vikros was tested for hidden effects of inbreeding (self-pollination), and no deviations from the standard characteristics of the original cultivar were revealed. To diversify the genetic basis of the rapeseed germplasm, the seeds of the original cultivar were treated with aqueous solution of ethyl methanesulphonate in concentrations of 0.2% for 16 h. All studied rapeseed plants were grown with the use of pre-grown seedlings: seeds were sowing in the greenhouse followed (30–40 days later) the outdoor planting of at least 50 seedlings. In mutant plants, the leading shoots were isolated at floral initiation stage to obtain self-pollinated seeds. At maturity, seed siliqua were collected from the leading shoots. The identification of the plants was performed according to the Manual of Brassica napus L. [ 48 ]. The progeny selection for morphological and agronomic characters was carried out in М2-М5 plants. Statistical data analysis was performed using standard functions of Microsoft Excel 2013. For each generation, at least 50 plants in every mutant line were analyzed.
Biochemical profile
The biochemical profile was analysed for 20 plants of each mutant line. The fatty acid composition and total oil content were determined in milled seeds (2 g from one plant, 15 plants of each line) using the gas chromatograph Kristall 2000M (Chromateck, Yoshkar Ola, RF) with Zebron ZB-FFAP Capillary GC Column 25m x 0.20mm x 0.30μm (Phenomenex, Torrance, USA) according to the manufacturer’s protocol. The content of protein was estimated photometrically using a biochemical flowing auto analyzer of chemical composition CIAK-K (Kinzh-Agro, Moscow, RF) according to the manufacturer’s protocol. Statistical data analysis was performed using standard functions of Microsoft Excel 2013.
DNA extraction
Total genomic DNA was extracted from green leaves and seedlings using the “Genomic DNA Purification Kit” (Thermo Fisher Scientific, Vilnius, Lithuania) according to the manufacturer’s protocol. The DNA concentration and purification degree were determined using the Implen Nano Photometer NP60 spectrophotometer (Implen, Munich, Germany). Fifteen individual plants of each mutant line were used to estimate the genetic heterogeneity.
Genome-specific PCR
Allelic forms of the B . napus FAD3 genes were identified by PCR amplifications of the gene fragments comprising wild-type and mutation sites followed the detection of the mutant alleles by the microsequencing method (SNaPshot) with locus-specific primers.
Target DNA fragments were amplified in two independent reactions with genome-specific primer pairs FAD3Af/FAD3Ar for the BnaA.FAD3 gene and FAD3Cf/FAD3Cr for the BnaC.FAD3 gene as it was described earlier [ 49 ]. Amplification was carried out on SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, USA) in following conditions: 4 min at 95°C followed by 30 steps with 30 s at 95°C, 30 s at 55°C and 30 s at 72°C, and with the final elongation step for 30 min at 72°C. Reaction mix included 100 ng of genomic DNA template, 1 μM dNTP, 1.5 mM MgCl2, 10x PCR-buffer (650 mM Tris-HCl, 166 mM (NH4)2SO4, 0,2% tween 20, pH 8.8), 0.25 μM each primer, and 1 U Taq polymerase (Primetech, Minsk, Belarus) in a total volume of 25 μl. PCR products were separated by electrophoresis in 1.5% agarose gel with an addition of ethidium bromide solution to a final concentration of 0.5 μg/ml at a voltage of 100 V with the use of 100 bp Plus DNA-ladder (Thermo Scientific, Vilnius, Lithuania). After amplification, post-PCR purification was performed as follows: 5 μl of the PCR product was incubated with 1 U of FAST alkaline phosphatase and 2 U of exoI (Thermo Fisher Scientific, Vilnius, Lithuania) for 1 h at 37°C, followed by 15 min at 80°C for enzyme inactivation.
SNaPshot analysis
The amplified on the first step fragments were used for the detection of FAD3 mutant and wild-type alleles by SNaPshot technique. In the SNaPshot analysis, previously described primers mutA-1f and mutC-45F [ 49 ] modified with a poly-A tail, were used. To discriminate these fragments, the s550 high density size standard for fragment analysis (Synthol, Moscow, RF) was used. Primer extension reactions were carried out independently for FAD3A and FAD3C in a final volume of 10 μl containing 2 μl exoI/FAST treated PCR product (5–50 ng DNA) as a template, 2 μl of the SNaPshot Ready Reaction Mix (Applied Biosystems, Foster City, USA) and 0.2 μM primer. The following amplification protocol was applied: 35 cycles of 10 s at 95°C, 5 s at 50°C and 30 s at 60°C. After the extension reaction, PCR products were treated with FAST alkaline phosphatase (1 unit per sample) for 1 h at 37°C. For electrophoresis, 0.5 μl of the purified primer extension reaction products were combined and mixed with 9 μl of Hi-Di (highly deionized) formamide and 0.5 μl of s550 size standard (Synthol, Moscow, Russia), denatured for 5 min at 95°C and separated by capillary electrophoresis on an ABI Prism 310 Genetic Analyser (Applied Biosystems, Foster City, USA) using POP6 polymer. Alleles of the FAD3A and FAD3C were scored using Gene Mapper 4.1 software (Applied Biosystems, Foster City, USA). The presence of polymorphic alleles was visualized by colour depending on the included in the SNaPshot-PCR product ddNTP which carried the corresponding fluorescent label:
- A–dR6G label–green
- C–dTAMRA label–black
- G–dR 110 label–blue
- T(U)–dROX label–red
Considering that the alleles of FAD3 genes differed from each other by one nucleotide (FAD3A –C; fad3A –T; FAD3C –G; fad3C –A), the following coloured peaks were visualized as a result of SnaPshot-PCR with a single locus-specific forward primer:
- FAD3A (wild-type allele)–black
- fad3A (mutant allele)–red
- FAD3C (wild-type allele)–blue
- fad3C (mutant allele)–green
Chromosome spreads
For chromosome spread preparation, rapeseed root tips (1–0.5 cm) were incubated (16–24 h) in ice-cold water with 1 μg/mL of 9-AMA (Sigma, St. Louis, USA) to inhibit chromosome condensation process and accumulate prometaphase chromosomes [ 50 ]. Then, the roots were treated in ethanol: glacial acetic acid fixative (3:1) for 48 h at room temperature and after that stored at −20°C until use. Chromosome spreads were prepared according to the technique described previously [ 51 ].
For meiotic chromosome preparation, young floral buds (prefoliation) were fixed in ethanol:acetic acid (3:1) fixative for 30 min at 4°C and then chromosome spreads were prepared as previously described [ 51 ]. After freezing in liquid nitrogen, the cover glasses were removed, and the slides were stored in 96% ethanol at −20°C until use.
DNA probe preparation and FISH
Following probes were used for FISH:
- pTa71 containing a 9 kb long repeated DNA sequence of common wheat including 18S- 5.8S-26S rDNA [ 52 ];
- pTa794 containing a 420 bp long repeated DNA sequence of wheat including 5S rDNA [ 53 ].
DNA probes were labelled directly with SpectrumRed or SpectrumAqua fluorochromes (Abbott Molecular, Wiesbaden, Germany) by nick translation according to manufacturer’s protocol. FISH procedure was performed as described previously [ 54 ]. After hybridization (16–20 h), the slides were washed twice with 0.1xSSC at 44 C for 10 min, twice with 2xSSC at 44 C for 5 min followed by a 5-min wash in 2xSSC and three washes in PBS for 3 min each at room temperature. Then the slides were dehydrated through a graded ethanol series and air dried.
DAPI-banding
After the FISH procedure, chromosome slides were stained with 0.1 μg/mL DAPI (4′,6-diamidino-2-phenylindole) (Serva, Heidelberg, Germany) in Vectashield mounting medium (Vector laboratories, Peterborough, UK). DAPI-banding analysis was used as an additional parameter for the identification of individual chromosomes [ 46 , 47 ].
Chromosome analysis
The slides were examined using Olympus BX61 epifluorescence microscope (Olympus, Tokyo, Japan) combined with a monochrome CCD camera (Cool Snap, Roper Scientific Inc., Tucson, USA). The captured images were processed with Adobe Photoshop 10.0 software (Adobe Systems Inc., Birmingham, USA). At least 30 plants of each line and 15 metaphase plates of each plant were analyzed. In karyotypes, the cytological numerical designation of the chromosomes of A and C subgenomes was according to Levan’s criterion [ 55 ]. Additionally, the identification of chromosomes and genome affiliation were performed based on the chromosome morphology, revealed chromosome markers as well as earlier described data [ 46 , 47 , 56 , 57 ]. The meiotic chromosome preparations were analyzed as described previously [ 51 ].
Analysis of pollen
The examination of pollen grains was performed with the use of a scanning electron microscope (SEM) JEOL JSM– 6380LA (accelerating voltage 20 kV, SEI mode) (Jeol, Tokyo, Japan). In each line, pollen grains were collected from six plants (three flowers from the main inflorescence). Fresh pollen was mounted on carbon adhesive tape. The analysis of pollen grains was performed with the use of SEM Control User Interface, Version 7.11 (Jeol, Tokyo, Japan). For each line, ten ocular views (250 x) of pollen grains were analysed. Statistical data analysis was performed using standard functions of Microsoft Excel 2013.
Morphological characterization
Within the M2-M3 progeny, a segregation of morphological traits was found, and plants of B . napus -like and B . rapa -like morphotypes displaying distinct morphological differences were revealed. In M4-M5 generations, the progeny of B . napus -type plants presented constant morphotypes. Within the progeny of the B . rapa -type line, further segregation of morphological traits was observed, and both B . rapa - and B . napus - (up to 12%) morphotypes were detected.
At the stage of the third pair of true leaves, these morphological differences became more evident. Plants of the B . rapa -like morphotype had tender, thin, round and puberulent leaf blades and bright green (non-glaucous) leaves, stems and siliqua ( Fig 1 ). The pubescence disappeared at the stage of the fifth pair of true leaves. Rapeseed-like plants had more coriaceous and smooth leaf blades and glaucous leaves, stems and siliqua ( Fig 1 ).
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Plants of B . napus cv. Vikros (a1), mutant plants of B . rapa -type (b1) and B . napus -type (c1) at the rosette vegetative growth stage; position and shape of siliqua in B . napus cv. Vikros (a2), in plants of B . rapa -type (b2-1) and B . napus -type (c2); a plant of B . rapa -type with a long hypocotyl (b2-2).
https://doi.org/10.1371/journal.pone.0221699.g001
In most mutant plants, we observed moderate decrease in the mean value of plant height compared to the original cultivar. However, this character was highly variable ( Table 1 ). The plants of B . rapa -type had longer hypocotyl ( Fig 1 ) and were more liable to lodging at the stage of early flower bud formation if compared with the original cultivar and rapeseed-type plants. Then, the leading shoot checked in growth, and the plant height in such plants became contingent on first-order shoot development. Basal first-order shoots in B . rapa -type plants, were well-developed and grew subopposite from the hypocotyl (vs. in B . napus -type plants, they were also well-developed but grew from the root neck). In plants of B . rapa -type, shoots III were also observed though side shoots II were less developed compared to B . napus -type ( Table 1 ). Siliqua in B . rapa -like plants were thinner and grew more vertical (vs. in B . napus -type plants, the angle was about 45°) ( Fig 1 ).
https://doi.org/10.1371/journal.pone.0221699.t001
All studied plants had yellow racemose inflorescences. In mutant plants, inflorescences were shorter and few-flowered. In plants of B . rapa -type, flowers were a little smaller and the flower colour was lighter than in rapeseed-like plants ( Fig 2 ). In all studied plants, the pollen grains were tricolpate (typical for Brassicaceae ) ( Fig 2 ). However, more imperfect and/or deformed pollen grains were revealed in plants of B . rapa -type compared to the original cultivar and rapeseed-like plants ( Fig 2 , Table 1 ). Also, in plants of B . rapa -type, the number of seeds per silique was more variable; seeds were red-brown, irregular-shaped and smaller in size; and seed productivity was less if compared with the plants of rapeseed-type ( Fig 2 , Table 1 ).
Inflorescences of B . napus cv. Vikros (a1), plants of B . rapa -type (b1) and B . napus -type (c1); SEM images of pollen grains in B . napus cv. Vikros (a2), in plants of B . rapa -type (b2) and B . napus -type (c2); seeds of B . napus cv. Vikros (a3), plants of B . rapa -type (b3) and B . napus -type (c3). Scale bar– 50 μm.
https://doi.org/10.1371/journal.pone.0221699.g002
Chromosomal structural variations in the EMS populations
In most studied maternal pollen cells of B . napus cv. Vikros, regular meiotic chromosome behavior with normal chromosome disjunction and nineteen bivalents (19 II ) was observed ( Fig 4A ). Besides, in the reduction division, few common meiotic abnormalities were detected. As an example, the occurrence of some chromosomes outside the metaphase spread is shown in Fig 4B . However, the cumulative percentage of these irregularities in maternal pollen cells was nonessential (~1.5%).
(a) A-I, 19 II ; (b) several chromosomes are localized outside the metaphase plate; (c) A-I, chromosome lagging; (d) A-I, chromosomal bridges; (e) M-I, 14 II +2 IV (short arrows)+2 I (long arrows); (f) A-I, chaotic disjunction and chromosome lagging; (g) three-polar configuration with chromatin agglutination; (h) asynchronous division within one meiocyte.
https://doi.org/10.1371/journal.pone.0221699.g004
In both constant and segregated populations of B . napus -type plants, common meiotic abnormalities were detected in 0.15–5.1% of the maternal pollen cells. For instance, chromosome lagging and chromosome bridges at anaphase I are shown in Fig 4C and Fig 4D , correspondingly. Besides, at anaphase II, the spindle function related abnormalities (asynchronous division and lagging) were also detected in maternal pollen cells.
In the B . rapa -type plants, the cumulative percentage of common meiotic abnormalities in maternal pollen cells was ranged from 0.15% to 11.8%. For example, univalents and quadrivalents in the reduction devision (M-1) as well as chaotic disjunction and chromosome lagging at A-I are presented in Fig 4E and Fig 4F , correspondingly.
In one M5 plant of B . rapa -type, multiple meiotic abnormalities (elimination of chromosome groups at anaphase-telophase I, micronuclei in dyads, chromosome elongation and chaotic chromosome distribution at metaphase II, chromatin agglutination, three-polar configurations and asynchronous division within one meiocyte) were revealed in 0.17%– 35.7% of the studied maternal pollen cells. For example, a three-polar configuration with chromatin agglutination and asynchronous division within one meiocyte are shown in Fig 4 g and Fig 4H , correspondingly.
In the original cultivar and most studied M2-M5 plants, rapeseed karyotypes with 2n = 38 chromosomes were observed. The exception was one M5 plant with 2n = 40 chromosomes (Figs 5 and 6 ).
Metaphase plates of B . napus cv. Vikros (A), M2 plant of B . napus -type (B), M3 plant of B . rapa- type (C), M4-4 plant of B . rapa -type (D), M-17 plant of B . napus- type (segregated) (E), M5-3 plant of B . rapa- type (F), M5-2 plant of B . rapa- type (G). The correspondent probes and their pseudo-colours are specified in the upper right-hand corner. DAPI-banding (blue). Scale bar– 5 μm.
https://doi.org/10.1371/journal.pone.0221699.g005
Karyograms of the metaphase plates shown in Fig 5 after DAPI-banding (blue) and FISH with 45S (green) and 5S rDNA (red). Scale bar– 5 μm.
https://doi.org/10.1371/journal.pone.0221699.g006
In karyotypes of the original cultivar, FISH analysis revealed separate 45S rDNA sites in the secondary constriction regions (subtelomere positions of the short arms) of two large chromosome pairs 7 and 8 (C subgenome) and also in the pericentromeric region of one middle-sized chromosome pair 2 (A subgenome). Separate 5S rDNA sites were detected in the pericentromeric and interstitial positions (the long arm) of one large chromosome pair 4 (C subgenome) and in the subtelomere region of the short arm of the smallest chromosome pair 10 (A subgenome). Co-localized 45S and 5S rDNA sites were found in the pericentromeric region of middle-sized chromosome pairs 1, 3 and 4 (A subgenome) and also in the secondary constriction region (subtelomere positions of the short arms) of the pair of a middle-sized chromosome pair 5 (A subgenome) (Figs 5 and 6 ).
In karyotypes of most studied mutant plants, patterns chromosomal distribution of 45S and 5S rDNA were similar to those observed in the original cultivar with the exception of one M3 plant of B . rapa -type having only separate 45S rDNA sites on chromosome pairs 4 (A subgenome); one M5 plant of B . rapa -type with double trisomy (2n = 40) and also one M5 plant of B . rapa -type with a homeologous substitution of one chromosome pair (4) between A and C subgenomes (Figs 5 and 6 ).
EMS is included among the so-called 'supermutagens' which can be used to generate the important recessive and dominant genomic mutations at a high rate and thereby create a basis for useful genetic variations required for plant breeding programs [ 23 , 58 – 59 ]. EMS mutagenesis is an effective approach to create mutations in genes of the polyploid species such as B . napus . These mutagens was found to induce non-lethal point DNA mutations which could be retained in the genome due to its capacity for self-pollination [ 60 ]. These induced genetic variations correlate to variability in agronomic and phenotypic traits in rapeseed mutant populations [ 61 – 63 ]. In the present study, EMS mutagenesis induced extensive morphological diversity among mutant progeny of canola B . napus cv. Vikros. As a result, we could successfully select EMS populations of B . napus - and B . rapa- morphotypes displaying distinct differences in morphological and agronomic traits. Within the progeny of the B . rapa -type line, further segregation of morphological traits was observed indicating that EMS had induced the heterozygous mutations in genomes of B . rapa -type plants, and both B . rapa - and B . napus - (up to 12%) morphotypes were revealed. As it was quite possible that the mutagenesis could result in genotypic differences between constant and segregated populations of B . napus -type, we performed comparative analysis among the EMS populations of different types. Currently, producing short-stem lines is a high-priority task, and due to breeding efforts for growth limitation of lateral meristems in joints of a plant stem, plant height is considered to be an important agronomic trait. The decrease in plant height was described earlier in several EMS mutagenized populations of crops including Brassica species [ 63 ]. In the studied EMS populations of different types, we observed moderate decrease in plant height and also high variability of this feature. These findings indicate that plant height could probably be reduced by a further selection process in the EMS progeny.
Also, plants of B . rapa -type and segregated B . napus -type plants were found to have more extensively developed shoots I and shoots II. Probably, due to high density of plant tillers, poor flowering, lower number of siliqua and lower level of seed productivity were observed in those plants compared to plants of the original cultivar and constant rapeseed-type.
Biochemical analysis showed that seeds of the studied mutant plants were high-protein and low in oil which makes them useful for feed production. Also, seeds of B . rapa- type plants had the highest crude fiber content, but this character was more variable compared to the protein contents in the seeds.
Different fatty acid components of rapeseed oil make it best suited to particular uses. Canola ‘00’ (low erucic acid and low glucosinolate) produces seeds that are used to generate excellent edible oil that is lower in saturated fat and higher in omega-3 fatty acids than most other commercially available oils [ 64 ]. These attributes have been shown to have a significant positive impact on human health, reducing diseases such as cancer, heart disease and some neurological disorders [ 65 , 66 ]. EMS mutagenesis can induce genetic changes in plants and modify the levels of fatty acids in seed oil [ 60 ]. In this study, however, the treatment of canola seeds with EMS at low concentration did not influence the contents of main fatty acids in canola seeds with the exception of a palmitic (C 16:0 ) acid which level was higher compared to the original cultivar. One of the rapeseed breeding goals is to obtain genotypes producing naturally stable oil. Particularly, a low content (≤10%) of the linolenic acid prevents oxidation and rancidification of seed oil which is important for healthy food production [ 60 , 67 ]. Besides, high stability of the oil with low linolenic acid content makes it an important source of raw material for biofuel production. Genetic analyses revealed that the fatty acid composition of rapeseed varied depending on the allelic composition of FAD3 genes as well as the ratio of mutant fad3a, fad3c alleles and FAD3, FAD3A, FAD3C wild-type alleles [ 49 , 67 ]. Moreover, single-nucleotide mutations detected in mutant rapeseed lines resulted in a decrease in the content of linolenic acid in rapeseed oil [ 39 , 49 ]. SNaPshot analysis using SNP markers is an effective approach for detecting mutant alleles of the FAD3 genes in B . napus [ 49 , 67 ]. In the present study, the performed SNaPshot analysis did not detect any single-nucleotide polymorphisms in FAD3 genes in both A and C subgenomes indicating the homozygous state of these genes in the studied lines. Considering also that the original canola cultivar and the plants of B . rapa - and B . napus- morphotypes had related meanings of linolenic (C 18:3 ) fatty acid contents (8–10%), our results showed that mutagenesis did not influence the stability of this essential fatty acid in the obtained mutant lines.
Chemical mutagens can influence the plant genome and cause the meiotic disorders manifested themselves as typical anaphase aberrations (chromosome fragments, bridges, lagging, etc.) as well as fragmentation, nondisjunction, chromosome stickiness and other abnormalities [ 17 , 68 ]. In most studied here maternal pollen cells of the original rapeseed cultivar and mutant lines, normal chromosome disjunction (19:19) was observed. However, typical meiotic abnormalities including chromosome fragments, chaotic chromosome disjunction and lagging at anaphase I; occurrence of some chromosomes outside the metaphase spread and bridges were also revealed. Chromosome nondisjunction, occurred at anaphase I, is considered to be a serious meiotic abnormality which resulted in chromosome loss as well as unequal distribution of genetic material. These disorders could appear due to the paracentric inversions as previously described in tomatoes and Nigella sativa [ 69 , 70 ].
Besides, deviations from the normal bivalent conjugation could be displayed as univalent and multivalent formation at metaphase I stage [ 71 ]. In this study, univalents at diakinesis were also detected in maternal pollen cells of the studied mutant plants. The mutagen-induced univalent formation was supposed to be a result of chromosome structure changes followed by the reduction of chiasma frequency due to restriction of pairing to homologs [ 72 ].
In the original B . napus plants, the cumulative percentage of meiotic irregularities in maternal pollen cells was nonessential (~1.5%). However, the percentage of cells with meiotic disorders was higher in the studied plants rapeseed-type (up to 5.1%) and B . rapa -type (up to 11.8%) compared to the original cultivar. In one M5 plant of B . rapa -type, multiple meiotic abnormalities including elimination of chromosome groups at anaphase-telophase I, micronuclei in dyads, chromosome elongation and chaotic chromosome distribution at metaphase II, chromatin agglutination, three-polar configurations and asynchronous division within one meiocyte were revealed. The analysis of meiotic chromosome behaviour indicated that in plants of the obtained EMS populations, various chromosome rearrangements could occur. Probably, the observed high level of phenotypic variability could also be related to these chromosomal variations. However, zygotes with chromosome abnormalities (appeared due to disorders during meiosis) were shown not always to produce viable seeds, and most meiotic abnormalities were eliminated before the tetrade stage and therefore, do not influence the pollen quality [ 73 ]. However, several meiotic irregularities, such as chromatin agglutination, as well as high level of abberrations (35.7%) revealed in this sample could reduce the quality and fertility of pollen and subsequently, result in reductions in seed yield.
The amphidiploid genome of B . napus consists of closely related A and C subgenomes [ 44 , 74 – 76 ] which display numerous deviations from parental Brassica species additivity [ 77 – 79 ]. Consequently, B . napus is considered to be an important model species to study the processes of genomic reorganizations in recently formed polyploids [ 15 , 80 – 81 ]. The examples of such processes could be different chromosomal rearrangements and intragenomic substitutions observed in natural and resynthesized rapeseed lines which could probably be related to the maintenance of genomic stability [ 15 , 45 ]. Also, it was previously shown that an enhanced genome instability in resynthesized rapeseed lines developed under the pressure of selection resulted in chromosome rearrangements or/and deletions and even elimination of the whole parental genome in hybrids in the succeeding generations [ 47 ]. Besides, intraspecific polymorphism in pattern of chromosomal distribution of 45S and 5S rDNA was previously described for B . napus [ 46 ]. In this study, the molecular cytogenetic analysis of the original B . napus cultivar and obtained mutant lines of B . rapa - and B . napus -morphotypes indicated a high degree of karyotypic stability despite the fact that the cumulative percentage of microsporocytes with meiotic disorders was higher in mutant plants compared to the original cultivar. FISH analysis showed that all the studied karyotypes in B . napus -type plants and most karyotypes in B . rapa -type plants did not differ in chromosome number, morphology and pattern of 45S and 5S rDNA chromosomal distribution from the original cultivar. However, among M3-M5 progeny of B . rapa -type, chromosomal reorganizations including variations in number of 45S and 5S rDNA, trisomy and substitutions between homeological chromosomes were also revealed. It should be noted that the observed chromosomal reorganizations correlated to the higher levels of different meiotic abnormalities, differences in plant morphology and also low seed productivity detected in B . rapa -type progeny, and this could be related to the EMS induced mutations. Different cytogenetical abnormalities induced by EMS mutagenesis were observed earlier in tomatoes and Nigella sativa [ 71 , 72 ]. Our findings demonstrate that rapeseed breeding via chemical mutagenesis could result in cytogenomic instability in the obtained mutant progeny, and therefore, should include karyotype examination.
Thus, molecular cytogenetic analysis of the original B . napus cv. Vikros and its EMS mutagenized progeny indicated that the processes of mutagenesis and also selection for morphological and agronomic traits did not induce changes in chromosomal structure of both constant and segregated mutant lines of B . napus- type, and these mutant lines could be a basis for further rapeseed improvement. The revealed structural chromosomal reorganizations in karyotypes of the mutant plants of B . rapa- type showed that it can be useful for the development of rapeseed forms with trisomy and also chromosome addition/substitution lines. Such aneuploidy lines are important for rapeseed breeding as they provide the opportunity to produce introgression lines and also offer the way to check heterologous gene expression and interaction between recipient genome and donor chromosomes in plants [ 82 – 84 ].
Conclusions
In the present study, EMS mutagenesis induced extensive diversity in morphological and agronomic traits among mutant progeny of canola B . napus cv. Vikros resulted in selection of EMS populations of B . napus - and B . rapa- morphotypes. The obtained unique data on phenotypic, biochemical and cytogenomic variability within these populations showed distinct differences among them. The mutant plants with abnormal karyotypes revealed within the EMS populations indicate that rapeseed breeding by chemical mutagenesis can induce chromosome instability in the mutant progeny, and therefore, it should include karyotype examination. Our findings demonstrate that EMS at low concentrations has great potential in rapeseed improvement.
Acknowledgments
The authors acknowledge Dr. N.N. Kozlov and Dr. V.L. Korovina (Laboratory of Genetic Resources of Fodder Plants, FWRC of Forage Production and Agroecology, Lobnya, Moscow region, RF) for providing us valuable plant material and Dr. A.G. Bogdanov (Laboratory of Electron Microscopy, Faculty of Biology, Lomonosov Moscow State University, Moscow, RF) for SEM technical assistance.
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Mason Creative Writing, coupled with Watershed Lit: Center for Literary Engagement and Publishing Practice, is a center of literary arts and publishing practice, and its graduates emerge as exceptional artists who are prepared for a variety of professional directions. Fiction concentration in the MFA in Creative Writing
George Mason University Virginia, United States Residential program Mason Creative Writing is a community of writers in Northern Virginia that encompasses a three-year residential MFA program and one of just 30 BFA programs in the country, both taught by highly acclaimed faculty.
Mason Creative Writing - GMU, Fairfax, Virginia. 524 likes · 15 talking about this. This is the official page for the creative writing program at George...
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Overview. Within the Creative Writing MFA of George Mason University, students in the Creative Writing program from George Mason University are members of a literary community that includes a student-organized program of readings, potluck dinners with faculty, three journals, a student-run publisher—Stillhouse Press—and the annual Fall for the Book literary festival.
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The data published on this site is licensed under the terms of Creative Commons 4.0 Attribution NonCommercial. ... Program Start date End date; National Security and Defense Council: 727/2022: 2022-10-19: 2032-10-19: National Agency on Corruption Prevention-2022-10-19-Data sources.
Overview Certificate Requirements Applying The Graduate Certificate in Publishing Practice is for current Mason graduate students, as well as current MFA in Creative Writing students, and industry professionals throughout the region who are not yet part of the George Mason University community.
At Mason, James teaches fiction writing, and this semester she is focusing on writing novels in ENGH 608 Novel Writing Seminar. When she came to Mason in 2016, James was mentored by fellow author Courtney Brkic, a professor in Mason's Creative Writing Program. Brkic said her mentorship of James was primarily about balancing teaching with her ...
The second program included an initial 5-min denaturation at 95 °C followed by 39 cycles of 1 min at 94 °C, 2 min at 46-51 °C (depending on the primer pair in use), 2 min at 72 °C and a final 10-min elongation at 72 °C (Chandra et al., 2013). The reproducibility of obtained results was attested in three-fold replication.
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The MFA in creative writing is a three-year residency program offering tracks in fiction, nonfiction, and poetry. Students in the program are members of a literary community that includes a student-organized program of readings, potluck dinners with faculty, three journals, a student-run publisher—Stillhouse Press—and the annual Fall for the Book literary festival.
Given our robust community, Mason Creative Writing is able to offer two paths of potential funding for MFA students: Graduate Teaching Assistantships and Graduate Professional Assistantships. Both GAs are funded with the same benefits and on equal terms. Each year, the MFA Program receives about 130 applications.
The phenotypic, biochemical and genetic variability was studied in M2-M5 generations of ethyl methansulfonat (EMS, 0.2%) mutagenized rapeseed lines generated from canola, '00', B. napus cv. Vikros. EMS mutagenesis induced extensive diversity in morphological and agronomic traits among mutant progeny resulted in selection of EMS populations of B. napus- and B. rapa-morphotypes. The seeds of ...