Acessibilidade / Reportar erro

Population structure and phylogenetic relationships in Brassica rapa L. subspecies by using isozyme markers

Estrutura populacional e relações filogenéticas em subespécies de Brassica rapa L. utilizando marcadores isoenzimáticos

Abstract

The present study aimed to assess population structure and phylogenetic relationships of nine subspecies of Brassica rapa L. represented with thirty-five accessions cover a wide range of species distribution area using isozyme analysis in order to select more diverse accessions as supplementary resources that can be utilized for improvement of B. napus. Enzyme analysis resulted in detecting 14 putative polymorphic loci with 27 alleles. Mean allele frequency 0.04 (rare alleles) was observed in Cat4A and Cat4B in sub species Oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. The highest genetic diversity measures were observed in subspecies dichotoma, accession CR 1585/96 (the highest average of observed (H0) and expected heterozygosity (He), and number of alleles per locus (Ae)). These observations make this accession valuable genetic resource to be included in breeding programs for the improvement of oilseed B. napus. The average fixation index (F) is significantly higher than zero for the analysis accessions indicating a significant deficiency of heteozygosity. The divergence among subspecies indicated very great genetic differentiation (FST = 0.8972) which means that about 90% of genetic diversity is distributed among subspecies, while 10% of the diversity is distributed within subspecies. This coincides with low value of gene flow (Nm = 0.0287). B. rapa ssp. oleifera (turnip rape) and B. rapa ssp. trilocularis (sarson) were grouped under one cluster which coincides with the morphological classification.

Keywords:
Brassica rapa L.; isozyme markers; F-statistics; genetic relationships

Resumo

O presente estudo teve como objetivo avaliar a estrutura populacional e as relações filogenéticas de nove subespécies de Brassica rapa L. representadas com 35 acessos, cobrindo uma ampla gama de áreas de distribuição de espécies usando análise isoenzimática, a fim de selecionar acessos mais diversos como recursos suplementares que podem ser utilizados para melhoria de B. napus. A análise enzimática resultou na detecção de 14 loci polimórficos putativos com 27 alelos. A frequência média de 0,04 alelo (alelos raros) foi observada em Cat4A e Cat4B, nas subespécies Oleifera CR 2204/79 e nas subespécies trilocularis CR 2215/88 e CR 2244/88. As maiores medidas de diversidade genética foram observadas na subespécie dicotômica CR 1585/96 (a média mais alta observada (H0) e heterozigosidade esperada (He) e número de alelos por locus (Ae). Essas observações tornam esse acesso um valioso recurso genético a ser incluído em programas de melhoramento de oleaginosas B. napus. O índice médio de fixação (F) é significativamente maior que 0 para os acessos à análise, indicando uma deficiência significativa de heterozigose. A divergência entre as subespécies indicou uma grande diferenciação genética (FST = 0,8972), o que significa que cerca de 90% da diversidade genética é distribuída entre as subespécies, enquanto 10% da diversidade é distribuída nas subespécies. Isso coincide com o baixo valor do fluxo gênico (Nm = 0,0287). B. rapa ssp. oleifera (nabo) e B. rapa ssp. trilocularis (sarson) foram agrupados conforme a classificação morfológica.

Palavras-chave:
Brassica rapa L.; marcadores de isoenzima; estatística F; relações genéticas

1. Introduction

Brassica rapa L. em. Metzg. (syn. Brassica. campestris L.) belongs to family Brassicaceae (Takuno et al., 2007TAKUNO, S., KAWAHARA, T. and OHNISHI, O., 2007. Phylogenetic relationships among cultivated types of Brassica rapa L. em. Metzg. as revealed by AFLP analysis. Genetic Resources and Crop Evolution, vol. 54, no. 2, pp. 279-285. http://dx.doi.org/10.1007/s10722-005-4260-7.
http://dx.doi.org/10.1007/s10722-005-426...
). It is consisted of various cultivated subspecies including B. rapa ssp. rapa L., B. rapa ssp. oleifera (DC.) Metzg., B. rapa ssp. pekinensis (Lour.) Hanelt, B. rapa ssp. chinensis (L.) Hanelt, subsp. dichotoma (Roxb.) Hanelt, subsp. trilocularis (Roxb.) Hanelt and ssp. Brassica rapa subsp. campestris (L.) A.R.Clapham, Brassica rapa f. biennis (Metzg.) Thell. and Brassica rapa f. annua (Metzg.) Thell. (Stace, 1997STACE, O., 1997. New flora of the british isles. Cambridge: Cambridge University Press.).

It is thought that B. rapa L. is originated in the mountainous areas near the Mediterranean Sea (Tsunoda, 1980TSUNODA, S., 1980, Eco-physiology of wild and cultivated forms in Brassica and allied genera. In: S. TSUNODA, K. Hinata, and C. Gómez-Campo, eds. Brassica crops and wild allies. Tokyo: Japan Scientific Societies Press, pp. 109-120.). However, the time of domestication is unknown. There are two main centres of origin for B. rapa L.: one is the Mediterranean and the other is the Afghanistan-Pakistan region (Sinskaia, 1928SINSKAIA, E.N., 1928. The oleiferous plants and root crops of the family Cruciferae Bull, Bulletin of Applied Botany. Genetics and Plant Breeding, vol. 19, no. 1, pp. 1-648.).

B. rapa, with the exception of the Indian yellow sarson form (subspecies trilocularis (Roxb.) Hanelt), is an obligate outcrosser due to the presence of self-incompatible genes (Kimber and McGregor, 1995KIMBER, D.S. and MCGREGOR, D.I. 1995. The species and their origin, cultivation and world production. In: D. S. KIMBER, and D. I. MCGREGOR, ed. Brassica oilseeds - production and utilization. Oxon, UK: CAB International, pp. 1-8.).

Subspecies of B. rapa L. species are widely cultivated as leaf and root vegetables, fodder and oilseed crops. In addition, they can be a weed of cultivated land and disturbed sites (Shahzadi et al., 2015SHAHZADI, T., KHAN, F.A., ZAFAR, F., ISMAIL, A., AMIN, E. and RIAZ, S., 2015. An overview of Brassica species for crop improvement. American-Eurasian Journal of Agricultural & Environmental Sciences, vol. 15, no. 8, pp. 1568-1573.). Some of the plants of B. rapa L. subspecies includes both annual and biennials forms, e.g. subsp. Chinensis, subsp. oleifera and B. rapa subsp. rapa L.; others are biennials e.g. subsp. pekinensis.

Genetic diversity is essential for long-term survival in time and in place as it supplies the species by the plasticity to cope with the new conditions brought about by the environment. It has a leading role in competition, symbiosis, parasitism, and the impact of climate and the effect of deficiencies (Tanhuanpää et al., 2016TANHUANPÄÄ, P., ERKKILÄ, M., TENHOLA-ROININEN, T., TANSKANEN, J. and MANNINEN, O., 2016. SNP diversity within and among Brassica rapa accessions reveals no geographic differentiation. Genome, vol. 59, no. 1, pp. 11-21. http://dx.doi.org/10.1139/gen-2015-0118. PMid:26694015.
http://dx.doi.org/10.1139/gen-2015-0118...
). The knowledge of the genetic diversity of plant genetic resources is essential for saving the present genetic diversity and for subsequent utilization in crop improvement. In Brassica genetic diversity study is major requirement for success in plant breeding and crop improvement (Shahzadi et al., 2015SHAHZADI, T., KHAN, F.A., ZAFAR, F., ISMAIL, A., AMIN, E. and RIAZ, S., 2015. An overview of Brassica species for crop improvement. American-Eurasian Journal of Agricultural & Environmental Sciences, vol. 15, no. 8, pp. 1568-1573.). Knowledge of genetic diversity in Brassica gene pool may be a valuable genetic source to overcome obstacles in genetic improvement (Bird et al., 2017BIRD, K.A., AN, H., GAZAVE, E., GORE, M.A., PIRES, J.C., ROBERTSON, L.D. and LABATE, J.A., 2017. Population Structure and Phylogenetic Relationships in a Diverse Panel of Brassica rapa L. Frontiers in Plant Science, vol. 8, no. 321.). Brassica rapa L is one of the valuable crop species in Brassicaceae family. It is known by its common names field mustard, turnip, and/or Chinese cabbage. It is characterized by its broader global distribution than most of other Brassica species (OECD, 2016ORGANIZATION FOR ECONOMIC COOPERATION AND DEVELOPMENT – OECD, 2016. Safety assessment of transgenic organisms in the environment. Paris: OECD Publishing. OECD Consensus Documents. Harmonisation of Regulatory Oversight in Biotechnology, vol. 5.). The assessment of genetic distances within B. rapa L. may help to broaden the genetic diversity in B. napus since it suffered a series of bottlenecks during its development and has reduced genetic diversity (Becker et al., 1995BECKER, H.C., ENGQVIST, G.M. and KARLSSON, B., 1995. Comparison of rapeseed cultivars and re-synthesized lines based on allozyme and RFLP markers. Theoretical and Applied Genetics, vol. 91, no. 1, pp. 62-67. http://dx.doi.org/10.1007/BF00220859. PMid:24169668.
http://dx.doi.org/10.1007/BF00220859...
; Cowling, 2007COWLING, W.A., 2007. Genetic diversity in Australian canola and implications for crop breeding for changing future environments. Field Crops Research, vol. 104, no. 1-3, pp. 103-111. http://dx.doi.org/10.1016/j.fcr.2006.12.014.
http://dx.doi.org/10.1016/j.fcr.2006.12....
). Moreover, landraces of B. rapa L. are adapted to a broad range of environments, e.g. cold or high temperatures, across a very wide geographic area (Dixon, 2007DIXON, G. R., 2007. Vegetable brassicas and related crucifers. United Kingdom: CABI. Crop Production Science in Horticulture Series, no. 14.) which means that they contain valuable genetic traits which can be used for B. napus improvement (Annisa et al., 2011ANNISA, GUO, Y., CHEN, S. and COWLING, W., 2011. Global genetic diversity of Brassica rapa. In: Proceedings of the 17th Australian Research Assembly on Brassicas (ARAB), 2011, Wagga Wagga, NSW. Australia: AOF, pp. 17-19.).

The aim of the present study was to assess of genetic diversity and population structure of Brassica rapa L. subspecies in order to select more diverse accessions as Supplementary Material resources that can be utilized for improvement of B. napus.

2. Material and Methods

2.1. Biological Material

Thirty- five accessions of B. rapa L. were obtained as donation from IPK gene bank Gatersleben Germany. The accessions belong to nine subspecies of B. rapa L. The represented subspecies are: chinensis, pekinensis, rapa, oleifera f. annua (annual turnip rape, summer turnip rape), Brassica rapa subsp. campestris (L.), oleifera f. biennis ((biennial turnip rape, winter turnip rape), dichotoma, oleifera, trilocularis. The origin and the accession number of these accessions are recorded as shown in Table 1.

Table 1
Subspecies names, accession code and origin of 35 accessions of Brassica rapa subspecies.

2.2. Isozymes extraction, electrophoresis and staining

The seeds of the studied accessions were surface sterilized in 70% v/v ethanol for 1 min before germination at 25ºC in sterilized Petri dishes with three moist filter papers. Three-day-old seedlings of each accession were macerated in 5 ml saline solution containing 0.8% NaCl and 0.2% NaNO3, then centrifuged at 12000 rpm for 15 minutes. Supernatants were collected in pre-chilled tubes and stored at -20ºC until use for electrophoretic separation of isozymes.

15 µL clear supernatants were mixed with equal volumes of loading buffer (50% glycerol containing 1% bromophenol blue), then applied directly on 7% vertical PAGE at 4°C in a Mini Protean III unit (BioRad, USA) according to the method of Manchenko (1994)MANCHENKO, G., 1994. Handbook of detection of enzymes on electrophoretic gels. Florida: CRC Press, 334 p.. Electrophoresis was carried out at 15 mA/gel for 60 min.

The electrophoresed gels were stained for acid phosphatase, catalase, α-esterase and peroxidase (Pasteur et al., 1988PASTEUR, N., PASTEUR, G., BONHOMME, F., CATALAN, J., and BRITTON-DAVIDSON, J., 1988. Practical isozyme genetics. England: Ellis Horwood Ltd.). The gels of acid phosphatase were incubated in 100 ml solution of 0.1 M sodium phosphate buffer, pH 5.1, at 37 0C for 3 to 5 h, then stained in solution formed of 10 mM I2 mixed with 14 mM KI developing white bands on a dark blue background. The chromatic or light brown bands appeared at the bottom of the gels were amylase bands. The gels of catalase were stained by immersing in 1:1 mixture of solutions 2% potassium ferricyanide and 2% ferric chloride after incubation in a solution of 3% H2O2 for about 15 min. The gels were then washed and gently agitated for a few minutes in water. Yellow bands of catalase activity appeared on a blue-green background. The gels of α-esterases were incubated at 37°C for 15 min in 100 ml staining solution consisted of 0.05 M phosphate buffer, pH 7.2, containing 1% α-naphthyl acetate and 50 mg Fast Blue RR until brown colored bands appeared. The stained gels were photographed as quickly as possible and stored in 3% acetic acid. The gels of peroxidase were incubated in 100 ml 0.05 M acetate buffer (pH 5.0 containing 65 mg benzidine dissolved in 1ml of ethanol. 2 ml of 0.1 M CaCl2 were added as co-enzyme. Finally, 2 ml of H2O2 were added as a substrate and incubated in refrigerator until dark brown bands appeared. Stained gels were washed with distilled water and fix in 50% glycerol (Soltis et al., 1983SOLTIS, D.E., HAUFLER, C.H., DARROW, D.C. and GASTONY, G.T., 1983. Starch gel electrophoresis of ferns: a compilation of grinding buffers, gel and electrode buffers and staining schedules. American Fern Journal, vol. 73, pp. 9-27.). At least 5 and generally 10 plants per accession were examined for isozyme patterns.

2.3. Data analysis

The data of isozymes were analyzed using POPGENE version 1.31 Microsoft Window-based Freeware for Population Genetic Analysis. The construction of a dendrogram was made based on Nei’s genetic distance using UPGMA (Nei, 1978NEI, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, vol. 89, no. 3, pp. 583-590. PMid:17248844.). The banding patterns were first encoded using Microsoft Excel for easier editing before being transformed into a POPGENE data file.

3. Results

3.1. Loci and alleles scored

Enzyme electrophoresis resulted in detecting 14 putative polymorphic loci with 27 alleles. The mean allele frequency ranged 0.04 in loci Cat4A and Cat4B to 0.62 in Per1B. The alleles having frequency 0.04 were termed rare alleles (as shown in Supplementary Table 1). These alleles were observed for Cat4A and Cat4B in sub species Oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. It also showed that the total number of alleles in each accession for the 14 loci ranged from 4 in subspecies chinensis accession RA 1637/94”K89 and subspecies rapa accessions BRA 337/79, BRA 1116/99 and BRA 1224/87 to 17 in subspecies chinensis accession BRA 116/ 80 with a mean of 6.8 (total = 27). The percent of polymorphic loci varied from 0.29 in accession RA 1637/94”K89 of subspecies chinensis and accessions BRA 337/79, BRA 1116/99 and BRA 1224/87 of subspecies rapa L. to 1.21 in accession BRA 116/ 80 of subspecies chinensis (as shown in Supplementary Table 1).

3.2. Genetic diversity and accession-level homozygosity

The mean number of alleles per locus (A) and the effective number of alleles per locus (Ae) varied respectively from 1.13 in subspecies chinensis, accession BRA 1014/85 to 1.63 in subspecies rapa, accession BRA 1321/91 with a mean of 1.35 and from 1.13 in subspecies chinensis, accession BRA 1014/85 to 1.75 in subspecies dichotoma, accession CR 1585/96 with a mean of 1.281 (as shown in Table 2).

Table 2
Accessions of Brassica rapa subspecies, mean allele number per locus (A), mean of effective allele number per locus (Ae), observed heterozygosity (Ho), expected heterozygosity (He), average fixation index (F), number of loci with a significant excess heterozygosity (HE), number of loci with a significant deficiency of heteozygosity (HD), non significant inbreeding coefficients (NS).

The average of observed heterozygosity (Ho) was 0.31, ranging from 0.09 (sub species chinensis accession K 9420/95, subspecies rapa accessions BRA 1116/99, subspecies Brassica rapa subsp. campestris (L.) accession CR 1578/93) to 1.75 (subspecies dichotoma, accession CR 1585/96) and the average of expected heterozygosity (He) was 0.24 ranging from 0.09 in sub species chinensis accession K 9420/95, subspecies rapa accessions BRA 1116/99 and subspecies campestris (L.) accession CR 1578/93 to 1.75 in subspecies dichotoma, accession CR 1585/96 (as shown in Table 2).

The average fixation index (F) is significantly higher than zero for the analysis accessions (as shown in Table 2) indicating a significant deficiency of heteozygosity. In general, it was found that the number of loci with significant deficiency of heterozygosity for all accessions was extremely higher than the number of loci with excessive heterozygosity. The non significant inbreeding coefficients (NS) were ranged between 1.00 in subspecies dichotoma accession CR 1585/96 to 7.00 in sub species oleifera biennis accessions CR 289/84 & CR 1586/87and subspecies Oleifera accession CR 2203/79 (as shown in Table 2).

3.3. Genetic structure and gene flow

The mean breeding index was significantly higher than zero (FIT = 0.8228), which means that there is excess of average heterozygotes in the studied accessions. A high and significant value was obtained for FST (The coefficient of genetic differentiation) with mean = (0.8972) suggesting the occurrence of random mating system for the studied accessions (as shown in Table 3). The mean value of FIS all over the analyzed loci was -0.7235 confirming the self- incompatibility of B. rapa. The estimate of gene flow based on Wright (1951)WRIGHT, S., 1951. The genetical structure of populations. Annals of Eugenics, vol. 15, pp. 323-354. equation was very low: NmW = 0.0287 which explained the low average of observed heterozygosity (0.31) and expected heterozygosity (0.24).

Table 3
Genetic divergence measures and gene flow of the studied accessions of Brassica rapa subspecies.

3.4. Genetic distance and dendrogram

Dendrogram constructed based on isozyme showed four main clusters at Nei’s genetic distance 0.60 (see Figure 1). Accessions of subspecies oleifera and trilocularis were collected with each other in cluster (C2). The accessions of subspecies oleifera biennis and oleifera annua were separated in clusters (C1) and (C2) respectively. The accessions of other subspecies (chinensis, pekinensis, rapa, dichotomy, campestris L.) were clustered in two clusters each.

Figure 1
UPGMA dendrogram showing the relationships among the 35 accessions of the studied accessions of Brassica rapa subspecies.

The matrix of eigenvectors and values of the principle components (PCs) resulting from the interaction of the isozyme data influenced 54.93% of the variability accumulated up to the first four components of the PCA (as shown Table 4). The accessions of subspecies rapa, dichotoma, oleifera and trilocularis were separated on PC1 and the accessions of oleifera biennis on PC2. The other accessions were separated on more than one access.

Table 4
Sub species of Brassica rapa, accessions numbers and values of the principal components for isoyme data.

4. Discussion

The surprising phenotypic diversity of B.rapa has made it a precious multi-use crop for food, fodder, and oil. Although the complex domestication history and intense selective breeding of B. rapa had an effective role in generating and shaping this diversity, they highly complicated the elucidation of population structure and evolutionary relationships in the species. With using isozyme data, we provide a more detailed analysis of population structure and the relationships of B. rapa subspecies.

4.1. Loci and alleles scored

The alleles with low mean allele frequency (0.04) or what were known as rare alleles were observed in Cat4A and Cat4B in sub species oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. The presence of this allele could be due to deleterious mutations or may be due to evolutionary relics (Sammour et al., 2019SAMMOUR, R.H., FAHMEE, S., MUSTAFA, A.-E. and TAHER, W., 2019. Isozyme analysis of genetic variability and population structure of Lathyrus sativus L. germplasm. Legume Research, vol. 42, no. 3, pp. 1-7. http://dx.doi.org/10.18805/LR-441.
http://dx.doi.org/10.18805/LR-441...
). The detection of rare allele in combination with low allelic frequency of other loci leads to the conclusion that the studied accessions had narrow genetic differentiation.

Although the studied accessions except the accessions of subspecies trilocularis are an obligate outcrosser due to the presence of self-incompatible genes, the mean allele frequency in general was low. This was attributed to breakdown and mismatch of the differently adapted gene complexes or what is known outbreeding depression. The great variation in allele frequency in studied subspecies could be attributed to these subspecies include many varieties or forms or cultivars. The selected forms of some subspecies e.g. chinensis exhibited significantly different morphological traits which made early botanists classified them as separate species (OECD, 2016ORGANIZATION FOR ECONOMIC COOPERATION AND DEVELOPMENT – OECD, 2016. Safety assessment of transgenic organisms in the environment. Paris: OECD Publishing. OECD Consensus Documents. Harmonisation of Regulatory Oversight in Biotechnology, vol. 5.).

4.2. Genetic diversity and accession-level homozygosity

There was wide variation in mean number of allele per locus (A) and effective number of allele per locus (Ae). Such wide variation was reported by Karam et al. (2014)KARAM, M.A., YASSER, S., MORSI, Y.S., SAMMOUR, R.H. and ALI, R.M., 2014. Assessment of genetic relationships within Brassica rapa subspecies based on polymorphism. International Journal of Current Microbiology and Applied Sciences, vol. 3, no. 3, pp. 1-10.. However, the subspecies which showed low and high number of allele per locus (A) and effective number of allele per locus in Karam et al. (2014)KARAM, M.A., YASSER, S., MORSI, Y.S., SAMMOUR, R.H. and ALI, R.M., 2014. Assessment of genetic relationships within Brassica rapa subspecies based on polymorphism. International Journal of Current Microbiology and Applied Sciences, vol. 3, no. 3, pp. 1-10. were different from that observed in the present study. This might be attributed to that Karam et al. (2014)KARAM, M.A., YASSER, S., MORSI, Y.S., SAMMOUR, R.H. and ALI, R.M., 2014. Assessment of genetic relationships within Brassica rapa subspecies based on polymorphism. International Journal of Current Microbiology and Applied Sciences, vol. 3, no. 3, pp. 1-10. carried out their study at population level and we carried out study at accessions level.

Low average of observed heterozygosity (Ho) and expected heterozygosity (He) which observed in sub species chinensis (accession K 9420/95), subspecies rapa (accessions BRA 1116/99) and subspecies campestris (L.) (accession CR 1578/93) might be attributed to these accessions at the edge of the subspecies distribution centre; the position at which population sizes gradually decrease as does genetic diversity or the origin of these accession could be associated with some sort of bottleneck event followed by the absence of inter-population gene flow (Meeus et al., 2012MEEUS, S., HONNAY, O. and JACQUEMYN, H., 2012. Strong differences in genetic structure across disjunct, edge, and core populations of the distylous forest herb Pulmonaria officinalis (Boraginaceae). American Journal of Botany, vol. 99, no. 11, pp. 1809-1818. http://dx.doi.org/10.3732/ajb.1200223. PMid:23092991.
http://dx.doi.org/10.3732/ajb.1200223...
; Surina et al., 2014SURINA, B., SCHNEEWEISS, G.M., GLASNOVIĆ, P. and SCHÖNSWETTER, P., 2014. Testing the efficiency of nested barriers to dispersal in the Mediterranean high mountain plant Edraianthus graminifolius (campanulaceae). Molecular Ecology, vol. 23, pp. 2861-2875.; Radosavljevic et al., 2015RADOSAVLJEVIC, I., SATOVIC, Z. and LIBER, Z., 2015. Causes and consequences of contrasting genetic structure in sympatrically growing and closely related species. AoB Plants, vol. 7, pp. plv106. http://dx.doi.org/10.1093/aobpla/plv106. PMid:26333826.
http://dx.doi.org/10.1093/aobpla/plv106...
).

The highest average of observed and expected heterozygosity, and the highest effective number of alleles per locus (Ae) were observed in subspecies dichotoma, accession CR 1585/96 from Canada. The variation in genetic structure between subspecies dichotoma, accession CR 1585/96 from Canada and other accessions of subspecies dichotoma from Asia could be due to Asian accessions were subjected to long term selection which narrow their genetic variation or/and the variation of geographical position of the accessions within subspecies distribution range or severe effects of small population sizes (Radosavljevic et al., 2015RADOSAVLJEVIC, I., SATOVIC, Z. and LIBER, Z., 2015. Causes and consequences of contrasting genetic structure in sympatrically growing and closely related species. AoB Plants, vol. 7, pp. plv106. http://dx.doi.org/10.1093/aobpla/plv106. PMid:26333826.
http://dx.doi.org/10.1093/aobpla/plv106...
).

The highest genetic diversity measures were observed in subspecies dichotoma, accession CR 1585/96 (the highest average of observed and expected heterozygosity, and number of alleles per locus (Ae)), These observations make subspecies dichotoma, accession CR 1585/96 valuable genetic resources to be included in breeding programs for the improvement of oilseed B. napus.

4.3. The population structure and gene flow

The population structure and gene flow were analyzed in the term of F statistic. Genetic divergence was quantified by computing F statistic as an indicator for genetic diversity and gene flow among subspecies. The inbreeding coefficient of the individuals within each subspecies was relatively low (FIS = -0.7235) which agreed with the self- incompatibility of B. rapa.

The coefficient of genetic differentiation (FST) was 0.8972 which was considered very great based on the guidelines suggested by Wright (1978)WRIGHT, S., 1978. Evolution and the genetics of populations. Chicago: University of Chicago Press. Variability within and among natural populations, vol. 4.. This guideline considered FST ranges 0.0 to 0.05, 0.05 to 0.15, 0.15 to 0.25 and above 0.25 as indicator for little, moderate, great and very great genetic differentiation respectively. The divergence among subspecies indicated very great genetic differentiation (FST = 0.8972) which means that about 90% of genetic diversity is distributed among subspecies, while 10% of the diversity is distributed within subspecies. This was coincided with low value of gene flow (Nm = 0.0287).

Although Brassica rapa was classified as obligate self-incompatible (Koch and Al-Shehbaz, 2009KOCH, M.A. and AL-SHEHBAZ, I.A. 2009. Molecular systematics and evolution. In: S. K. GUPTA, ed. Biology and breeding of crucifers. Boca Raton: Taylor & Francis Group, pp. 1-18. http://dx.doi.org/10.1201/9781420086096.ch1.
http://dx.doi.org/10.1201/9781420086096....
) the inbreeding coefficient of the individuals in the entire studied populations (within all subspecies) was relatively high (FIT = 0.8228). This can be attributed to the geographic isolation of the individuals of the studied subspecies. As a result of ongoing breeding depending on local preferences in different parts of the world, B. rapa has been undergone selection that increased genetic variation within the species (Gomez Campo, 1999GOMEZ CAMPO, C., 1999. Developments in plant genetics and breeding. Amsterdam: Elsevier. Biology of Brassica coenospecies, vol. 4. ; Koch and Al-Shehbaz, 2009KOCH, M.A. and AL-SHEHBAZ, I.A. 2009. Molecular systematics and evolution. In: S. K. GUPTA, ed. Biology and breeding of crucifers. Boca Raton: Taylor & Francis Group, pp. 1-18. http://dx.doi.org/10.1201/9781420086096.ch1.
http://dx.doi.org/10.1201/9781420086096....
). Variation in genetic structure was observed among accessions of the same subspecies and was attributed to severe selection, domestication and low gene flow (Snowdon et al., 2007SNOWDON, R., LÜHS, W. and FRIEDT, W., 2007. Brassica oilseeds. In: R. J. SINGH, ed. Genetic resources, chromosome engineering and crop improvement: oilseed crops. Boca Raton: Taylor & Francis Group, pp. 195-230.).

4.4. The relationships among B. rapa subspecies

The subspecies were differentiated at Nei s genetic distance 0.60 into four main clusters, each cluster contained more than a subspecies confirming that Brassica rapa had a polyphyletic origin (Tanhuanpää et al., 2016TANHUANPÄÄ, P., ERKKILÄ, M., TENHOLA-ROININEN, T., TANSKANEN, J. and MANNINEN, O., 2016. SNP diversity within and among Brassica rapa accessions reveals no geographic differentiation. Genome, vol. 59, no. 1, pp. 11-21. http://dx.doi.org/10.1139/gen-2015-0118. PMid:26694015.
http://dx.doi.org/10.1139/gen-2015-0118...
). The accessions of each of oleifera, oleifera annua, oleifera biennis, trilocularis were collected with each other in a specific cluster. This could be due to the accessions of each of these subspecies collected from same sub-geographic region, which did not have a high fluctuation in environmental and ecological factors: the factors that may cause change/modifications in isozymes markers (Horáček et al., 2009HORÁČEK, J., GRIGA, M., SMÝKAL, P., and HÝBL, M., 2009. Effect of environmental and genetic factors on the stability of pea (Pisum sativum L.) isozyme and DNA markers. Czech Journal of Genetics and Plant Breeding, vol. 45, no. 2, pp. 57-71.). Conversely, the accessions of other subspecies were distributed between two clusters exhibiting a wide variation in genetic structure, e.g. subspecies chinensis, pekinensis, rapa, dichotomy, campestris L. The variation in the genetic structure of these subspecies based on isozyme analysis coincides the distinct variation of the morphotypes of these subspecies (OECD, 2016ORGANIZATION FOR ECONOMIC COOPERATION AND DEVELOPMENT – OECD, 2016. Safety assessment of transgenic organisms in the environment. Paris: OECD Publishing. OECD Consensus Documents. Harmonisation of Regulatory Oversight in Biotechnology, vol. 5.).

It was reported B. rapa L. classified into two main groups based on morphological and restriction fragment length polymorphism (RFLP) markers: one group consists of ssp. rapa and ssp. oleifera in Europe and another is the group of leafy vegetables, such as ssp. pekinensis, ssp. chinensis and ssp. nipposinica in East Asia (Takuno et al., 2007TAKUNO, S., KAWAHARA, T. and OHNISHI, O., 2007. Phylogenetic relationships among cultivated types of Brassica rapa L. em. Metzg. as revealed by AFLP analysis. Genetic Resources and Crop Evolution, vol. 54, no. 2, pp. 279-285. http://dx.doi.org/10.1007/s10722-005-4260-7.
http://dx.doi.org/10.1007/s10722-005-426...
). However, our results based on isozyme analysis did not support this classification. The discrepancy between our results and Takuno et al. (2007)TAKUNO, S., KAWAHARA, T. and OHNISHI, O., 2007. Phylogenetic relationships among cultivated types of Brassica rapa L. em. Metzg. as revealed by AFLP analysis. Genetic Resources and Crop Evolution, vol. 54, no. 2, pp. 279-285. http://dx.doi.org/10.1007/s10722-005-4260-7.
http://dx.doi.org/10.1007/s10722-005-426...
conclusion was attributed to Takuno et al. (2007)TAKUNO, S., KAWAHARA, T. and OHNISHI, O., 2007. Phylogenetic relationships among cultivated types of Brassica rapa L. em. Metzg. as revealed by AFLP analysis. Genetic Resources and Crop Evolution, vol. 54, no. 2, pp. 279-285. http://dx.doi.org/10.1007/s10722-005-4260-7.
http://dx.doi.org/10.1007/s10722-005-426...
did not include ssp. rapa and ssp. oleifera from East Asia in their studies (Tsunoda, 1980TSUNODA, S., 1980, Eco-physiology of wild and cultivated forms in Brassica and allied genera. In: S. TSUNODA, K. Hinata, and C. Gómez-Campo, eds. Brassica crops and wild allies. Tokyo: Japan Scientific Societies Press, pp. 109-120.).

B. rapa ssp. oleifera (turnip rape) and B. rapa ssp. trilocularis (sarson) were grouped in one cluster. Similar relationship was observed by Song et al. (1991)SONG, K.M., SUSUKI, J.Y. and SLOCUM, M.K., 1991. A linkage map of Brassica rapa (syn. B. campestris) based on restriction fragment length polymorphism loci. Theoretical and Applied Genetics, vol. 82, pp. 296-304. based on RFLP and Karam et al. (2014)KARAM, M.A., YASSER, S., MORSI, Y.S., SAMMOUR, R.H. and ALI, R.M., 2014. Assessment of genetic relationships within Brassica rapa subspecies based on polymorphism. International Journal of Current Microbiology and Applied Sciences, vol. 3, no. 3, pp. 1-10. based on isozyme. The grouping of these two subspecies coincides with the morphological classification suggested by Inaba and Nishio (2002)INABA, R. and NISHIO, T., 2002. Phylogenetic analysis of the Brassiceae based on the nucleotide sequences of the S-locus related gene SLR1. Theoretical and Applied Genetics, vol. 105, no. 6, pp. 1159-1165. http://dx.doi.org/10.1007/s00122-002-0968-3. PMid:12582894.
http://dx.doi.org/10.1007/s00122-002-096...
that sarson had been derived from turnip rape and was selected and developed in India.

The separation of dichotoma and trilocularis with each other on PC1 confirmed the suggestion of Bird et al. (2017)BIRD, K.A., AN, H., GAZAVE, E., GORE, M.A., PIRES, J.C., ROBERTSON, L.D. and LABATE, J.A., 2017. Population Structure and Phylogenetic Relationships in a Diverse Panel of Brassica rapa L. Frontiers in Plant Science, vol. 8, no. 321. for collecting of these two subspecies in a separate subpopulation by using a high-throughput GBS method that leverages next-generation sequencing and multiplexing of RRLs. However, the suggestion of Bird et al. (2017)BIRD, K.A., AN, H., GAZAVE, E., GORE, M.A., PIRES, J.C., ROBERTSON, L.D. and LABATE, J.A., 2017. Population Structure and Phylogenetic Relationships in a Diverse Panel of Brassica rapa L. Frontiers in Plant Science, vol. 8, no. 321. to assign each of subspecies chinensis and pekinensis to a subpopulation, since these two species had many morphotypes, great variation in genetic structure based on isozyme (Karam et al., 2014KARAM, M.A., YASSER, S., MORSI, Y.S., SAMMOUR, R.H. and ALI, R.M., 2014. Assessment of genetic relationships within Brassica rapa subspecies based on polymorphism. International Journal of Current Microbiology and Applied Sciences, vol. 3, no. 3, pp. 1-10.) and RFLP analysis (Takuno et al., 2007TAKUNO, S., KAWAHARA, T. and OHNISHI, O., 2007. Phylogenetic relationships among cultivated types of Brassica rapa L. em. Metzg. as revealed by AFLP analysis. Genetic Resources and Crop Evolution, vol. 54, no. 2, pp. 279-285. http://dx.doi.org/10.1007/s10722-005-4260-7.
http://dx.doi.org/10.1007/s10722-005-426...
).

These two subspecies and other subspecies of B. rapa need more study on a big collection of accessions covering their distribution area and using more than one molecular markers covering majority of their genome to assess genetic diversity and relationships within and among them.

5. Conclusion

In conclusion, enzyme electrophoresis resulted in detecting 14 putative polymorphic loci with 27 alleles. The rare alleles, alleles with mean frequency 0.04 were observed in Cat4A and Cat4B in subspecies Oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. The average fixation index (F) is significantly higher than zero for the analysis accessions indicating a significant deficiency of heteozygosity. The highest genetic diversity measures were observed in subspecies dichotoma, accession CR 1585/96 (the highest average of observed (H0) and expected heterozygosity (He), and number of alleles per locus (Ae)) which made this accessions valuable genetic resources to be included in breeding programs for the improvement of oilseed B. napus. Low heterozygosity in some accessions might be due to these accessions at the edge of the subspecies distribution centre; the position at which population sizes gradually decrease as does genetic diversity or origin of these as these accession could be associated with some sort of bottleneck event followed by the absence of inter-population gene flow. B. rapa ssp. oleifera (turnip rape) and B. rapa ssp. trilocularis (sarson) were grouped in one cluster which coincides with their morphological classification. These two subspecies and other subspecies of B. rapa need more study on a big collection of accessions covering their distribution area and using more than one molecular markers covering majority of their genome to assess genetic diversity and relationships within and among them.

Supplementary Material

Supplementary material accompanies this paper.

Supplementary Table 1. Allele frequencies at the 14 analyzed enzyme loci for 35 accessions of Brassica rapa subspecies.

This material is available as part of the online article from http://www.scielo.br/bjb

Acknowledgements

This project was supported by King Saud University, Deanship of Scientific Research, College of Science, Research Center, Saudi Arabia.

  • (With 1 figure)
  • Erratum

    In the article “Population structure and phylogenetic relationships in Brassica rapa L. subspecies by using isozyme markers”, DOI: https://doi.org/10.1590/1519-6984.226889, published ahead of print on Aug 26, 2020 in the Brazilian Journal of Biology, on page 1, in the section “*e-mail”:
    Where it reads:
    redasammour54@gmail.com
    It should be read:
    rsammour@ksu.edu.sa

References

  • ANNISA, GUO, Y., CHEN, S. and COWLING, W., 2011. Global genetic diversity of Brassica rapa In: Proceedings of the 17th Australian Research Assembly on Brassicas (ARAB), 2011, Wagga Wagga, NSW. Australia: AOF, pp. 17-19.
  • BECKER, H.C., ENGQVIST, G.M. and KARLSSON, B., 1995. Comparison of rapeseed cultivars and re-synthesized lines based on allozyme and RFLP markers. Theoretical and Applied Genetics, vol. 91, no. 1, pp. 62-67. http://dx.doi.org/10.1007/BF00220859 PMid:24169668.
    » http://dx.doi.org/10.1007/BF00220859
  • BIRD, K.A., AN, H., GAZAVE, E., GORE, M.A., PIRES, J.C., ROBERTSON, L.D. and LABATE, J.A., 2017. Population Structure and Phylogenetic Relationships in a Diverse Panel of Brassica rapa L. Frontiers in Plant Science, vol. 8, no. 321.
  • COWLING, W.A., 2007. Genetic diversity in Australian canola and implications for crop breeding for changing future environments. Field Crops Research, vol. 104, no. 1-3, pp. 103-111. http://dx.doi.org/10.1016/j.fcr.2006.12.014
    » http://dx.doi.org/10.1016/j.fcr.2006.12.014
  • DIXON, G. R., 2007. Vegetable brassicas and related crucifers United Kingdom: CABI. Crop Production Science in Horticulture Series, no. 14.
  • GOMEZ CAMPO, C., 1999. Developments in plant genetics and breeding Amsterdam: Elsevier. Biology of Brassica coenospecies, vol. 4.
  • HORÁČEK, J., GRIGA, M., SMÝKAL, P., and HÝBL, M., 2009. Effect of environmental and genetic factors on the stability of pea (Pisum sativum L.) isozyme and DNA markers. Czech Journal of Genetics and Plant Breeding, vol. 45, no. 2, pp. 57-71.
  • INABA, R. and NISHIO, T., 2002. Phylogenetic analysis of the Brassiceae based on the nucleotide sequences of the S-locus related gene SLR1. Theoretical and Applied Genetics, vol. 105, no. 6, pp. 1159-1165. http://dx.doi.org/10.1007/s00122-002-0968-3 PMid:12582894.
    » http://dx.doi.org/10.1007/s00122-002-0968-3
  • KARAM, M.A., YASSER, S., MORSI, Y.S., SAMMOUR, R.H. and ALI, R.M., 2014. Assessment of genetic relationships within Brassica rapa subspecies based on polymorphism. International Journal of Current Microbiology and Applied Sciences, vol. 3, no. 3, pp. 1-10.
  • KIMBER, D.S. and MCGREGOR, D.I. 1995. The species and their origin, cultivation and world production. In: D. S. KIMBER, and D. I. MCGREGOR, ed. Brassica oilseeds - production and utilization Oxon, UK: CAB International, pp. 1-8.
  • KOCH, M.A. and AL-SHEHBAZ, I.A. 2009. Molecular systematics and evolution. In: S. K. GUPTA, ed. Biology and breeding of crucifers Boca Raton: Taylor & Francis Group, pp. 1-18. http://dx.doi.org/10.1201/9781420086096.ch1
    » http://dx.doi.org/10.1201/9781420086096.ch1
  • MANCHENKO, G., 1994. Handbook of detection of enzymes on electrophoretic gels Florida: CRC Press, 334 p.
  • MEEUS, S., HONNAY, O. and JACQUEMYN, H., 2012. Strong differences in genetic structure across disjunct, edge, and core populations of the distylous forest herb Pulmonaria officinalis (Boraginaceae). American Journal of Botany, vol. 99, no. 11, pp. 1809-1818. http://dx.doi.org/10.3732/ajb.1200223 PMid:23092991.
    » http://dx.doi.org/10.3732/ajb.1200223
  • NEI, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, vol. 89, no. 3, pp. 583-590. PMid:17248844.
  • ORGANIZATION FOR ECONOMIC COOPERATION AND DEVELOPMENT – OECD, 2016. Safety assessment of transgenic organisms in the environment Paris: OECD Publishing. OECD Consensus Documents. Harmonisation of Regulatory Oversight in Biotechnology, vol. 5.
  • PASTEUR, N., PASTEUR, G., BONHOMME, F., CATALAN, J., and BRITTON-DAVIDSON, J., 1988. Practical isozyme genetics England: Ellis Horwood Ltd.
  • RADOSAVLJEVIC, I., SATOVIC, Z. and LIBER, Z., 2015. Causes and consequences of contrasting genetic structure in sympatrically growing and closely related species. AoB Plants, vol. 7, pp. plv106. http://dx.doi.org/10.1093/aobpla/plv106 PMid:26333826.
    » http://dx.doi.org/10.1093/aobpla/plv106
  • SAMMOUR, R.H., FAHMEE, S., MUSTAFA, A.-E. and TAHER, W., 2019. Isozyme analysis of genetic variability and population structure of Lathyrus sativus L. germplasm. Legume Research, vol. 42, no. 3, pp. 1-7. http://dx.doi.org/10.18805/LR-441
    » http://dx.doi.org/10.18805/LR-441
  • SHAHZADI, T., KHAN, F.A., ZAFAR, F., ISMAIL, A., AMIN, E. and RIAZ, S., 2015. An overview of Brassica species for crop improvement. American-Eurasian Journal of Agricultural & Environmental Sciences, vol. 15, no. 8, pp. 1568-1573.
  • SINSKAIA, E.N., 1928. The oleiferous plants and root crops of the family Cruciferae Bull, Bulletin of Applied Botany Genetics and Plant Breeding, vol. 19, no. 1, pp. 1-648.
  • SNOWDON, R., LÜHS, W. and FRIEDT, W., 2007. Brassica oilseeds. In: R. J. SINGH, ed. Genetic resources, chromosome engineering and crop improvement: oilseed crops Boca Raton: Taylor & Francis Group, pp. 195-230.
  • SOLTIS, D.E., HAUFLER, C.H., DARROW, D.C. and GASTONY, G.T., 1983. Starch gel electrophoresis of ferns: a compilation of grinding buffers, gel and electrode buffers and staining schedules. American Fern Journal, vol. 73, pp. 9-27.
  • SONG, K.M., SUSUKI, J.Y. and SLOCUM, M.K., 1991. A linkage map of Brassica rapa (syn. B. campestris) based on restriction fragment length polymorphism loci. Theoretical and Applied Genetics, vol. 82, pp. 296-304.
  • STACE, O., 1997. New flora of the british isles Cambridge: Cambridge University Press.
  • SURINA, B., SCHNEEWEISS, G.M., GLASNOVIĆ, P. and SCHÖNSWETTER, P., 2014. Testing the efficiency of nested barriers to dispersal in the Mediterranean high mountain plant Edraianthus graminifolius (campanulaceae). Molecular Ecology, vol. 23, pp. 2861-2875.
  • TAKUNO, S., KAWAHARA, T. and OHNISHI, O., 2007. Phylogenetic relationships among cultivated types of Brassica rapa L. em. Metzg. as revealed by AFLP analysis. Genetic Resources and Crop Evolution, vol. 54, no. 2, pp. 279-285. http://dx.doi.org/10.1007/s10722-005-4260-7
    » http://dx.doi.org/10.1007/s10722-005-4260-7
  • TANHUANPÄÄ, P., ERKKILÄ, M., TENHOLA-ROININEN, T., TANSKANEN, J. and MANNINEN, O., 2016. SNP diversity within and among Brassica rapa accessions reveals no geographic differentiation. Genome, vol. 59, no. 1, pp. 11-21. http://dx.doi.org/10.1139/gen-2015-0118 PMid:26694015.
    » http://dx.doi.org/10.1139/gen-2015-0118
  • TSUNODA, S., 1980, Eco-physiology of wild and cultivated forms in Brassica and allied genera. In: S. TSUNODA, K. Hinata, and C. Gómez-Campo, eds. Brassica crops and wild allies Tokyo: Japan Scientific Societies Press, pp. 109-120.
  • WRIGHT, S., 1951. The genetical structure of populations. Annals of Eugenics, vol. 15, pp. 323-354.
  • WRIGHT, S., 1978. Evolution and the genetics of populations Chicago: University of Chicago Press. Variability within and among natural populations, vol. 4.

Publication Dates

  • Publication in this collection
    26 Aug 2020
  • Date of issue
    Jul-Sep 2021

History

  • Received
    01 Aug 2019
  • Accepted
    01 Feb 2020
Instituto Internacional de Ecologia R. Bento Carlos, 750, 13560-660 São Carlos SP - Brasil, Tel. e Fax: (55 16) 3362-5400 - São Carlos - SP - Brazil
E-mail: bjb@bjb.com.br