VEGETOS: An International Journal of Plant ResearchOnline ISSN: 2229-4473
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Research Article, Vegetos Vol: 30 Issue: 2

Isolation of Male Sterile and Maintainer Lines from NorthIndian Onion (Allium cepa L.) Populations with the Aid of PCRBased Molecular Marker

Geetika Malik1*, Ajmer Singh Dhatt2 and Ajaz Ahmed Malik3

1Division of Vegetable Science and Floriculture, ICAR-Central Institute of Temperate Horticulture, Srinagar, Kashmir-190007, India

2Department of Vegetable Science, Punjab Agricultural University, Ludhiana, Punjab-141004, India

3Division of Vegetable Science, Sher-e-Kashmir University of Agricultural Sciences and Technology, Shalimar, Kashmir

*Corresponding Author : Geetika Malik
Division of Vegetable Science and Floriculture, ICAR-Central Institute of Temperate Horticulture, Srinagar, Kashmir-190007, India
Tel: 0194 230 5044
E-mail: [email protected]

Received: March 14, 2017 Accepted: May 03, 2017 Published: May 05, 2017

Citation: Malik G, Dhatt AS, Malik AA (2017) Isolation of Male Sterile and Maintainer Lines from North-Indian Onion (Allium cepa L.) Populations with the Aid of PCR-Based Molecular Marker. Vegetos 30:2. doi: 10.5958/2229-4473.2017.00142.2

Abstract

Isolation of Male Sterile and Maintainer Lines from NorthIndian Onion (Allium cepa L.) Populations with the Aid of PCRBased Molecular Marker

Marker assisted selection (MAS) using mitochondrial DNA based marker cytochrome b (cob) was integrated with phenotypic evaluation to isolate male sterile and maintainer lines from openpollinated onion varieties adapted to North Indian agro-climatic region. Cytotype (N/S) determination by cob marker followed by morphological and microscopic study of pollen discovered male sterile plants (Smsms) at frequencies of 0.015 in Punjab Naroya, 0.020 in Punjab Selection, and 0.006 in Punjab White. The progeny scoring of test-crosses between male sterile and N-cytoplasmic plants isolated the maintainers (Nmsms) at frequencies of 0.133 in Punjab Naroya, 0.231 in Punjab Selection and 0.182 in Punjab White. As a novel approach, Trait Recovery Programme was demonstrated to reduce the population size required to recover a male sterile plant by 91.08% in Punjab Naroya, 92.99% in Punjab Selection and 97.66% in Punjab White. For recovering a maintainer, 10% reduction in Punjab Naroya and 9.10% in Punjab Selection was calculated. However, no reduction was observed in Punjab White. This analysis also validated that in a randomly mating onion population, frequency of recessive ms allele squared is equal to the frequency of male sterile plants among S-cytotype and frequency of maintainers among N-cytotype (fms2 = fSmsms/fS = fNmsms/fN)....

Keywords: Onion; S-cytoplasm; Male sterile; Maintainer; Marker assisted selection

Introduction

Onion (Allium cepa L.) exhibits cytoplasmic-genic male sterility (CGMS), which involves the interaction of male sterile cytoplasm with nuclear fertility non-restorer allele [1,2]. The resultant male sterility has been exploited to produce onion F1 hybrids in many parts of the world. Unfortunately, in India, the development of F1 hybrids by public sector has been meager. The predominance of open-pollinated varieties has resulted in low productivity (16.12 MT/ha in 2013- 14) and non-uniform bulb quality. As a result, the domestic supply of onion is insufficient and export is limited. Onion is India’s most important export oriented vegetable that earned 3,170 Crore INR in 2013-14, which comprised 22 per cent of foreign exchange earned by total horticultural crops and 44 per cent by fresh and preserved vegetables [3]. However, to catch up with the productivity potential of 47-67 MT/ha, as achieved in Republic of Korea, USA, Spain and Japan [4] and extend export, F1 hybrid development is advisable. However, to achieve that, male sterility is a pre-requisite because hand emasculation and pollination is not possible in very large number of minute flowers in onion umbels. Thus, the exploitation of male sterility calls for the establishment of male sterile parent (A-line; Smsms) and its maintainer (B-line; Nmsms). Conventionally, it takes 4-8 years to identify cytoplasm (S or N) and nuclear non-restorer locus (Ms/ms) for isolating these lines, followed by 5-6 back-crosses (10-12 years) to transfer genetic background of B-line into A-line, if they are from different genetic backgrounds. This is a very cumbersome and resource consuming approach. Therefore, a strategy that significantly reduces resource input is highly desirable for onion improvement programmes in developing nations likes India.

As an alternative to above approach, PCR based molecular markers can determine cytotype of individual plants of a population at seedling stage [5-8]. It is also very efficient in screening sufficiently large number of plants that increases the probability of isolating both male sterile (Smsms) and maintainer (Nmsms) lines from same population/genetic background. Thus the back-cross programme is circumvented. Thirdly, expenditure of resources on populations lacking the S-cytoplasm is avoided. Considering these advantages, the PCR based molecular marker cytochrome b [8] was used to aid in development of male sterile and maintainer lines in three adapted populations of North India. This is the first report on successful large scale isolation of CGMS lines from adapted OP populations of the country with the help of marker assisted selection.

Additionally, a software based approach for limiting the population size necessary for recovering CMS lines has been proposed for reducing the effort and resources. The genetic relationship between different genotypes proposed in onion populations of other parts of the world was also confirmed for the Indian populations under study.

Material and Methods

The study was conducted from 2009 to 2013 at the Department of Vegetable Science, Punjab Agricultural University, Ludhiana, India. Approximately 2000 seedlings of each of the three openpollinated populations, namely, Punjab Naroya, Punjab Selection and Punjab White adapted to North Indian agro-climatic conditions were screened for identification and isolation of male sterile (Smsms) and maintainer (Nmsms) genotypes. The identification was done by combining MAS for cytotype and phenotypic assessment for nuclear fertility restorer locus (Ms/ms).

Marker assisted selection for cytotype

DNA isolation: The DNA was extracted with Shaky Prep method. In this method, 200 μl Shaky buffer (2 ml 50 mM Tris pH 9, 400 μl 5 mM EDTA and 38 ml double distilled water for 40 ml batch) and one stainless steel ball were added in each well of the plate. The crushing of the leaves was immediately done on a tissue-lyzer. The plate was then centrifuged at 3500 rpm for 10 minutes at 4ºC temperature in Centrifuge 5810 R (Eppendorf Corp., Germany). The supernatant from each well was diluted in 1:3 ratio with distilled autoclaved water to constitute DNA templates for polymerase chain reaction (PCR). This method was significantly faster than the traditional CTAB method of DNA isolation. Ninety-six crude DNA samples were extracted together and made ready for use as template for PCR in approximately 20 minutes. This simple high through-put method was found most suitable for marker assisted selection (MAS) of large number of plants.

DNA amplification: The PCR was performed in 20 μl reaction mixture containing 2 μl of DNA template, 4 μl of 5 X PCR buffer (GREEN GO TAQ ® FLEXI BUFFER, Promega Corp., USA), at MgCl2 concentration of 1.5 mM. The three primers (Cob-N, Cob-S and Cob-common) were added at the final concentration of 0.25 μM each, dNTP at 0.15 mM and Taq polymerase (GO TAQ® DNA POLYMERASE, Promega Corp., USA) at 0.25 units per microlitre.

The temperature profile for the PCR amplification comprised initial denaturation at temperature of 94°C for 2 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 53°C for 2 minutes and extension at 72°C for 2 minutes. The final extension was given at 72°C for 10 minutes.

Electrophoresis and gel documentation of DNA products

The PCR products were resolved by horizontal electrophoresis in 2% agarose gel. The 8 g of agarose powder (Amresco Llc, USA) was added to 400 ml of 1 X TAE and heated at 900 W for 6 minutes in a microwave oven. After 5 minutes of cooling, 18 μl of ethidium bromide (HiMedia lab. Pvt. Ltd) was added and properly mixed. The solution was poured into electrophoresis tray with 28-well combs. The apparatus was then left undisturbed for 30 minutes for solidification of the gel. The tray was then placed in electrophoresis tank containing 1800 ml 1 X TAE buffer. The 8 μl of each PCR product was loaded in the wells along with 8 μl 50 bp ladder (Thermo Scientific Inc., USA) for the reference bands. The electrophoresis was run at a potential difference of 5 V/cm of the gel length for 2 hours. The gel was documented in Alpha Imager® HP high performance imaging system (Fisher Scientific Ltd., UK).

Phenotypic evaluation for nuclear fertility restorer locus

Raising bulbs and morphological scoring to identify male sterile plants: The bulbs of identified S and N-cytoplasmic plants were harvested individually. In the first week of December 2011 bulbs of known S and N-cytoplasm were planted in paired rows at 60 cm x 60 cm spacing. At the initiation of bolting, individual populations were caged for protection from foreign pollen. At the onset of dehiscence, each S-cytoplasmic plant was morphologically scored for sterility (Smsms) on the basis of viable pollen absent/present in the anthers. The plants showing the presence of yellowish brown or greenish yellow pollen and positive acetocarmine test were designated as male fertile (SMs_) and those without visible color and with negative acetocarmine test as male sterile. After confirmation, the fertile plants from S-cytoplasmic line were rogued out and male sterile were retained for crossing with individual N-cytoplasmic plants (N_ _) to develop test cross progenies for identification of maintainers.

Test-crossings and progeny scoring to identify maintainer plants: Soft paint brushes were used for cross-pollination of sterile plant and selfing of corresponding fertile plant of each pair. Utmost care was taken to avoid contamination from pollen of other plants, thus hands and brushes were properly sterilized with 99.5% acetone GR (Lobachemie Pvt. Ltd.) after each crossing. The crossed seed on male sterile and selfed seed on corresponding male fertile plant of each pair were separately harvested and labeled. The cleaned seeds of all pairs were stored in paper bags at cool and dry place. For progeny scoring the crossed seed from male fertile parent of each pair was sown by following seed-to-seed method to save one year. In this approach, seed was sown early in first week of September 2012 and transplanted in first week of November. In the progeny of each test cross 25 plants were transplanted. To know fertility restoration and non-restoration status of N-cytoplasmic parent, every progeny was scored upon dehiscence of the flowers. The N-cytoplasmic parent whose 100% progeny showed absence of pollen was identified as maintainer, while, those with 100% fertile progeny was designated fertility restorer. The plants whose progenies included both male sterile and fertile plants were categorized as heterozygotes. The selfed seed of each pair was also sown in first week of November 2012 and transplanted in first week of January 2013 for development of bulbs.

Predicting frequencies of male sterile and maintainer plants and reducing population size: With the help of MAS, relationships image and Trait Recovery Program [9], it was demonstrated how the frequencies of male sterile and maintainer plants in an OP population of onion can be predicted and the population size required for isolation of A and B lines be reduced.

Results and Discussion

Marker assisted isolation of male sterile and maintainer plants

The gel documentation of DNA amplification of 1992, 1989 and 1982 seedlings of Punjab Naroya, Punjab Selection and Punjab White, respectively, revealed 187, 151 and 50 S-cytoplasmic seedlings in respective populations. The representation of gel documentation and frequencies of S and N cytoplasms among these populations are given in Plate 1 and Table 1, respectively.

Plate 1: DNA amplification of single plants from Punjab Naroya with ‘cob’ marker for cytotype identification M: 50 bp DNA ladder, S: S-cytotype (414 bp) Others: N-cytotype (180 bp).

Population Frequency of S-cytoplasm Frequency of N-cytoplasm
Punjab Naroya 0.094 0.906
Punjab Selection 0.076 0.924
Punjab White 0.025 0.975

Table 1: Frequencies of S and N cytoplasms estimated by ‘cob’ marker in different populations of onion.

The morphological scoring of S-cytoplasmic seedlings by observing pollen visually and microscopically (Plates 2 and 3) at flowering, followed by progeny scoring of individual test-crosses between male sterile and N-cytoplasmic plants gave the frequencies of male sterile and maintainer plants in all the three populations. The results are presented in Table 2.

Plate 2: Non-viable pollens from a male sterile plant of Punjab Naroya unstained with acetocarmine (magnification 40X).

Plate 3: Viable pollens from a male fertile plant of Punjab Naroya stained with acetocarmine (magnification 40X).

PUNJAB NAROYA
  Sterile (Smsms) Fertile
(SMs_ , N_ _ )
Test crosses Maintainer (Nmsms) Heterozygous
(NMsms)
Restorer
(NMsms)
NUMBER 30 1962 30 4 24 2
FREQUENCY (f) 0.015 0.985 - 0.133 0.800 0.067
PUNJAB SELECTION
  Sterile (Smsms) Fertile
(SMs_ , N_ _ )
Test crosses Maintainer (Nmsms) Heterozygous
(NMsms)
Restorers
(NMsms)
NUMBER 39 1950 39 9 28 2
FREQUENCY (f) 0.020 0.980 - 0.231 0.718 0.051
PUNJAB WHITE
  Sterile (Smsms) Fertile
(SMs_ , N_ _ )
Test crosses Maintainer
(Nmsms)
Heterozygous (NMsms) Restorer
(NMsms)
NUMBER 11 1971 11 2 8 1
FREQUENCY (f) 0.006 0.994 - 0.182 0.727 0.091

Table 2: Number and frequencies of male sterile, maintainer, heterozygous male fertile and restorer plants obtained by combining MAS and phenotypic evaluation.

In this study 30, 39 and 11 male sterile plants and 4, 9 and 2 maintainer plants were discovered in Punjab Naroya, Punjab Selection and Punjab White, respectively. The seeds of these plants are being increased for development of A and B lines for use in future F1 hybrid development and research purpose.

This study strongly suggests MAS for cytotype on a sample before planting large populations for morphological screening for male sterile plants or carrying out test crosses of random plants with an A line to isolate maintainers, as male sterile plants can’t be isolated from purely N-cytoplasmic populations and maintainers can’t be recovered from purely S-cytoplasmic populations. The cytoplasmic nature of any onion population, if confirmed conventionally, will take at least 2 years in contrast to 15-20 days with MAS. Secondly, the identification of S and N cytoplasmic seedlings reduces the number of bulbs to be handled/ stored for further selection of male sterile and maintainer plants at flowering, as for Smsms isolation, only S-cytoplasmic plants will be carried forward. Similarly, for Nmsms, only N-cytoplasmic plants will be grown further and S-cytoplasmic rogued out. This saves resources and reduces storage losses.

Efficiency of MAS in reducing the population size for isolating a male sterile/maintainer plant

In addition to aiding the identification of appropriate population and reducing the plants/ bulbs to be handled, the cytoplasm linked molecular markers can further save resources in isolating male sterile/ maintainer plants. It is possible to directly deduce the frequency of ms allele from the frequencies of S and N cytoplasms (as revealed by MAS) and that of male sterile plants (revealed by observing viable pollen within 1-year by following seed-to-seed flowering approach under Northern conditions of India). This calculated frequency of ms allele will lead to the prediction of frequency of maintainers in the population without carrying out testcrosses, as shown in Table 3.

Population fSmsms fS fmsimage fN fNmsms
(fms2 x fN )
Punjab Naroya 0.015 0.094 0.400 0.906 0.145
Punjab Selection 0.020 0.076 0.513 0.924 0.243
Punjab White 0.006 0.025 0.500 0.975 0.244

Table 3: Frequencies of maintainers in different populations predicted with aid of MAS for cytotype.

The predicted frequencies can be used to estimate the number of open-pollinated (OP) and N-cytoplasmic plants required to recover one maintainer as proved in the present study. The frequencies of maintainer (fNmsms) plants in different populations predicted in Table 3 were used in Trait Recovery Program to estimate the number of OP plants required to isolate maintainer. Further, the number of N-cytoplasmic plants required to isolate one maintainer were also estimated. The difference in the number of estimated OP plants and the N-cytoplasmic plants is the reduction in population size required to isolate maintainers. Based on TRP, the minimum number of OP and N-cytoplasmic plants required and per cent reductions in Punjab Naroya, Punjab Selection and Punjab White are shown in Table 4.

Population Frequency of maintainers Minimum no. of OP plants required to identify one maintainer Minimum no. of N plants required to identify one maintainer Reduction in no. of plants required (%)
Punjab Naroya 0.145 20 18 10.00
Punjab Selection 0.243 11 10 9.10
Punjab White 0.244 11 11 0.00

Table 4: Calculated per cent reduction in the number of plants required to isolate maintainers with the aid of marker assisted selection for cytotype.

The perusal of table reveals that marker assisted selection for cytotype can reduce the investment in development of maintainer (B) line in a population, where male sterile plants are available. The reduction in case of Punjab Naroya and Punjab Selection directly denotes the decrease in the number of test-crosses required to isolate a maintainer, as only the N-cytoplasmic plants will be carried forward till flowering and used for test crossing with a male sterile (A) line. It is also noticed that the per cent reduction in number of plants was in proportion to the S-cytoplasmic plants in the population. As in the present study, the proportion of S-cytoplasm was found to be 9.4% in Punjab Naroya 7.6% in Punjab Selection and 2.5% in Punjab White, the reduction in number of plants required to identify one maintainer was highest in Punjab Naroya (10 %), followed by Punjab Selection (9.1%), whereas, in Punjab White no difference was observed for requirement of OP and N-cytoplasmic plants. In India, the onion cultivars are predominantly open pollinated, which are primarily N-cytoplasmic in nature [10,11], therefore, reduction in population size for isolation of maintainers was found to be the modest, whereas, the US cultivars like Mountain Denvers, B2215C and Brigham Yellow Globe possess high proportions of S-cytoplasm, hence, show drastic reduction [6].

In populations where N-cytoplasm is present in high proportions, the probability of finding maintainers is higher than that of male sterile plants. In such cases, frequency of maintainer plants can be estimated by test crossing with a known CMS line in 2 years by following Seed-to-Seed approach. This frequency can then be used to predict the frequency of male sterile plants in the population, as described in Table 5.

Population fNmsms fN fmsimage fS fSmsms
( fms2 x fS )
Punjab Naroya 0.133 0.906 0.383 0.094 0.014
Punjab Selection 0.231 0.924 0.500 0.076 0.019
Punjab White 0.182 0.975 0.432 0.025 0.005

Table 5: Frequencies of male sterile plants in different populations predicted with aid of MAS for cytotype.

The calculated frequency estimates the minimum number of S-cytoplasmic plants required to isolate at least one male sterile plant in the respective populations, as shown in Table 6. The per cent reduction will be obtained by comparing this number with the number of OP plants required.

Population Frequency of sterile plants Minimum no. of OP plants required to identify one male sterile Minimum no. of S-cytoplasmic plants required to identify one male sterile Reduction in no. of plants required (%)
Punjab Naroya 0.014 213 19 91.08
Punjab Selection 0.019 157 11 92.99
Punjab White 0.005 598 14 97.66

Table 6: Calculated per cent reduction in the number of plants required to isolate male sterile plants with the aid of MAS for cytotype.

A significant reduction in number of plants required to isolate one male sterile plant from a population already characterized with cytoplasm specific molecular markers has been demonstrated in the table. With this information in hand, only the required number (as calculated above for each population) of S-cytoplasmic seedlings has to be managed for getting male sterile plants. The identified male sterile plants from the respective populations can directly be crossed with previously identified maintainer plants for the establishment of A and B lines. This approach is worth exploiting in those onion populations, which have low frequency of S-cytoplasm. As in the present study, the three populations possess S-cytoplasm with very low frequencies of 0.025 (Punjab White), 0.076 (Punjab Selection) and 0.094 (Punjab Naroya) compared to those of 0.40, 0.37 and 0.85 in varieties like Sapporo-Ki, Brigham Yellow Globe and Texas Grano 1015Y, respectively [6,12]. It is also noticed that the per cent reduction in number of plants was proportionate to the N-cytoplasmic plants in the population. Thus, the prior knowledge of S-cytoplasmic seedlings at nursery stage using DNA markers can significantly reduce the labor and cost involved.

This approach can be employed for development of A and B lines in any adapted population having maintainers in plenty and male sterile plants in scarcity. If population is primarily N-cytoplasmic, the probability of getting maintainers is high, but it requires 5-6 backcrosses viz. 10-12 years for transfer of male sterility from known source in biennial crop like onion. However, with application of molecular markers for cytoplasmic type as shown in this experiment, this time period can be reduced to just 2-3 years.

Relationship between the proportion of male sterile among S-cytoplasmic and maintainers among N-cytoplasmic plants

A random mating OP population of onion possessing N and S cytoplasms is assumed to have equal proportion of male steriles among S-cytoplasmic and maintainers among N-cytoplasmic plants. This proposition was tested on the populations studied in this experiment wherein, the proportion of male sterile to S-cytoplasmic plants (PSmsms/S) was estimated by scoring for sterility of S-cytoplasmic plants identified with MAS in each population. Similarly, proportion of maintainers in N-cytoplasmic plants (PNmsms/N) obtained by progeny scoring of the test crosses in each population was estimated and presented in Table 7 for the comparison.

Population PSmsms/S PNmsms/N
Punjab Naroya 0.160 0.147
Punjab Selection 0.258 0.250
Punjab White 0.220 0.187

Table 7: The proportion of male sterile plants among S-cytoplasmic and maintainers among N cytoplasmic plants.

To determine the significance of differences between the proportions, Z-test was used and results are presented in Table 8. The non-significance of differences indicates that the random mating within the onion populations carrying S and N cytoplasms causes the frequency of male sterile plants in S-cytotype to be approximately equal to that of the maintainers in N-cytotype. These findings confirm the earlier suggestion [6].

Number of male sterile plants and their proportion in S-cytoplasmic plants
  Punjab Naroya Punjab Selection Punjab White
Number (n1) 30 39 11
Proportion (p1) 0.160 0.258 0.220
Number of maintainers and their proportion in N-cytoplasmic plants
Number (n2) 265 459 361
Proportion (p2) 0.147 0.250 0.187
Z value 0.191NS 0.110NS 0.275NS
NSNon-significant

Table 8: Z-test for testing the significance of difference between the proportions.

Conclusions

Using the molecular marker based selection; CMS lines were isolated from three open pollinated populations Punjab Naroya, Punjab Selection and Punjab White adapted to North India. With the help of MAS and Trait Recovery Programme, a method has been suggested to predict the frequency of maintainers in any open pollinated onion population without performing test crosses and to calculate the reduced population size to be grown to isolate the maintainers. Same method is suggested for isolation of male sterile plants too. The perusal of frequencies of S-cytoplasm, N-cytoplasm, male sterile and maintainer plants in these populations supported the suggestion that proportion of male sterile among S-cytoplasmic plants is equal to the proportion of maintainers among N-cytoplasmic plants of a randomly mating onion population.

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