THE SEARCH FOR THE R1-NJMAIZE GENE ... - BioInfo Publication

International Journal of Agriculture S c i e n c e s ISSN: 0975-3710 & E-ISSN: 0975-9107, Volume 7, Issue 9, 2015, pp.-698-702. Available online at http://www.bioinfopublication.org/jouarchive.php?opt=&jouid=BPJ0000217

THE SEARCH FOR THE R1-NJ MAIZE GENE MARKER ENABLING THE SELECTION OF IN VIVO PRODUCED HAPLOID FORMS

MIKOŁAJCZYK S., TOMKOWIAK A., WEIGT D., BRODA Z. AND KURASIAK – POPOWSKA D.* Department of Genetics and Plant Breeding, Poznan University of Life Sciences, Dojazd 11, 60-632 Poznan, Poland *Corresponding Author: Email : [email protected] Received: September 28, 2015; Revised: October 12, 2015; Accepted: October 19, 2015 Abstract- The aim of this study was an attempt to identify the polymorphic product, which is characteristic of haploid maize lines, produced by inductor fertilisation. The plant material used for the research consisted of 6 haploid lines of maize produced by pollination with an inductor, which had an anthocyanin marker on the seeds, and it also consisted of 3 diploid forms. The ploidy level of the lines of suspected haploids under analysis was verified by means of a flow cytometer and cytological investigations. 280 oligonucleotide primers were tested in the research. On the basis of RAPD-PCR reactions one characteristic PCR product differentiating haploid from diploid plants was identified. Probably this product is related with the R1–nj gene expression. The gene is responsible for generation of the anthocyanin pigment in the embryo and endosperm of haploid maize forms and it may be the genetic marker of the trait under analysis. Keywords: Maize, in vivo, Inductor, Haploid, RAPD Citation: Mikołajczyk S., et al. (2015) The Search for the R1-Nj Maize Gene Marker Enabling the Selection of in Vivo Produced Haploid Forms, International Journal of Agriculture Sciences, ISSN: 0975-3710 & E-ISSN: 0975-9107, Volume 7, Issue 9, pp.-698-702. Copyright: Copyright©2015 Mikołajczyk S., 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. Introduction Modern maize growing is based on the use of heterosis. Inbred lines with a high level of homozygosity are obtained for this purpose [1]. The use of in-vitro culture techniques, such as androgenesis (anther cultures and isolated microspores cultures) is an alternative to maize inbreeding. Thanks to these techniques fully homozygous lines can be recovered in much shorter time. However, the efficiency of the process is low and strongly related with the genotype of donor plants that it encourages research on new methods, which will enable quick and effective creation of homozygous lines. Breeders are hopeful about the method of in vivo haploid induction by pollinating maternal plants with the pollen of an inductor, stimulating the ovum to the haploid development of the embryo [2]. DHs are adopted as a routine method in commercial maize breeding programs in North America [3] and Europe [4]. whereas in Poland they have been produced recently. To induce maternal haploids the donor plant is pollinated with an inductor. Usually it is a specific maize line or population [5, 6]. The efficiency of in vivo haploid induction largely depends on the inductor. Nevertheless, the percentage of haploids produced is also influenced by the genotype of maternal lines and environmental conditions [7]. In order to identify the presumable maternal haploids the system which enables differentiation between haploids and diploids is used. It is possible thanks to the R1-nj gene, whose expression causes production of the pigment in the embryo and endosperm. The system of a dominant anthocyanin marker was described in detail in the studies by Nanda & Chase [8] and by Chase [9]. The R1-nj gene expression was found to be largely dependent upon the maternal genotype, which makes the identification of

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presumable haploids from the flint forms very difficult, because some of them have the genes inhibiting the anthocyanin synthesis, i.e. C1-I, C2-Idf and In1-D [10, 11, 12]. This is an essential subject of study since meaning of flint forms of cultivated maize is growing in Central Europe. The occurrence of these genes in dent forms is much less frequent, so the identification of haploids is more reliable in them. The aim of this study was an attempt to identify the polymorphic product, which is characteristic of haploid lines of maize, produced in vivo as a result of pollination with an inductor. Materials and methods The plant material applied in experiment was received from The Plant Breeding and Acclimatization Institute (IHAR) - National Research Institutein Radzików (Poland) and consisted of 6 haploid maize lines produced by crossing maternal plants with an inductor, so they were obtained with the in vivo technique, 3 diploid forms, were used as controls [Table-1]. The haploid forms were selected on the basis of the presence of the morphological marker, i.e. an phenotypic marker on the caryopses, resulting from the R1–nj gene expression [Fig-1]. The ploidy level of the plants was cytologically assessed in all the lines under analysis. Mitotic divisions in the cells of the roots collected from seedlings were observed. The plant material was fixed in Carnoy's fixative (for example: 60 ml of ethanol CH3CH2OH, 30 ml of trichloromethane CHCl3 and 10 ml of acetic acid CH3CO2H) [13]. Fragments of the roots were stored in 70% ethanol at 4oC. The chromosomes were stained with the acetocarmine method [14]. Moreover the ploidy of the plants was confirmed by measuring the nuclear DNA content by laser flow cytometry. The tests were prepared according to the procedure by Śliwińska and Steen [15] with

International Journal of Agriculture Sciences ISSN: 0975-3710 & E-ISSN: 0975-9107, Volume 7, Issue 9, 2015

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The Search for the R1-Nj Maize Gene Marker Enabling the Selection of in-Vivo Produced Haploid Forms net, with the eyelet diameter of 50 µm. The obtained filtrate was analysed with a Partec CCA flow cytometer. In each test the relative DNA content was analysed in about 5000 isolated nuclei. A linear scale was applied for all measurements and the histograms were analysed with the Partec DPAC V2.1 computer software.

slight modifications [16]. The 2C genome of diploid, highly homozygous lines (MWD 31, MWD 40, MWF 26) were used as an internal standard, were MWD means Dent Corn, and MWF Flint Corn. For the analysis, young leaves of 50 plants per line of maize was fragmented with a razor blade on a Petri plate in the presence of 1 ml of a lysis buffer (0.1M Tris, 2.5mM MgCl 2.6H2O, 85mM NaCl, 0.1% Triton X-100), containing a fluorescent stain, i.e. 4',6-diamidino-2phenylindole (DAPI; 2 µg/ml). Next, the suspension was filtered through a nylon

1

2

3

4

.

5

6

7

8

9

Fig-1. Caryopses of diploid controls of maize of maize without morphological marker (1-MWD 31, 2-MWD 40, 3-MWF 26) and haploid lines with an anthocyanin marker visible on the caryopses (4-SH 17/2007, 5-SH 19/2007, 6-SH 20/2007, 7-SH 21/2007, 8-SH 27/2007, 9-SH 30/2007)

Table-1. The haploid lines and diploid maize forms analysed in the experiment No.

Presumable haploid lines

Diploid forms

1

-

MWD 31

2

-

MWD 40

3

-

MWF 26

4

SH 17/2007

-

5

SH 19/2007

-

6

SH 20/2007

-

7

SH 21/2007

-

8

SH 27/2007

-

9

SH 30/2007

-

DNA extraction and amplification For molecular analysis, DNA was extracted from ten plants for each line. The plants were equally mixed (bulked). The method of random search for RAPD (Random Amplified Polymorphic DNA) markers based on a typical PCR (Polymerase Chain Reaction) was used for molecular analysis. The genomic DNA from hybrid forms and parental components of maize was isolated with an extraction kit (Genomic Mini AX Plant, A&A Biotechnology)by means of the column-based method. The RAPD-PCR reaction was carried out in a 12.5l mixture, which consisted of deionised water, 1M Tris HCl (pH 8.3), 2mM MgCl 2, 2mM dNTP, 5pmol primer, 5UTaq polymerase, DNA extract – 25ng The DNA was amplified with a BIOMETRA T3 thermocycler (POLYGEN). The electrophoresis of amplification products was carried out in 1.5% agarose gel, which consisted of 1.5 g of agarose, 100ml of TBE1x buffer (10.8g of Tris base; 5.5g of hydrogen borate H3BO3; 4ml of 0.5M diaminoethane-tetraacetic acidEDTA pH 8.0) and 1l of EtBr (ethidium bromide).280 oligonucleotide primers (Operon Technologies INC) were tested. The highest number of polymorphic products was obtained for 18 primers [Table-2]. . Results The investigation of ploidy level of the maize lines under analysis with a laser flow cytometer revealed that all the lines with an anthocyanin marker on the seeds were haploids. On average the total content of nucleic DNA in the haploid plants was 2.7 pg, whereas the average DNA content in the nuclei of diploid forms was 5.4 pg [Fig-2]. Ploidy of individual plants belonging to the analyzed


population was variable [Table-3]. The SH 17/2007 line showed the highest level of 1C (haploid) plants, 91.7%. The lowest level of 1C plants (41.6%) was found in SH 20/2007. The ploidy level of the forms under analysis was cytologically confirmed [Fig- 3, 4]. This study was an attempt to identify the polymorphic product or striated profile, which is characteristic of haploid lines. Upon the analysis of 280 oligonucleotide primers of the RAPD-PCR reaction the characteristic product differentiating haploid plants from diploid forms was identified. The marker became visible when the OPA 12 primer with the 5’-TCGGCGATAG-3’sequence was used and it was present only in the haploid forms [Fig.-5]. Discussion Doubled haploids are a useful plant material both in basic genetic investigations and in breeding programmes. The characteristic feature of such plants is their homozygosity in all loci [17]. The use of doubled maize haploid plants enables faster creation of parental components for heterosis breeding than in conventional breeding. The relatively new technique, which consists in the pollination of diploid parental plants with an inductor, stimulating ova to develop a haploid embryos, is a promising method of production of haploid plants [18]. The identification of maize haploids at the phase of seeds is crucial to the application of the technology of maternal haploids in breeding. For many years the system based on the presence of anthocyanin coloration has been applied for that purpose. It was first described in the 1960s.The dominant anthocyanin marker, which was described in the study by Nanda and Chase [8] and in the study by Chase [9], is used to identify the haploid. Kernels of the maize with presence of the morphological marker were showed in the Fig. 1. This marker was described for the identification of seeds with the haploid embryo. This system is based on the R1-nj gene, whose expression enables distinction between haploids and diploids through the production of pigment in the embryo and endosperm. It is known that the expression of the R1-nj gene is strongly dependent on the form of the parent and makes it difficult to identify a putative haploid form of the flint maize type, because some of them have genes that inhibit the synthesis of the anthocyanin gene (C1-I, C2-Idf gene and In1-D gene); [10, 11, 12], and are less common forms of the maize dent type. This process results in different effectiveness, depending on the maternal plant genotype and environmental conditions. Therefore, it is necessary to check the induced plants for their homozygosity before using them for further breeding works. A genetic marker, which is an anthocyanin mark appearing on the seeds of haploid plants, is used for this purpose. The R gene has four basic types of alleles in the Rlocus: R-r, r-g, r-r, r-g. The R-nj gene, which is analysed in this study, also belongs to the R locus.


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Mikołajczyk S., Tomkowiak A., Weigt D., Broda Z. and Kurasiak – Popowska D. Table-2 Applied primers with the number of RAPD markers obtained and the percentage of polymorphic markers for each primer. No. of primer

OPA 04 OPA 07 OPA 09 OPA 12 OPA 18 OPA 19 OPB 01 OPB 03 OPB 07 OPB 18 OPC 05 OPD 07 OPD 13 OPD 16 OPD 19 OPE 01 OPE 04 OPE 19

Primer sequence [5’-3’]

Total no. of products

No. of polymorphic products

Percentage of polymorphic products [%]

11 5 8 7 7 2 9 14 2 11 6 7 11 8 9 12 8 6

10 5 7 7 7 1 8 14 1 11 6 6 9 8 9 11 8 5

90,9 100,0 87,5 100,0 100,0 50,0 88,8 100,0 50,0 100,0 100,0 85,7 81,8 100,0 100,00 91,6 100,0 83,3

AATCGGGCTG GAAACGGGTG GGGTAACGCC TCGGCGATAG AGGTGACCGT CAAACGTCGG GTTTCGCTCC CATCCCCCTG GGTGACGCAG CCACAGCAGT GATGACCGCC TTGGCACGGG GGGGTGACGA AGGGCGTAAG CTGGGGACTT CCCAAGGTCC GTGACATGCC ACGGCGTATG

Fig-2. The result of the measurement of the DNA content in the haploid plant SH 19/2007, which was obtained after the pollination of the maternal line SH 19/2007 with an inductor – compared with the diploid model – the Trophy cultivar right peak. The pollinator inducing haploids has the R-njR-nj genotype and the maternal form should be recessive allele arrangement: r-njr-nj. However, the R-nj gene expression may be inhibited by inhibitor genes, e.g. by C1-l which control the expression of genes responsible for the anthocyanin biosynthesis in aleurone and scutellar tissue [19]. In this system of genes the morphological marker, i.e. the anthocyanin mark on the caryopses of haploid plants, may not develop. For this reason, the selection of haploids by means of the morphological marker is not fully effective. As a result of conducted cytological analyses 91.6% of haploid plants of genotype SH17/2007 were demonstrated the presence of the morphological marker [Table-3], [Fig.-1] and [Fig.-2]. The R-nj gene, which is an allele of the R gene, is responsible for the anthocyanin pigmentation of the aleurone layer and it is used for the selection of haploids after pollination with an inductor. Nanda & Chase [8] observed that the R-nj gene causes the synthesis of anthocyanins with different intensification,


which depends on the genetic background, and those authors were the first to use it for the selection of maternal haploids. The caryopses where the R-nj gene expression takes place in the endosperm and where it is absent in the embryo are classified as haploid [3, 20]. The presumable maize haploids were selected on the basis of the morphological marker which is the effect of the activity of the aforementioned R-nj gene. The elimination of non-homozygous plants from breeding is possible by application of the methods detecting the polymorphism of the isoenzymatic proteins or polymorphism of the deoxyribonucleic acid, what allows to find differences between individual loci [17]. The system of the R1-nj gene-based marker is routinely used to identify maize maternal haploids. However, visual assessment of the expression of this gene is very laborious Additionally the system of kernels identification based on the morphological marker may be ineffective. Many seeds identified morphologically


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The Search for the R1-Nj Maize Gene Marker Enabling the Selection of in-Vivo Produced Haploid Forms Table-3 Total DNA content in plants obtained as a result of pollination by haploid inducer Genotype

SH17/2007 SH19/2007 SH20/2007 SH21/2007 SH27/2007 SH30/2007 MWD31 MWD40 MWF26

No. of analyzed plants

50 50 50 50 50 50 10 10 10

Percentage of content of the genome DNA [%] 1C 2C 91,67 62,5 41,66 75,0 90,9 87,5 -

Fig-3. The chromosomes of the diploid plant with the MWD 40 genotype.

8,33 37,5 58,34 25,0 9,1 12,5 100,0 100,0 100,0

Average content DNA [pg] Haploid plants [pg] 2,779 2,864 2,805 2,835 2,794 2,768 -

Diploid plants [pg] 5,407 5,248 5,393 5,411 5,424 5,471 5,442 5,380 5,425

revealed that the amount of haploids ranged from 41.6% to 91.6%, whereas the others were diploids which were erroneously visually identified as haploids. Therefore, this study was an attempt to identify the marker which is characteristic of haploid lines with the R-nj gene. RAPD markers were used for this purpose. Thanks to the analysis of the polymorphism of the generated reaction products in combination with phenotype observations the R-nj gene marker, which codes the pigment synthesis, was found. The conversion of the RAPD method into the SCAR (Sequence-Characterised Amplified Region) specific marker system will be the next stage of the research and it will enable wider application of this marker for the selection of haploid plants Due to the fact that environmental conditions strongly influence the effectiveness of induction marker genes B1 and P11 are also used for the identification of maize maternal haploids. The genes cause sunlight-independent purple pigmentation in the plant tissue, which enables the identification of haploids at the stage of a four-day-old seedling [12, 18]. At present the maize haploid identification system after pollination with an inductor is supplemented with the high kernel oil content [21]. Li et al. [23] used the inductor - CAUHOI line with the R1-nj marker and high content of oil. The haploidic caryopses which were obtained from the hybridisation of inductor CAUHOI and maternal line ZD958 had significantly lower content of oil in seeds (37.50 g/kg) than hybrid caryopses (60.03 g/kg) [23]. Li et al. [23] proposed a model accounting for the mechanism of induction of maize maternal haploids.

Fig-4. The chromosomes of the haploid plant with the SH 17/2007 genotype. as containing haploid embryos were diploids (from the 8.3% up to the 58.3%) [Table-3]. Performing additional analyses (cytological, analysis of the content DNA or applying of DNA markers) is raising costs of selection kernels with haploid embryos.The system fails in the presence of allele C1-l, which inhibits the expression of the R1-nj marker [12, 18, 21]. In view of the unreliability of the R1nj gene-based haploid identification system all caryopses are sown in fields and they are visually assessed before florescence. Haploids are shorter and they have thin stalks and erect, narrow leaves [22]. The research findings confirm the fact that the R1-nj gene-based maize haploid identification system is unreliable. The cytometric analysis of the offspring exhibiting the presence of the marker


Fig-5 The electrophoretic distribution of the PCR products obtained by means of RAPD molecular markers with the OPA12 primer. The consecutive paths correspond to the following genotypes: MWD31, MWD40, MWF26, SH17/2007, SH19/2007, SH20/2007, SH21/2007, SH27/2007, SH30/2007.


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Mikołajczyk S., Tomkowiak A., Weigt D., Broda Z. and Kurasiak – Popowska D. They indicated that after pollination with an inductor at different stages, after double or single fertilisation, the genetic material of the pollinating line may be incompletely eliminated and the haploids contain the introgression of the inductor genome. This mechanism may account for the fact why the results revealed the presence of a specific product obtained after the application of starter OPA 12 with sequence 5’-TCGGCGATAG-3’ only in the identified maize haploids [Fig-.5]. Those observations were confirmed in the study by Zhang et al. [24], who analysed the F1 generation received to the effect of the crossing of maternal line Hua24 and inductor HZ11, where 28 SSR markers were applied. They observed that the genetic composition of all the haploids was similar to the maternal line, but some of them had heterozygous stripes from the paternal parent (from 3.6% to 15%).

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Conclusions The effectiveness of application of maternal haploids in maize breeding depends on the effectiveness of identification of haploids, which is not reliable. Therefore, researchers search for an alternative to the R1-nj marker system (identification based on the high kernel oil content, marker genes B1 and Pl1). The findings may be used to make another system of identification of maize maternal haploids based on the product of RAPD-PCR reaction with starter OPA 12 after conversion to a SCAR marker References [1]

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