Parental Bias in Segregation Distortion of RAPD Markers from Solanum Anther-Derived Plants

A diploid Solanum clone 9507-04 was used to grow anther-derived plantlets that were putatively monoploid. Sixteen RAPD primers were used to observe the RAPD profiles from anther-donor and anther-derived materials. A total of 56 RAPD markers were scored and each marker was identified according to the primer’s designation and size (kilo base pairs) of the amplified fragment. Of 56 RAPD markers assayed, 44.6% did not follow the expected 1:1 Me ndelian segregation (χ 2 >4.26, P<0.05) in the anther-derived progeny. The selected six morphological traits also segregated in anther-derived plants including the nine that retained 3-4 of the parental leaf characters. The 12 distortion–causing RAPD markers (χ2=8.89-17.64, P<0.003) with above 84% presence in the progeny were retained in all above nine plants. Such preference of parental markers in potato anther-derived progeny may indicate a linkage with androgenesis.


Introduction
Anther-culture of diploid potatoes can be used for the production of monoploid plants [1,2,3].However, the gametic cells respond variedly to the culture conditions leading to different levels of tissue development in anther-derived progeny [1,4].Such variations in anther-culture response are attributed to actions of gametophytic active genes, sporophytic active genes, or gameto-sporophytic interactions [5].Therefore, potato anther-derived progeny has been reported to give distorted segregation in 29 to 70% of the molecular markers [6,7,8].Gamete selection, (sub) lethal alleles, anther-culture technique, and regeneration pressure have been suggested as possible causes of distorted segregation of molecular markers [9,10].RAPD (random amplified polymorphic DNA) markers can be used without any prior genome information for the genetic analyses of anther-derived monoploids [11,12].The issue of dominant inheritance of RAPD markers [13] can be circumvented by using gametophytic tissue, since a heterozygous genotype (for an allelic pair) segregates into gametes carrying dominant and recessive markers [14].RAPDs have been markers of choice for genetic analyses of tissue cultured potatoes [15,16,17].This report aims to examine the segregation of some RAPD markers and morphological traits in anther-derived progeny of the diploid 9507-04 parent.

Plant materials
The diploid Solanum clone 9507-04 (50% S. tuberosum and 50% wild: S. chacoense, Argentinian wild potato species) was kindly provided by Dr. Hielke De Jong, Potato Research Centre, Agriculture and Agri-Food Canada.Solanum clone 9507-04 produced abundant anther-derived plantlets under anther-culture conditions of Aziz et al. [18].The leaves silhouette and amount of anthocyanin as well as morphological features of primary/terminal leaflet were identified as described [19,20,21].The putative monoploid anther-derived material from the clone 9507-04 was chosen for the RAPD analysis.

DNA isolation and amplification
Up to 50mg of the leaf tissue was homogenized in 300μl of the extraction buffer [22] with a pellet pestle (Mandel Scientific Co.).The mixture was incubated at 65 o C for 90 min followed by two chloroform/isoamyl alcohol (24:1) extractions.The DNA was precipitated by an equal volume of cold isopropanol and resuspended in 30μl of distilled water.Quantification of the DNA preparation was made by comparing with DNA Mass Ladder (Life Technologies) under gel electrophoresis conditions.Ten ng DNA template or distilled sterile water as control was used in polymerase chain reaction (PCR) mixtures.The PCR mixture (25μl in 0.2ml tube) also included 0.2μM primer, 50μM each of dATP, dCTP, dGTP, dTTP, 1 unit Ampli-Taq DNA polymerase (Perkin Elmer) and 1X PCR buffer (10mM TrisHCl, pH 8.3, 50mM KCl, 2mM MgCl 2 , 0.001% gelatin) that was overlaid with a drop of fresh mineral oil.After an initial four min denaturation at 94 o C in a PTC-100 Thermal Controller (MJ Research), the amplification reactions were carried out using 45 cycles (default ramp time) of 1 min denaturation at 94 o C, 1 min annealing at 36 o C, and 2 min extension at 72 o C. All PCR products were then subjected to 10 min extension at 72 o C followed by soak at 4 o C until recovery.In each set of amplification reactions one control was a PCR mixture excluding any template whilst the other contained DNA from the anther donor (Figure 1).The PCR products (10μl of the reaction mixtures) were separated by electrophoresis (3.5Vcm -1 for 3 h) on 1.4% agarose gel stained with ethidium bromide (0.5μgml -1 ) in 0.5X TBE (0.045M Tris, 0.045M Boric acid, 0.001M EDTA) buffer.
The gels were photographed using a UV transilluminator (Fotodyne, Inc.).Sizes of the RAPD markers were determined by comparing with a DNA Mass Ladder.

Statistical analysis
Reproducible RAPD-PCR products in the parent were used as markers.Parental RAPD markers were then scored for presence or absence in the anther-derived progeny.For each RAPD marker goodness-of-fit to expected segregation of 1:1 was tested by Chi-square analysis using SYSTAT version 7.0 (SPSS Inc., USA).Segregation ratios that differed from the expected value (significant at P=0.05 or less) were classified as distorted.

Results and discussion RAPD markers' identification
Sixteen RAPD primers (Table 1 1).Therefore, these markers can conveniently be used for a standard comparison with RAPD profiles from other related genotypes.

Leaf morphology of anther-derived plants
The mature leaves of potato are described as a pinnately compound [21] and six of the leaf characters were used to identify progeny of anther-donor parent (Table 2).The leaf morphological traits of parent segregated in anther-derived plants thus differing from each other.The leaf silhouette was either open or medium.Amount of leaf anthocyanin and pubescence varied from light to medium or heavy.The shape of terminal or primary leaflet was found elliptical, narrowly ovate or broadly ovate.The tip of these leaflet were classified as acuminate, acute or cuspidate.Also the leaflet bases were found as cordate, obtuse, acute or truncate.Such morphological differences reflected the segregation of parental and non-parental type genetic characters in the progeny.
The anther-derived progeny revealed assorted non-parental leaf traits.Androgenesis has been reported to induce genomic changes in potato [27].Out of the 20 anther-derived plants generated, nine were found similar to the parent.These plants showed retention of three or four of the parental leaf characters (Table 2).Segregation studies in monoploid regenerants would also be useful in genetic analysis since they are expected to be developed from individual microspores.Therefore, such studies can also be used to identify extra-chromosomal and/or physiological factors affecting the survival of monoploids or dihaploid.

Segregation distortion of RAPD markers
Parental RAPD markers were scored for presence or absence of a DNA band in the anther-derived progeny.Segregation of the RAPD markers was examined against the expected 1:1 ratio by Chi-square analysis.Of 56 markers assayed (Table 1), 44.6% did not segregate according to expected 1:1 Mendelian ratio (χ 2 4.26,P0.05).
Anther-derived progeny is reported to give much higher percentage of skewed markers as compared to that from the populations of selfed progenies [7].Segregation distortion can be caused by the gamete selection, i.e., only a few selected microspores may survive in the mature anthers [28].During anther culture, the genotypes which are culture-responsive could be further selected, causing an even greater distortion in the segregation of molecular markers [7,10].The marker UBC291-1.4showed a distorted ratio in anther-derived progeny for 'presence of band: absence of band'=21:2 (χ 2 =15.696,P<0.005).
Interestingly, the same marker also showed the distorted segregation in the pollen population [29].It appears that UBC291-1.40 is selected against during pollen maturation and selected for anther culture.This marker therefore may be related to alleles essential for the survival of culture responsive microspores.Segregation distortion can occur due to the selection pressure in the pollen maturation process, and additional selection pressures during anther-culture conditions may contribute further to the distorted segregation of the markers.From 25 distorted markers (Table 1) 13 RAPDs were either unevenly present or absent in anther-derived progeny (χ2=4.26-11.56,P=0.001-0.05).
Non-random chromosome segregation during meiosis is another possible explanation for the distorted segregation observed [30].The other 12 RAPD markers showed distorted segregation by being retained in more than 84% of the progeny (χ2=8.89-17.64,P<0.003).Thus being distortion-causers [31], most of these RAPDs have been used as seed-markers for a linkage mapping approach.Incidentally this group of 12 distortion-causing markers was found intact in the nine plants (Table 2) having mostly parental traits.Segregation distortion during doubled haploid production is likely to be due to a range of factors including selection during the process of gamete culture and plantlet regeneration [32].Simultaneous presence of group parental markers in anther-derived progeny reported in this study may indicate a linkage with androgenesis in potatoes.Androgenic fitness has an important role in the prevalence of markers with segregation distortion and linkage of such markers to specific genes controlling in vitro androgenesis [ 33].Further studies of segregation of molecular markers at various pollen maturation and microspore regeneration stages would help to identify the genetic factors involved in these processes.presence in monolploids *The letters on left side of the hyphen represent the name of the RAPD primer (UBC/OP); dig its on the right show the size (kbp) of the amplified fragment. 1Categories for RAPD markers are based on Chi-square test for 1:1 segregation of the parental DNA band presence vs. absence in the progeny.

[ 19 , 20 , 21 ]
of parent in the nine anther-derived plants that showed retention of 3-4 traits of the anther donor.

Figure 1 :
Figure 1: Groups of four lanes showing PCR amplifications with a rando m amplified poly mo rphic DNA (RAPD) primers: the two lanes at the extreme left present the template free reactions and the DNA profiles of the parental clone 9507-04 respectively; the two lanes at the extreme right show the segregation of parental RAPD markers in the selected anther-derived progenies.Lanes a-d, primer OPC-07; lanes e-f, primer OPC-15; lanes i to l, primer UBC184.Lane m presents a 2.0 kbp (200 ng) to 0.1 kbp (10 ng) DNA Mass Ladder (Life Technologies).At the left margin relative positions of the DNA markers are shown.The parental RAPD markers were identified by primer name and their sizes (kbp), to be scored as present or absentin the anther derived progeny.

22, 23, 3, 24, 25,8]
) reported to generate polymorphic markers from potato [Their recommended nomenclature of RAPDs was used in this study.The 56 RAPD markers in this study were designated according to the name of primer and size (kbp) of the amplified fragment (Table

Table 2 :Leaf morphological characters of anther-derived plants* from Solanum clone 9507-04
* The segregating leaf morphological characters