Effects of space flight factors on genetic diversity of Buchloe dactyloides seeds

The objective of this research was to investigate the effects of space flight factors on Buchloe dactyloides “Jingyin No.3” seeds. After the retrieval, basic turf characters of plants were tested. Among the 100 plants tested, 21 showed great change on phenotype characters, including leaf blade length and width, height, stem diameter, number of tillers, number and length of stolon, length of stolon inter node, leaf color and extent of leaf turning yellow. 33 primers were screened in inter-simple sequence repeats (ISSR) analysis to evaluate DNA variation between mutations and their ground controls. Results show that 15.6 reliable bands were generated by 7 primers, of which 12.9 (80.9%) were polymorphic. Based on the study, we can conclude that the space flight factors could induce inheritable mutagenic changes on B. dactyloides seeds, and do further research to demonstrate these changes in genetic material of the mutants.


INTRODUCTION
Genetic diversity is an important precursor in studies of many species because the amount and distribution are likely to affect the evolutionary potential of species and/or populations (Futuyma, 1986).
In recent years, the number of molecular assays available for application in this area has increased dramatically.Genetic variation of wide Buchloe dactyloides accessions and nature populations had been evaluated by randomly amplified polymorphic DNA (RAPD) (Huff and Smouse, 1993;Wu, 1991), microsatellites simple sequence repeats, (SSRs), intersimple sequence repeats (ISSR) and sequence-related amplified polymorphism (SRAP) markers (Budak and Shearman, 2004a, b).
To obtain new germplasms by the recoverable satellite launch, a great number of phenotypes have been observed and compared between the spaceflight mutants and ground control.It showed the space treatment could promote these traits such as height, leaf blade width, number of leaves, germination percentage of seed, seedling emergence and growth in field.Yu et al. (2007) found that in rice flown on recoverable satellites, the height of the mutants was 31% taller than that of control.Wang et al. (2004) reported germination potential and germination percentage had been promoted by 17.1 and 4.8%.Soga et al. (2002) reported that elongation growth of Arabidopsis hypocotyls was stimulated under microgravity conditions in space, which was similar to the results for wheat coleoptiles, lettuce hypocotyls, and garden-cress hypocotyls grown in space (Halstead and Dutcher, 1987).Besides the phenotype observation, Fourier Transform Infrared Spectroscopy (FTIR), (Yang et al., 2006), the detection technology of peroxidase isoenzyme pattern (Liu, 1999) and molecular markers, including AFLP (Pu et al., 2006), RAPD (Mei et al., 1998;Ren et al., 2009), SCAR (Wang et al., 2008), SSR (Zhou et al., 2001;Fang et al., 2010) and so on, were also used to space-flown varieties and ground control.
ISSR has been described as a powerful technique to evaluate genetic diversity among closely related species and to detect similarities between and within species levels (Davila et al., 1998;Ghariani et al., 2003;Moreno et al., 1998;Pasakinskiene et al., 2000;Zietkiewicz et al., 1994).Buchloe dactyloides, as one of the oldest gramineae grasses in North America, which has been primarily used as the forage for almost 100 years, also plays an important role in landscaping and environmental sustenance in North China (Riordan et al., 1998).But the study on genetic diversity of B. dactyloides populations after seeds have been flown on satellite by ISSR molecular markers has never been reported.
To uncover the molecular nature of mutation induced by space flight factors, some of B. dactyloides plants with morphological changes were chosen for ISSR analysis in the present study.Our objectives were to detect polymorphisms between mutation plants and ground controls or within mutated individuals based on ISSR.

Plant materials
Dry seeds of B. dactyloides were provided by the Grassland Research Institute, China Agricultural University.

Space flight treatment
The seeds were grouped into two portions: One was kept at 7°C for ground control; the other was prepared for space flight.The seeds were carried into space aboard on a recoverable seed breeding satellite "Practice 8" for a 15 days flight from September 9 to 24 in 2006.The flight conditions were as follows: The distances for perigee and apogee were 180 and 469 km, respectively; orbit obliquity was 63°, and cabin temperatures at flight stage were 7.21-20.72°C.Heavy particle rate was 4.44 particles/cm 2 days.Average space radiation dose for plant seeds at linear energy transfer (LET) space was 4.79 mGy.

Field observation
A single seed from each collected singled spike was planted into an individual pot in the greenhouse, with an approximate temperature 22°C and 16 h photoperiod.Then both space flight and ground control seedlings were planted in the field.Field observation was carried out to evaluate morphological changes of plants developed from flight seeds.Ten types of basic turf characters were observed such as leaf blade length and width, height, stem diameter, number Bao-Yi et al. 12813 of

DNA extraction and ISSR polymerase chain reaction (PCR) amplification
Fresh young leaves were powdered in liquid nitrogen and genomic DNA was extracted using the CTAB method described by Doyle (1991).The isolated genomic DNA was diluted to 10 ng/ml and stored at ¯20°C for ISSR amplification.After purification, DNA samples were quantified using Gene-Quant spectrophotometer (Pharmacia) and their quality was evaluated by electrophoresis on 0.8% agarose gel according to Sambrook et al. (1989).
In a preliminary study, 33 primers provided by University of British Columbia (UBC) were screened for PCR amplification (Table 1).Finally, 7 ISSR primers that generated clear, reproducible banding patterns were selected for further analysis.
The effects of Mg 2+ , dNTP, DNA templates, primers and DNA polymerase on the amplification were tested, and the determined optimal reaction system of ISSR for B. dactyloides was as follows: 1×Taq polymerase buffer, 0.3 mmol/L dNTP, 1.0U Taq DNA polymerase, 0.6 µmol/L Primers, 1.0 mmol/L Mg 2+ , 50 ng of template DNA in the total 25 µl reaction volume.Amplification was performed in a PTC thermocycler (Bio-Rad, USA) under the following cycle profile: an initial step of 3 min at 94°C, followed by 35 cycles, each one including 30 s at 94°C for denaturation, 30 s at 48 to 60°C (depending on the used primer) for annealing and 1 min at 72°C for elongation.A 10 min step at 72°C is programmed as a final extension.The amplification products were separated through electrophoresis on 12.0% polyacrylamide gel electrophoresis (PAGE) and stained with silver staining as described by Bassam et al. (1991).

Data analysis
Firstly, significant analysis and cluster analysis were conducted to the field study data using the SAS8.2 software.Moreover, unequivocally scorable and consistently reproducible amplified ISSR bands were scored as present (1) and absent (0), each of which was treated as an independent character regardless of its intensity.Smeared and weak bands were excluded.Fragments of the same molecular weight were considered as the same locus.The following genetic diversity parameters were calculated: the percentage of polymorphic bands (PPB), the polymorphic information content (PIC), total genetic diversity of materials i and j (Nij), genetic diversity of material i (Ni), genetic diversity of material j (Nj), Nei's (1973) coefficient of genetic differentiation among populations ( ), genetic distance (GD=1-GS).A UPGMA (Unweighted Pair Group Method with Arithmetic Mean) dendrogram was constructed based on the matrix of Nei's (1978) unbiased genetic similarity coefficients (NTSYS-pc version 2.11x, Rolf, 2000) (Table 1).

Morphology observation
After space flight, some characters of the B. dactyloides accessions changed significantly including the leaf blade width, stem diameter, height, leaf color (SPAD), while the others (leaf blade length, number of tillers, number and length of stolon, length of stolon inter node, extent of leaf turning yellow of B. dactyloides) were without significant change (Table 2).
Compared with the ground control, the leaf blade width decreased by 11.60% (P=0.034), that is the leaf became slimmer after been carried with a mean of 1.81 mm.Meanwhile, both the stem diameter (15.63% less than ground control) and the height (7.08% less than ground control) declined after the space flight, with a mean of 0.96 mm (P=0.010) and 26.69 cm (P=0.005)respectively.Moreover, the SPAD value averaged out to 22.52 (P=0.002),higher than ground control, indicating the leaf color darkened significantly (Figure 1).
Selected the mutants among the 21 spaceflight B. dactyloides accessions, which had significant difference in leaf blade length and width, stem diameter, height, number of stolon and leaf color (SPAD) than CK.
Leaf blade length of 1 plant increased significantly while that of 6 plants shortened by comparison with CK.It   had more stolon and 15 mutants had larger SPAD value than CK.

ISSR polymorphism
In the study, the 7 selected primers were applied for PCR amplification of 21 spaceflight B. dactyloides accessions (Figure 2), generated 15.6 reliable bands of which 12.9 (80.9%) were polymorphic.It also demonstrated the good discriminatory power of the markers identified (Table 3).

Cluster analysis
The UPGMA dendrogram based on ISSR obviously revealed the genetic relationships of 21 spaceflight accessions which were been divided into four main clustering groups based on GS value (0.602) (Figure 3).The first cluster (A) included 9 accessions (No.1,3,6,7,10,13,16,17 and 18), of which the GS value ranged from 0.614 to 0.773.The second cluster (B) included 8 accessions (No.2,4,5,8,9,11,14 and 19), of which the GS value ranged from 0.659 to 0.750.The third cluster (C) included 3 accessions (No.15,20 and 21), of which the GS value ranged from 0.636 to 0.727.The fourth cluster (D) included 1 accession (No.12).On the other hand, the cluster analysis based on 6 characters (leaf blade length and width, stem diameter, height, number of stolon and leaf color) with significant difference was also been operated on each single mutants and ground controls accordingly.As a result, the result of cluster analysis based on genetic similarity coefficients did not accord with the one base on phenotype perfectly as a whole.
For example, accessions (No.6 and 16) which had a great GS and a small GD had a major difference in characters including the extent of leaf turning yellow, number and length of stolon while without significant change in number of tillers, stem diameter, length and width of leaf blade.On the contrary, accessions (No.12 and 13) which had a small GS and a great GD had a similar performance in stem diameter, width of leaf blade and leaf color (SPAD).
But there were still few similarities between these two cluster analyses.For instance, accessions (No.15 and 20) in the third cluster(C) (Figure 3) were also be clustered in the same group in the UPGMA-derived dendrogram of B. dactyloides materials regarding height and leaf color (SPAD) variation.That is, they had the same significant changes in the two characters.However, the similarity had not been seen in the cluster analysis based on the other four characters.
In order to find out the relationship between the phenotype and genetic ISSR data, correlation analysis was conducted, and we calculated that the related coefficient was only 0.1874.It revealed the results of morphological analysis has been greatly influenced on external environment and anthropic factor.Consequently, the phenotype and ISSR data of B. dactyloides had no definitely corresponding relations.

DISCUSSION
Space inducement, as a new developed method, has been applied broadly in the research of plenty of crops to find out the truth about what change it brings about, comparing the spaceflight group with the ground control and explain the mechanisms inside.
Firstly, significant morphology mutations were clearly Secondly, based on the study, we can conclude that the spaceflight B. dactyloides mutants changed greatly on several characters and expressed high polymorphism by ISSR with the PPB 80.9% by 7 primers after having been carried to the outer space.It verified these changes not only from appearance but also in the inheritable genotype.Similar results were reported in Cistanche deserticola that 9 out of the 94 ISSR primers were showed high polymorphism, and a total of 118 bands were generated, of which 80 were polymorphic (Wu et al., 2011).The degree of polymorphism exhibited by ISSR banding patterns in their study is considerably high (82.20%)compared to that of the RAPDs reported by Cui et al. (2004) (47.37%) and by Dang et al. (2003) respectively.What is more, in our study,  got higher average PIC (0.85) than that of polymorphism analysis in spaceflight alfalfa (0.6366) by using SSR molecular (Fan et al., 2010).Therefore, ISSR markers were definitely been regarded as an effective tool to reveal the genetic diversity of spaceflight B. dactyloides mutants.
In addition, it should be noted that, in our study, the result of PCR amplication showed part of mutants had less bands than the ground control, which implied that spaceflight mutants had occur deletion under the space radiation environment (Figure 2).This probably is connected with the mutative traits.Reports indicated that cosmic radiation could bring about breakage or deletion of chromosome DNA in the same time microgravity disturbed the DNA repair pathway (Gartenbach et al., 1994).Vibration and linear acceleration, which occurred at the launch and return of satellite, also caused chromosome aberrations, and brought the breakage of large fragment of genomic DNA with the control (Anikeeva et al., 1979;Vaulina and Kostina, 1975).So, a lot of chromosome rearrangement during the carry of B. dactyloides seeds had occurred such as duplication, translocation, inversion, insertion and deletion.The same conclusions were reached in studies on molecular analysis of space flight mutants of rice, kidney bean, tomato (Zhou et al., 2001;Zhang Jian et al., 2000;Lu Jinying et al., 2005).
Thirdly, the result of cluster analysis based on genetic similarity coefficients did not accord with the one base on phenotype perfectly.That is, both the morphological and genetic variation had a considerably complicated relationship.An agreement was reached with the similar conclusion dawn by Fan et al., (2010).She found that alfalfa materials C-01, C-28, D-49, A-07 all with high DNA polymorphism while the former three had obvious change in phenotype and the latter one (A-07) was not seen any variation.Consequently, it revealed that cosmic radiation could bring about great mutations that indicated the great prospect of providing brand-new excellent germplasms for breeding but there is no connection between the phenotype and DNA polymorphism.
Further, they had already been used in grasses, for example: alfalfa (Medicago sativa L.) and yellow tall fescue (Festuca arundinacea) (Fang et al., 2010;Hu et al., 2004).Both of them had something in common that they came to the conclusions based on their studies of consecutive generations from SP1 to further selected progenies.Therefore, to understand the mutagenic effects of space flight on plants and lay a solid foundation of breeding, it is better to conduct long-term research for reasonable data and unshakable evidence.More work is being conducted to select the stable mutants with favorable traits and provide a valuable material in developing new cultivars.

Figure 1 .
Figure 1.Change morphological characters.CK and SM represented check (ground control) and spaceflight mutant, respectively.(A) is variety No.15 , whose leaf color is darker than CK.(B) is the leaf blade width change of variety No.16 whose leaf gets slimmer.(C) showed the plant type changed in height; SM is higher than CK.
tillers, number and length of stolon, length of stolon inter node, leaf color and extent of leaf turning yellow.21 B. dactyloides individuals with great changes in phenotype were selected among the 100 spaceflight B. dactyloides single plants.Leaves of the 21 mutations and 21 plants from ground control were collected for DNA extraction.

Table 1 .
Information of ISSR primers used in the experiment.

Table 2 .
Effects of spaceflight on basic turf characters of B. dactyloides.
1 SM: Spaceflight Mutant;2 CK: Check; 3 P-value: Probability value, which represents a decreasing index of the reliability of a result;4 SEM: Standard Error of the Mean, a measure of how far your sample mean is likely to be from the true population mean;5 SPAD: Soil Plant Analysis Development, which is corresponding to relative chlorophyll concentration.

Table 3 .
The results of ISSR amplication.

Table 4 .
GS value of the 21 spaceflight B. dactyloides accessions based on ISSR.