Development of a SCAR Marker for Rapid Identification of New Kentucky Bluegrass Breeding Lines

As a commonly used turfgrass, Kentucky bluegrass (Poa pratensis L.) (KBG) has many commercially available cultivars for production. After several years of screening, two new lines were obtained (‘KBG03’ and ‘KBG04’), which have high tolerance to summer. The study showed that the two lines revealed similar morphological characteristics, with light green leaf color, narrow leaf blade, high plant height and light 1,000-grain weight. A total of 400 random amplified polymorphic DNA (RAPD) primers and 256 sequence-related amplified polymorphism (SRAP) primer combinations were screened among the two lines and other 4 imported commercial cultivars. The percentages of polymorphic sites were 65.5% (RAPD) and 22.6% (SRAP) respectively. By cluster analysis of RAPD and SRAP data, the dendrogram at a similarity of 0.29 gave two main clusters, of which one group had 4 commercial cultivars, and the other had the two new breeding lines. Furthermore, one specific band of ‘KBG04’ was successfully converted into a dominant sequence characterized amplified region marker (SCAR196). Then the SCAR marker was verified by 39 KBG DNA samples, including imported varieties, domestic varieties and self-breeding lines of our laboratory, and it exhibited high consistency with the original RAPD polymorphic amplification. The results showed that the SCAR marker can be used to distinguish the new line ‘KBG04’ from numerous KBG germplasms, which would be useful for cultivar identification and property rights protection in the future.


Introduction
Kentucky bluegrass (Poa pratensis L.) (KBG) is a cool-season, attractive truf grass widely used on home lawns, golf courses and athletic fields throughout temperate regions of the world (Beard, 1973).Due to its allopolyploid origin, a complex series of polyploidy and aneuploidy exist among KBG (Huff, 2003), whereas chromosome numbers range from 24 to 124 (Love and Love, 1975).KBG produces seed both asexually (apomixis) and sexually.As to the level of apomixis, different KBG genotypes vary considerably (Meyer, 1982).Ecotype selection and intraspecific hybridization have been demonstrated as successful breeding methods for developing cultivars for the commercial turf market.
In face of a large number of KBG varieties, many domestic studies focused on the identification of traditional botanical characteristics, comparison of some physiological and biochemical characterization of stress (heat, drought, disease etc.) resistance.Due to the complex genetic relationship among the varieties, the identification of germplasms, relying just on morphological traits, is becoming more and more difficult.It is well known that the phenotypic traits are usually influenced by environmental and physiological factors and the number of the traits is so limited.As the DNA molecular markers are more reliable and precise over traditional phenotypic markers, the use of an unlimited number of molecular markers may become a powerful tool for KBG cultivars identification.
As a relatively new marker system, sequence-related amplified polymorphism (SRAP) was first demonstrated by Li and Quiros in Brassica oleracea in 2001.SRAP marker system has many merits over others, such as simplicity, reasonable throughput rate, not crop-specific, numerous co-dominant and clear highintensity bands etc. (Aneja et al., 2012).Above all, SRAP markers Sequence characterized amplified region (SCAR) marker is an efficient molecular marker system, reported by Paran and Michelmore in 1993.For its reliable, specific, sensitive and rapid detection characteristics, it has been widely used in many plants on genetic breeding researches.However, this technology applied in identification of turfgrass germplasms was seldom reported.This study was designed to identify the important agronomic traits and genome-specific molecular markers (SRAP and RAPD) of the new breeding lines.Then a SCAR marker was derived from the polymorphic band and tested in more KBG germplasms.The SCAR marker will be useful for cultivar identification and property rights protection.

Plant material
Two clones ('KBG03' and 'KBG04'), resistant to summer-stress, were selected from about 133 thousand initial single plants of 18 germplasms in turf trial plots.The plot in experiment was under natural and artificial stresses of spring inoculation with one Rhizoctonia Solani Kühn isolate, the casual fungus of brown patch disease, and summer water logging between March 2006 and October 2010 (Yuan et al., 2014).According to the records of selecting procedure, 'KBG03' was originated from 'Midnight' turf test plots and 'KBG04' from 'Evergreen'.Because the similar field visual quality of the two new lines, a combination of the two has been used, as a mixed population (KBG03+KBG04) registered as a new cultivar named 'Huhe 2', which was involved in the identification.
preferentially amplify open reading frames (ORFs) and thus the information given by SRAP markers is more in accordance with the morphological diversity of the germplasms (Ferriol et al., 2003).SRAP markers have been widely used in several crop plants for genetic diversity analysis (Comlekcioglu et al., 2010;Ferriol et al., 2003;Zhao et al., 2009), genetic linkage map construction (Yuan et al., 2008;Xue et al., 2010;Xie et al., 2011), genes tagging (Devran et al., 2011;Zhang et al., 2011) etc.There are also some reports on turfgrass research.SRAP markers are useful for estimating genetic diversity and phenetic relationships among a wide range of cool-and warm-season turfgrass species (Budak et al., 2004a).Budak et al. (2004b) also evaluated genetic diversity in 53 buffalograss germplasms using 34 SRAP primer pairs.Wang et al. (2011) assessed genetic relationships of bermudagrass (Cynodon dactylon var.dactylon) from different countries by 30 SRAP primer combinations.Huang et al. (2012) examined the genetic variation of 12 populations of Hemarthria compressa (Poaceae) using SRAP markers.
Besides the SRAP markers, many more types of molecular markers were used in turfgrass genetic diversity analysis and germplasm identification.For example, using amplified fragment length polymorphism (AFLP) markers, Talebi-Badaf et al. (2006) analyzed the genetic diversity of five grass species with five cultivars from each.Wang et al. (2010) successfully identified 32 clonal turf bermudagrass cultivars by 11 simple sequence repeat (SSR) markers.Using 14 inter-simple sequence repeat (ISSR) primers, Farsani et al. (2012) classified 27 bermudagrass (Cynodon dactylon) genotypes from different parts of Iran into six main clusters.Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic diversity of 19 trufgrass genotypes (mainly including 16 Poa pratensis) (Fard et al., 2012).PCR reactions for RAPD and SRAP markers were performed in 10-μl mixture volume [40 ng genomic DNA, 5 pmol of each primer, 100 μmol/L dNTPs, 1×Taq buffer, 1.5 mmol/L MgCl2, and 0.5 unit of Taq DNA polymerase (TaKaRa, Talien, China)].RAPD PCR reaction was as follows: 94 °C for 3 min, followed by 35 cycles at 94 °C for 10 s, an annealing temperature depending on different primers for 40 s and 72 °C for 45 s, a final extension at 72 °C for 6 min.The amplification products were separated on 1.5% agarose gels.The SRAP PCR reaction was as follows: 94 °C for 3 minutes, followed by the first 8 cycles at 94 °C for 10 s, 37 °C for 30 s and 72 °C for 1 minute.Then, the annealing temperature was raised up to 48 °C for another 35 cycles.The last cycle was run at 72 °C for 6 minutes.The SRAP products were separated in 4% denatured polyacrylamide gels.The DNA bands were visualized by AgNO3 solution (Bassam et al., 1991).All PCR reactions were confirmed at least twice.

Cloning and sequencing the unique fragment of new lines
The unique amplicons of new lines were converted into SCAR markers.The specific fragments were excised from 1.5% agarose gels and retrieved with the Gel Extraction Kit (DV805A, TaKaRa, Talien, China).The target bands were ligated into the pMD18-T vector (D101A, TaKaRa, Talien, China).The positive clones bearing DNA of the expected size were sequenced in both directions by Sangon Biotech (Shanghai, China).

SCAR primer design and amplification
Primers for the SCAR marker were designed using Primer Premier5.0software.One polymorphic SCAR marker was obtained, named as SCAR196 (Forward primer sequence: 5' CCCCCTCCTCCTCTAATAGATATTG 3'; Reverse primer sequence: 5' TCCTCCCCAATATACTTAGGTGATC 3').The conditions of the SCAR amplification was as follows: 94 °C for 3 minutes, followed by 38 cycles at 94 °C for 10 s,56 °C for 30 s and 72 °C for 45 s and a final extension at 72 °C for 6 minutes.

Plant culture and morphological characteristics observation
In March 2012, 1,000-grain weight of the K1-K6 materials was measured in four replications, before seeding.The seeds were sowed into plastic containers filled with horticultural medium [peat:perlite=7:3(v:v)].The containers were 7.6 cm in diameter and 20 cm in height, with holes pierced at the bottom for drainage.The containers of grass were first kept in a greenhouse under natural conditions, for about eight months, in Shanghai, China.Plants received a monthly application of 100 ml fullstrength Hoagland's nutrient solution (Hoagland and Arnon, 1950) to maintain adequate nutrient levels.Turf was cut every two weeks at 6 cm height with a hand-clipper.
In December 2012, plants were transplanted into plots of clayey loamy soil (Soil Survey Staff, 1990) on research farm of School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.From each germplasm 120 plants were randomly sampled for transplanting.The individual plants were spaced at 35 cm distance in rows placed at 50 cm apart.In May 2013, matured plant characteristics were observed before and after seed harvesting.Sixty single plants (replication) were sampled randomly for the measurements of plant height, flag leaf length, flag leaf width, and the second internode length.

RAPD and SRAP analysis
The amounts of 30 flag leaves were collected from each germplasm and frozen for subsequent extraction of genomic DNA with the CTAB method (Clark, 1998).
A total of 400 RAPD primers and 256 SRAP primer combinations were used to screen polymorphisms in the 6 materials.RAPD markers were chosen according to the sequences of NAPS Unit standard primers (University of British Columbia, Canada).SRAP markers were from Li andQuiros (2001), Ferriol et al. (2003), Li et al. (2003) and Wang et al. mean separations were performed with the Fisher's protected least significance difference test at P = 0.05 (Steel and Torrie, 1980).Genetic relationships among the six KBG genotypes (K1-K6) were analyzed with the RAPD and SRAP markers.Polymorphic bands were scored as present (1) or absent (0) among the six genotypes in a binary matrix.The data were analyzed using the Numerical Taxonomy and Multivariate Analysis System (NTSYSpc) version 2.10 (Exeter Software, Setauket, NY) and dendrogram was constructed based on the unweighted pair group method (UPGMA).Simple matching coefficient was used to calculate genetic similarity among the genotypes.

Comparison of matured plants' morphological traits
In different cultivation environments, the seed plumpness of the same genotype may not always be consistent.Imported cultivars were also planted and their seeds were harvested in Shanghai so that the data of their 1,000-grain weight could be more comparable with the domestic cultivars and lines.
Analysis of variance of 1,000-grain weight showed significant difference among the six genotypes (K1-K6).Averaged 1,000grain weight values and significant differences were shown in Fig. 1.The imported seeds were significantly heavier than those of the same genotypes harvested in Shanghai.The 1,000-grain weight of 'Evergreen' and 'Midnight' harvested in Shanghai was only 74% and 65% of that produced in the U.S., respectively.Therefore, Shanghai is not suitable for KBG seed production.There was no significant difference among the 1,000-grain weight of these four imported commercial cultivars' seeds produced in Shanghai.Nevertheless, the 1,000-grain weight of 'KBG03', 'KBG04' and their mixed population (≤0.2 g) were all lighter than those of 'Langara' and 'Evergreen' (produced in Shanghai).
Mean phenotypic values and least significant differences of the mature plant characteristics are presented in Table 3.In all traits, significant differences among these germplasms were detected.The mean plant height, flag leaf length, flag leaf width and second internode length of the new lines ranged from 91.0-93.5 cm, 4.1-4.4cm, 0.28-0.3cm, and 20.5-22.2cm, respectively.The plant height values of 'KBG03' and 'KBG04' were significantly higher than 'Avanlenche', 'Langara' and 'Midnight', and the flag leaf width of 'KBG04' was the narrowest of all.It showed that, within the studied germplasms, the newly selected lines 'KBG03' and 'KBG04' were very similar, with light 1,000grain weight, high plant height and narrow flag leaf width.

RAPD and SRAP polymorphism
Among the six germplasms, 400 RAPD primers were tested for the polymorphic bands; 266 of the 400 primers (66.5%) could amplify 1,284 clear and reproducible bands (Fig. 2).The number of fragments for each primer varied from 1 to 11.From these 266 primers, 841 polymorphic loci were detected at a frequency of 3.2 marker bands per primer.
Cluster analysis 433 reproducible and unambiguous polymorphic bands (327 SRAPs + 106 RAPDs) were analyzed for genetic similarity coefficient (Fig. 4).The dendrogram grouped the six germplasms  into 2 clusters at the 0.29 similarity level.The first cluster included four imported commercial cultivars, while the second cluster was comprised of the new breeding lines ('KBG03' and 'KBG04'), which were grouped at a high level of similarity.

Specificity test using the SCAR marker
The RAPD marker No.196 was converted into SCAR196.Among the six KBG materials (K1-K6), the polymorphic bands amplified by the SCAR marker were identical to those of its corresponding RAPD marker (Fig. 2, Fig. 5).
To test the specificity of the SCAR marker, genomic DNA from 39 KBG materials (including the six materials aforementioned) were used.The polymorphic product was of 1587 bp; it appeared only in Y37 ('KBG04') and Y38 ('Huhe 2 (KBG03+KBG04)') and none in the others.The results showed that the specific band could be amplified by SCAR196 when the sample was 'KBG04'.

Discussions
Kentucky bluegrass is facing the serious threat of heat, high humidity or drought, diseases and insects during summer and early autumn (warm seasons) in Shanghai, China.Through a long screening progress, two newly selected lines were obtained ('KBG03' and 'KBG04') that showed better resistance to warm season stress than original cultivars ('Midnight' and 'Evergreen') under moderately low maintenance conditions, in multi-plot demonstration for several years in Shanghai (Yuan et al. 2014).At present, the mixed population of two lines has been authorized by Shanghai Crop Variety Certification Committee, named as 'Huhe 2'.Besides high stress tolerance, it has light green color, a rapid vertical growth rate and fine-leaf texture.
In this study, two newly bred lines were selected from two commercial varieties, 'Midnight' and 'Evergreen'.But interestingly, it seemed that little comparability between the new lines and their original cultivars existed, neither in phenotype nor in molecular marker polymorphism.However, these two lines were very similar.For seed production and commercialization purposes, the commercial KBG cultivars should be highly apomictic, but hybridization in a facultative apomictic system, is still possible.These aberrant plants stand out from their parent plants, so they could be selected and reproduced asexually.Through the turf comparative trial with control cultivars, new lines might appear.Of course, it is also possible that false seeds were mixed into the varieties during the process of transportation or planting.Yet, from 39 materials collected and adaptation tested in fields during the breeding processing periods, no materials seemed similar with the new lines in phenotypes and stress tolerance.In order to accurately confirm the relationship between the KBG materials, the further study would screen the polymorphism of these germplasms (or even more) by the polymorphic markers obtained from this study.
The SRAP marker system was proved to be very useful in genetic relationship analysis among and within buffalo grasses [Buchloe dactyloides (Nutt.)Englem.]( Budal et al., 2004c).Among closely related cultivars, SRAPs performed high polymorphism (95%), and its average discriminating power was the highest among the four systems (RAPD, ISSR, SSR and SRAP).Huang et al. (2012) also observed that the SRAP markers were more efficient than ISSR markers.The current work is the first time to detect the polymorphism among KGB germplasms by SRAP markers.As expected, more clear and reproducible bands per primer pair were generated by SRAP than RAPD.However, the percentage of polymorphic products in different studies was quite distinct, such as 72.7% in 69 C. pepo accessions with 11 primer combinations (Ferriol et al., 2003), 43.7% in 68 Qingke accessions with 20 primer combinations (Yang et al., 2010), 82.5% in 10 tartary buckwheat accessions with 30 primer combinations (Li et al., 2009), 71.1% in 23 mulberry accessions with 12 primer combinations (Zhao et al., 2009) etc.The present study revealed only 22.6% with 269 primer combinations in 6 KBG genotypes.Obviously, the polymorphism frequency was related to the primer number, material number and species.
It was found that the specific band of 'KBG04' amplified by the SCAR196 (developed from the RAPD amplicon) was distinct.By a BLAST search on the sequence in the NCBI, regrettably there was no significant similarity found in nucleotide databases.Considering the characteristic of SRAP marker system, hopefully more SCAR markers would be developed from SRAP bands.Not only because that SRAP-SCAR marker is an ideal tool for rapid germplasms identification, but also because more information about coding sequences could be obtained for further studies.Besides the limited unique bands of the new lines detected, low success rate of SCAR marker conversion (Horejsi, et al., 1999;Paran and Michelmore, 1993) also resulted in only one SCAR marker achieved for new line identification in this study.Actually, another SCAR marker derived from RAPD was obtained, but its amplified product was also detected in some other varieties (data not shown).
Although a SCAR marker reported here could clearly distinguish the new line 'KBG04' from others, it is hard to assure its specificity in more germplasms.So, more KBG materials would be collected from the world-wide, and then specificity tested by the SCAR marker.Meanwhile, more specific SCAR markers for the new lines, which are converted from SRAP markers, should be developed.With a combination use of more markers for cultivar identification, the result could be more accurate and reliable.Commercially, these molecular markers would also be available to KBG varieties protection.

Table 1 .
Source of the cultivars and lines Codes begin with Y in front are materials for Test 2, while K for Test 1 2 Imported varieties were distributed by: Bright, Beijing Bright Turf & Forage Co., Ltd.; Barenbrug China, Beijing Barenbrug International Co., Ltd.; Clover, Beijing Clover Turf & Forage Co., Ltd. and TopGreen, Beijing TopGreen Seed Co., Ltd.Domestic cultivars were distributed by: GRI IMAA&AHS, Grassland Research Institute of Inner Mongolia Acadermy of Agriculture & Animal Husbandry Science; ISF&ER HAAS, Institute of Soil Fertilizer and Environment Resource of Heilongjiang Acadermy of Agricultural Science; SABS SJTU, School of Agriculture and Biology Science of Shanghai Jiaotong University 81 (2005) (Table 2).All the primers were synthesized by Sangon Biological Engineering Technology and Service Co. Ltd, Shanghai.

Table 2 .
List of SRAP primers used in the present study

Table 3 .
Comparison between several mature plant characteristics of different KBG materials(cm)