# Advances towards a Marker-Assisted Selection Breeding Program in Prairie Cordgrass, a Biomass Crop

Authors: K. R. Gedye; J. L. Gonzalez-Hernandez; V. Owens; A. Boe
Journal: International Journal of Plant Genomics
 (2012)

---

## Abstract

Prairie cordgrass (Spartina pectinata Bosc ex Link) is an indigenous, perennial grass of North America that is being developed into a cellulosic biomass crop suitable for biofuel production. Limited research has been performed into the breeding of prairie cordgrass; this research details an initial investigation into the development of a breeding program for this species. Genomic libraries enriched for four simple sequence repeat (SSR) motifs were developed, 25 clones from each library were sequenced, identifying 70 SSR regions, and primers were developed for these regions, 35 of which were amplified under standard PCR conditions. These SSR markers were used to validate the crossing methodology of prairie cordgrass and it was found that crosses between two plants occurred without the need for emasculation. The successful cross between two clones of prairie cordgrass indicates that this species is not self-incompatible. The results from this research will be used to instigate the production of a molecular map of prairie cordgrass which can be used to incorporate marker-assisted selection (MAS) protocols into a breeding program to improve this species for cellulosic biomass production.


---

## Body

## 1. Introduction

Recent world issues associated with fuel consumption and supply have turned attention towards biofuel production, especially cellulosic biofuel. Perennial grasses provide an optimal source of cellulosic biomass due to their high yield potential. Prairie cordgrass (Spartina pectinata Bosc ex Link) is a perennial indigenous grass of North America and can be found as a native from Texas to near the Arctic Circle [1]. Ongoing studies on prairie cordgrass in comparison with switchgrass (Panicum virgatum L.) indicate that prairie cordgrass could produce more biomass than switchgrass [2]. Furthermore, results from the comparison of prairie cordgrass and switchgrass performed by Boe and Lee in 2007 [2] indicated that prairie cordgrass has a wider environmental amplitude and is adapted to poorly drained wet areas which can have high salinity and be poorly aerated, regions not suitable for the production of conventional crops such as maize (Zea mays) [2, 3]. These results are indicative of the potential of prairie cordgrass as a source of biomass for cellulosic biofuel production.A research program at South Dakota State University (SDSU) is underway to develop native prairie cordgrass into a viable cellulosic biomass crop. The development of a new crop species requires a multidisciplinary approach; examining and validating each step before commercialization can occur. These steps include, but are not limited to, the assembling of a germplasm collection, the development of an accelerated breeding program, and the optimization of cultivation practices. A fundamental step to accelerate the breeding of prairie cordgrass is to determine and optimize a crossing protocol. prairie cordgrass has been intrinsically believed to be a protogynous outcrossing species, based on the mode of reproduction of its maritime relative smooth cordgrass (Spartina alterniflora) [4, 5]. From work performed upon other members of the Spartina genus, it has been conjectured that prairie cordgrass will have a similar method of reproduction. In the majority of other Graminaceae species, breeding is performed via the initial emasculation of the floret to ensure that only cross pollination can occur. Prairie cordgrass has an inflorescence composed of between 0 and 31 short paracladia and 11–13 long paracladia [6], bearing a total of 10–80 fertile spikelets, of single florets [7]. Physical emasculation of prairie cordgrass is essentially impractical, but this technique may not be necessary as with protogyny and ascertaining the appropriate timing, directed cross pollination is feasible. Self pollination has not been identified previously in prairie cordgrass and assumptions have been made that prairie cordgrass may be self incompatible, if this is the case then the classic mapping technique of producing recombinant inbred lines will be fundamentally impossible and alternate strategies will need to be utilized.An initial stage of the prairie cordgrass project is to develop a molecular map of the species. Only limited molecular analyses have been performed upon prairie cordgrass, notably in contrast to its maritime relativeS. alterniflora. In the National Centre for Biotechnology Information (NCBI), only 57 prairie cordgrass sequences have been deposited (predominantly regions of the nuclear and organelle genomes utilized for diversity analysis, that is, Waxy (AY508655 and AF372461), ITS (AF019843, AJ489796, and EF153082) and the chloroplast trnL-trnF intergenic spacer region (EF137568, EU056305, and AF372625)). A recent publication on the analysis of the transcriptome of prairie cordgrass has increased the available knowledge on the genome of the species [8]. Constructed from the analysis of the expression sequence tags (ESTs) and identified from the transcriptome of prairie cordgrass, a total of 26,302 contigs and 71,103 singletons were assembled with all sequence information available as supplemental data [8]. Additional molecular analysis of genetic diversity of prairie cordgrass in natural populations in Minnesota with amplified fragment length polymorphism (AFLP) has been performed [9].An optimal marker system for the development of a molecular map of prairie cordgrass are SSRs or microsatellites. Microsatellites are highly utilized molecular markers and have been developed for the majority of agronomically and economically important crop species [10]; their efficacy arises predominantly due to their reproducibility, codominant inheritance, and abundance [11]. Modern techniques allow the rapid identification of microsatellite regions in species which have limited sequence information, and by using proprietary genomic library screening techniques, microsatellites have been developed for numerous non-model organisms, encompassing insects [12], birds [13], fish [14], mammals [15], and plants, including other species in the Spartina genera [16]. Previous studies on other members of the Spartina genera have developed 35 markers for microsatellite loci in S. alterniflora [16, 17]. Of these S. alterniflora markers, three have been found to amplify in prairie cordgrass [16]. SSRs have been used extensively in numerous crop species, in wheat (Triticum aestivum L.), and another Poaceae species; SSRs have been used to enhance molecular maps begun with restriction fragment length polymorphism (RFLP) and to identify genes of interest [18]. Furthermore, Wilde et al. in 2007 [19] found that the incorporation of MAS with SSR markers into a traditional breeding program can result in a substantial increase in the incorporation of Fusarium head blight resistance, a quantitative trait, in wheat with the shortest possible time. The results of Wilde et al. [19] are indicative of the potential of MAS for the breeding of prairie cordgrass.This paper details the first investigation into initial breeding and crossing of prairie cordgrass, specifically the production of F1 individuals and the validation of a crossing protocol. Furthermore, the characterization of 35 microsatellite loci in prairie cordgrass from genomic DNA is discussed. The validation of the microsatellite loci occurred in a prairie cordgrass germplasm collection and a reciprocal cross as an initial step in the development of a molecular map, which can be further utilized during marker-assisted selection of lines with traits desirable for the improvement of prairie cordgrass as a biofuel crop.


## 2. Materials and Methods

### 2.1. Genomic Library Construction

Genomic DNA was extracted from four lines of prairie cordgrass using the method as described by Karakousis and Langridge in 2003 [20], with minor modifications. A mixture of the four random prairie cordgrass clones DNA, totaling > 100 μg, was sent to Genetic Identification Services (GIS) (http://www.genetic-id-services.com/) for the development of genomic libraries. The genomic libraries were enriched for the four simple sequence repeat (SSR) motifs (CA)n, (GA)n, (AAG)n, and (CAG)n. Subsamples of 100 clones were sequenced and primers were designed to flank the SSR regions using DesignerPCR, version 1.03 (Research Genetics, Inc.) and PRIMER 3 [21]. The SSR regions were classified according to Jones et al. [22] as being pure repeats (i.e., [N1N2]X), compound repeats (i.e., [N1N2]X[N3N4]Y), and interrupted repeats (i.e., [N1N2]XN3[N4N5]Y).


### 2.2. Plant Material

Wild germplasm has been collected from throughout the mid-western states of the United States, creating a core germplasm collection to be utilized in prairie cordgrass breeding (unpublished data). A geographically diverse sample of sixteen plants, collected from South Dakota, North Dakota, Minnesota, and Iowa, was characterized using the identified SSR loci. A sample from the closely related speciesS. spartinae was also included to examine cross species amplification. The germplasm collection was grown in standard greenhouse conditions.Crosses between and amongst these genotypes were performed. Crossing between two plants of prairie cordgrass was performed in the following manner; two inflorescence at the appropriate stage of development were placed inside a crossing bag for seven days, producing F1 plants (Figure 1). A reciprocal cross between two genotypes (designated RR2 and RR21) from the Red River morphotype was produced. The reciprocal cross produced 45 F1 plants from the RR21 × RR2 directed cross and 49 F1 plants from the RR2 × RR21 directed cross, a total of 94 plants. Two genotypes identified as clones (designated SP1.1B and SP1.2A) from the SSR evaluation were crossed via the same technique, producing a total of 46 F1 individuals; a total of 20 F1 plants were produced when SP1.1B was used as the maternal parent and 26 F1 plants when SP1.2A was used as the maternal parent. All F1 plants and their respective parents were grown in standard greenhouse conditions.Figure 1
Two prairie cordgrass heads undergoing crossing, contained within a single bag.


### 2.3. DNA Extraction and Evaluation of SSR Primers

DNA was extracted from all examined plants using the method as described by Karakousis and Langridge in 2003 [20], with minor modifications. Evaluation of the primers was performed in a PCR reaction consisting of 2 U Taq DNA polymerase (Promega GoTaq), 1 × PCR Buffer (Promega GoTaq), 2 mM MgCl2, 0.6 mM dNTPs, and 0.6 mM of each primer, with a final volume of 20 μL [23]. PCR reactions were performed in a BioRad MyCycler (BioRad, Hercules, CA). Initially a gradient of annealing temperatures was used to determine optimal Ta, as these primers were designed to be utilized in a prairie cordgrass breeding program, a robust and high-throughput protocol was developed. Specific thermocycling conditions for the primers used in this study were as follows: an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 1 minute, 53 or 55°C for 1 minute, 72°C for 1 minute, followed by an extension step of 72°C for 10 minutes, and a 10°C hold. PCR product was visualized on 8% nondenaturing PAGE; bands were scored and sized by comparison to a 100 bp ladder using AlphaEaseFC  Software (Alpha Innotech, San Leandro, CA).


### 2.4. SSR Markers Analysis

Polymorphic bands in the two examined populations described above were scored on a presence(1) or absence (0) basis. An estimation of genetic distance was calculated with Nei and Li’s algorithm [24] and the resulting matrix was clustered with unweighted pair group method with arithmetic mean (UPGMA) [25]. Analysis was performed using multivariate statistics package (MVSP) [26].


## 2.1. Genomic Library Construction

Genomic DNA was extracted from four lines of prairie cordgrass using the method as described by Karakousis and Langridge in 2003 [20], with minor modifications. A mixture of the four random prairie cordgrass clones DNA, totaling > 100 μg, was sent to Genetic Identification Services (GIS) (http://www.genetic-id-services.com/) for the development of genomic libraries. The genomic libraries were enriched for the four simple sequence repeat (SSR) motifs (CA)n, (GA)n, (AAG)n, and (CAG)n. Subsamples of 100 clones were sequenced and primers were designed to flank the SSR regions using DesignerPCR, version 1.03 (Research Genetics, Inc.) and PRIMER 3 [21]. The SSR regions were classified according to Jones et al. [22] as being pure repeats (i.e., [N1N2]X), compound repeats (i.e., [N1N2]X[N3N4]Y), and interrupted repeats (i.e., [N1N2]XN3[N4N5]Y).


## 2.2. Plant Material

Wild germplasm has been collected from throughout the mid-western states of the United States, creating a core germplasm collection to be utilized in prairie cordgrass breeding (unpublished data). A geographically diverse sample of sixteen plants, collected from South Dakota, North Dakota, Minnesota, and Iowa, was characterized using the identified SSR loci. A sample from the closely related speciesS. spartinae was also included to examine cross species amplification. The germplasm collection was grown in standard greenhouse conditions.Crosses between and amongst these genotypes were performed. Crossing between two plants of prairie cordgrass was performed in the following manner; two inflorescence at the appropriate stage of development were placed inside a crossing bag for seven days, producing F1 plants (Figure 1). A reciprocal cross between two genotypes (designated RR2 and RR21) from the Red River morphotype was produced. The reciprocal cross produced 45 F1 plants from the RR21 × RR2 directed cross and 49 F1 plants from the RR2 × RR21 directed cross, a total of 94 plants. Two genotypes identified as clones (designated SP1.1B and SP1.2A) from the SSR evaluation were crossed via the same technique, producing a total of 46 F1 individuals; a total of 20 F1 plants were produced when SP1.1B was used as the maternal parent and 26 F1 plants when SP1.2A was used as the maternal parent. All F1 plants and their respective parents were grown in standard greenhouse conditions.Figure 1
Two prairie cordgrass heads undergoing crossing, contained within a single bag.


## 2.3. DNA Extraction and Evaluation of SSR Primers

DNA was extracted from all examined plants using the method as described by Karakousis and Langridge in 2003 [20], with minor modifications. Evaluation of the primers was performed in a PCR reaction consisting of 2 U Taq DNA polymerase (Promega GoTaq), 1 × PCR Buffer (Promega GoTaq), 2 mM MgCl2, 0.6 mM dNTPs, and 0.6 mM of each primer, with a final volume of 20 μL [23]. PCR reactions were performed in a BioRad MyCycler (BioRad, Hercules, CA). Initially a gradient of annealing temperatures was used to determine optimal Ta, as these primers were designed to be utilized in a prairie cordgrass breeding program, a robust and high-throughput protocol was developed. Specific thermocycling conditions for the primers used in this study were as follows: an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 1 minute, 53 or 55°C for 1 minute, 72°C for 1 minute, followed by an extension step of 72°C for 10 minutes, and a 10°C hold. PCR product was visualized on 8% nondenaturing PAGE; bands were scored and sized by comparison to a 100 bp ladder using AlphaEaseFC  Software (Alpha Innotech, San Leandro, CA).


## 2.4. SSR Markers Analysis

Polymorphic bands in the two examined populations described above were scored on a presence(1) or absence (0) basis. An estimation of genetic distance was calculated with Nei and Li’s algorithm [24] and the resulting matrix was clustered with unweighted pair group method with arithmetic mean (UPGMA) [25]. Analysis was performed using multivariate statistics package (MVSP) [26].


## 3. Results

### 3.1. Germplasm and Crossing

The crossing of the germplasm collection was successful with the production of a total of 14,813 putatively viable seeds from 110 crosses, performed in lines and collected from 48 distinct collection locations (Table1). Numerous nonviable seeds were produced from each cross. A seed was determined to be viable if the actual seed (endosperm and embryo) was visible within the glumes (Figure 2). The number of viable seeds produced per head varied from 6 to 642, with an average of 134 seeds per cross. The crosses can be grouped into two, dependent upon the parents, those that were outcrossed and those that were selfed. Crosses that were designated “out” were between genotypes that were geographically diverse, while crosses designated “self” were produced from members of the population collected at the same location. The average number of seeds produced from outcrossed individuals was 143, with a range of 12 to 250, while selfed individuals produced an average of 112 seeds per cross, with a range of 6 to 642 (Figure 3).Table 1
Summary of crosses performed amongst the core prairie cordgrass germplasm collection. The type of cross is designated “Out” for crosses between geographically diverse members and “Self” for members of the population collected at the same location.
Female parent
Male parent
Number of crosses
Type
Total no. of seeds
SP1
SP21
2
Out
126
SP1
SP71
1
Out
71
SP21
SP30
1
Out
51
SP21
SP31
6
Out
484
SP21
SP4
2
Out
248
SP21
SP42
1
Out
78
SP21
SP66
2
Out
233
SP21
SP7
1
Out
82
SP24
SP30
1
Out
180
SP29
SP22
1
Out
146
SP29
SP30
1
Out
42
SP3
SP77
1
Out
282
SP30
SP31
1
Out
164
SP30
SP52
1
Out
246
SP31
SP21
4
Out
408
SP31
SP30
1
Out
28
SP31
SP32
1
Out
38
SP31
SP46
3
Out
211
SP31
SP5
1
Out
81
SP31
SP66
1
Out
46
SP31
SP79
1
Out
198
SP32
SP21
1
Out
173
SP32
SP31
2
Out
310
SP32
SP45
1
Out
296
SP32
SP66
1
Out
240
SP32
SP67
1
Out
193
SP32
SP78
1
Out
94
SP35
SP78
1
Out
279
SP41
SP42
1
Out
6
SP41
SP45
1
Out
86
SP44
SP54
1
Out
312
SP44
SP59
1
Out
274
SP45
SP31
1
Out
282
SP45
SP49
1
Out
225
SP45
SP67
1
Out
138
SP49
SP40
1
Out
203
SP49
SP45
1
Out
176
SP49
SP66
2
Out
237
SP5
SP31
1
Out
189
SP5
SP78
1
Out
407
SP53
SP67
2
Out
43
SP54
SP44
1
Out
145
SP54
SP59
2
Out
81
SP56
SP58
1
Out
235
SP65
SP57
1
Out
70
SP66
SP31
1
Out
93
SP67
SP32
1
Out
99
SP67
SP45
2
Out
583
SP67
SP53
1
Out
185
SP7
SP46
1
Out
160
SP72
SP1
1
Out
125
SP72
SP30
2
Out
459
SP75
SP13
1
Out
59
SP77
SP72
1
Out
163
SP77
SP79
1
Out
184
SP78
SP35
1
Out
94
SP78
SP79
1
Out
113
SP82
SP14
1
Out
642
SP21
SP21
13
Self
1470
SP22
SP22
1
Self
110
SP31
SP31
13
Self
1377
SP40
SP40
1
Self
126
SP41
SP41
2
Self
393
SP53
SP53
1
Self
12
SP74
SP74
2
Self
229Prairie cordgrass seeds photographs taken over a light box. Illuminating prairie cordgrass with light box is used to distinguish the presence of a developed embryo in the seeds. (a) Non-viable seeds. (b) Viable seeds.
(a)
(b)Figure 3
Summary graph of results of crosses between members of the core germplasm collection of prairie cordgrass. White bars indicate self crosses and black bars indicate out crosses.


### 3.2. Microsatellite Characterization

Of the total 100 genomic clones sequenced, 70 contained microsatellite motifs, 26 from the (CA)n enriched library, 25 from the (GA)n enriched library, 3 from the (AAG)n enriched library, and 16 from the (CAG)n enriched library. All 70 loci were examined and 35 were amplified with the standardized conditions (Table 2). The repeat SSR structure that occurred most frequently was the pure repeat (27), followed by the compound repeat (7) and only one interrupted repeat sequence was found (Table 2).Table 2
Summary and description of primer sequences designed for SSR regions identified in prairie cordgrass and their amplification products.
Locus
Library
Primer sequence (5′3′)
SSR motif
Expectedsize (bp)
Sizerange  (bp)
T
a
(°C)
GenBank ID
SPSD001
(
CA
)
n
F: GCTGCTCCTCTTCTCTGTCT
(CA)13
180
212–386
55
GQ354531
R: ACGGCACACTTAGTTTTCTG
Perfect
SPSD002
(
CA
)
n
F: GCACTGTTTGGTGATGCTC
(CA)13
249
100–655
55
GQ354532
R: CTGACGCAAGGTTGATGAG
Perfect
SPSD009
(
CA
)
n
F: ATGGTTTCACAAGTCGGAAGT
(GT)15
166
215–432
55
GQ354533
R: CAGGGCTGCCTACAAGATG
Perfect
SPSD010
(
CA
)
n
F: AACCAAAAGGATAGACCCTA
(CA)28
168
127–310
53
GQ354534
R: ACGAAATATGTGACCGATAC
Perfect
SPSD011
(
CA
)
n
F: CTAACGTATGTCGTTCATGTGG
(GT)11
135
147–251
53
GQ354535
R: AAGGCGATTTTAAGAGGCTAAG
Perfect
SPSD013
(
CA
)
n
F: GGGATGCTTTGTAGATAAGAAA
(CA)41
157
85–173
55
GQ354536
R: TCTTCCTCTTTACTCTGTCACC
Perfect
SPSD016
(
CA
)
n
F: CCGACTACGAGCCACATT
(CA)14
155
156–268
53
GQ354537
R: GTTCCACACATACGAAGGAGA
Perfect
SPSD019
(
CA
)
n
F: CCTGCTTACTCTTACTCCGTC
(CA)13(GA)7
134
220–365
53
GQ354538
R: ACCCTTTTTTCTTTTGGTCTC
Compound
SPSD020
(
CA
)
n
F: ATGAGACGATAGCAGGATGAC
(CA)23
178
146–288
55
GQ354539
R: AGCAGATTACGATTCAGATGG
Perfect
SPSD025
(
CA
)
n
F: CAGTCCATGCAACTCAGAAGTA
(CT)10(CA)38
269
204–748
55
GQ354540
R: AACCTGATAGAAGTGGTCATGC
Compound
SPSD026
(
CA
)
n
F: GTGGAATCAACAACACCAGA
(GA)13(GT)20
209
196–539
55
GQ354541
R: GTCGCTTTAGCCCGTAAG
Compound
SPSD003
(
GA
)
n
F: ATGGAAACTGTCTGGAACTGAC
(CT)28
294
231–263
55
GQ354542
R: AGCAATAACCACAGAAGAGACC
Perfect
SPSD027
(
GA
)
n
F: TCAAACAATGGCGGAGAAG
(CT)17
214
188–224
55
GQ354543
R: CTGGCTCCACCTCTTTGG
Perfect
SPSD004
(
GA
)
n
F: GTTGCTCGGTTCCAGTTG
(GA)22
178
133 & 164
55
GQ354544
R: CGCCACACAAAAGTAGCC
Perfect
SPSD031
(
GA
)
n
F: TCGCACTTTTGATTCTCTTTAC
(CT)20
188
155–346
53
GQ354545
R: TGGATGGATTAGGTTACTGTTG
Perfect
SPSD032
(
GA
)
n
F: CTCTCGCCCATTGCTACTTA
(CT)13
196
146–193
53
GQ354546
R: CCATTGCTATGTTGTTTGAGC
Perfect
SPSD034
(
GA
)
n
F: CAGGTCTACGGAGGTCACTAC
(CT)12
160
144–306
53
GQ354564
R: TCAAAAGAAGAGCACATACACA
Perfect
SPSD036
(
GA
)
n
F: TTCACCACACCACTTTATCC
(CT)29
271
222–260
53
GQ354547
R: GGAAGCAACAAACATTGATG
Perfect
SPSD039
(
GA
)
n
F: CTTTCAGATAGCTCCACTGATC
(GA)12
206
193–448
55
GQ354548
R: AGCAATAACTGTGCATACCTCT
Perfect
SPSD040
(
GA
)
n
F: AATCGAAGTAGCAGACACCAAC
(GA)11
214
190–416
55
GQ354549
R: CATGCGTTTTTCACTCATGTAG
Perfect
SPSD041
(
GA
)
n
F: CCCAACGATGATTTCTCTTG
(GA)26
135
104–180
53
GQ354550
R: TCACGGTAACACGATTAGTCC
Perfect
SPSD042
(
GA
)
n
F: ACCTCCCACTCGTTGCTAC
(CT)19
211
131–257
53
GQ354551
R: GCCATTGCTCTGTTGTTTG
Perfect
SPSD043
(
GA
)
n
F: GTTCAAATGCGAACAAATCAG
(GA)30
277
231–252
53
GQ354552
R: ATTCGATCTCACATGCAACAC
Perfect
SPSD044
(
GA
)
n
F: AGCTATATGACCCGAGACTGTG
(GA)20
275
254–276
55
GQ354553
R: GGAATGGTCCCATACTTAATCC
Perfect
SPSD045
(
GA
)
n
F: AACGGAGGAAGTAATAAATAGC
(CT)16
241
214–426
53
GQ354554
R: AGCACACACTAGCAAGGAC
Perfect
SPSD046
(
GA
)
n
F: CAGGTTTATCAGTGAAGACATC
(TA)9(GA)12
278
57–426
53
GQ354555
R: GAGGTTCTTAAAGGAACATAGC
Compound
SPSD047
(
GA
)
n
F: CCACCTTCCTTGGATACAC
(CT)17
194
156–326
53
GQ354556
R: CCACAACTACCACCTC
Perfect
SPSD005
(
AAG
)
n
F: TGAACCAACATAACCTACCTG
(TTC)19
297
192–452
55
GQ354557
R: CCACACTAAACCGAAACTTG
Perfect
SPSD048
(
AAG
)
n
F: AAGGGCATAGTTTCAACCAAG
(AAG)10
197
88
55
GQ354565
R: CTTTTGCTTGTTCATCAACATG
Perfect
SPSD007
(
CAG
)
n
F: AATCCTTCGCCTATCCTACAC
(CTG)8(CT)9
168
146–519
55
GQ354558
R: TTCACACAGCAGACAGAACTG
Compound
SPSD008
(
CAG
)
n
F: GCAAGAACAGACTCAAGAGC
(CAG)9(CAA)4(CAG)4(CAA)2
276
144–309
55
GQ354559
R: CTGCTGCTGAAGTAAAAGTTG
Compound
SPSD049
(
CAG
)
n
F: TGGATTGTTTCCTGATACTCCA
(TTG)5(CTG)5
299
303–667
53
GQ354560
R: CCATAAATTGCTGCATTATTCC
Compound
SPSD050
(
CAG
)
n
F: GAAGCAGAAAACACAGTATTGC
(CTG)5
225
225–505
53
GQ354561
R: TTGCTGGAATTTAACCTATCTG
Perfect
SPSD053
(
CAG
)
n
F: ACGCCTTCTTCACTCCAAC
(CTG)8
208
166–316
53
GQ354562
R: GCCACCAGTTTTCATCACC
Perfect
SPSD056
(
CAG
)
n
F: GTTCTCCAAAGTCTCCTCCT
(TCC)6TTC(TCC)2
197
79–230
53
GQ354563
R: ATCTTTACCTTCCTTCTGGG
InterruptedOnly two primers produced monomorphic profiles from the analysis of the sixteen lines, SPSD004 from the (GA)n library and SPSD048 from the (AAG)n library. The remaining primers produced between 1 and 12 scorable bands. Of the examined primers, 11 were amplified between 1 and 7 bands in S. spartinae (Table 2).All sequences were examined using BLASTn [27] on the National Center for Biotechnology Information (NCBI) server for similarities to other recorded sequences. Only two of the prairie cordgrass sequences displayed homology (e≤e-20) to recorded sequences, SPSD003 to six sequences from rice, wheat, and field mustard (Brassica rapa) (accession numbers: AP008214.1, AP005495.3, NM_001067901.1, AK111788.1, EU660901.1, and AC189183.2) and SPSD050 to five sequences all from rice (accession numbers: CR855236.1, AP008210.1, AL606607.3, NM_001059819.1, and AK108706.1) [25].


### 3.3. Crossing Population

A total of 94 individuals from the RR2 × RR21 and the RR21 × RR2 crosses were examined with 35 SSR primers. The amplified bands were scored based on presence or absence, the resulting data was analyzed with Nei and Li’s coefficient producing a similarity matrix (data not shown), and the data was clustered with UPGMA producing a dendogram (Figure4). The results of the dendogram indicate that each individual was a cross between the two parents. When the population derived from the cross between the two plants (SP1.1B and SP1.2A) and determined to be clones was examined, bands present in both parents were found to segregate in the progeny (data not shown), indicative of sexual recombination; this result provides validation that successful crossing occurred.Figure 4
Unrooted dendogram clustered with UPGMA of the genetic association of the F1 progeny from a cross between prairie cordgrass lines RR21 and RR2, from the analysis of SSR regions with Nei and Li’s Coefficient of genetic similarity.


## 3.1. Germplasm and Crossing

The crossing of the germplasm collection was successful with the production of a total of 14,813 putatively viable seeds from 110 crosses, performed in lines and collected from 48 distinct collection locations (Table1). Numerous nonviable seeds were produced from each cross. A seed was determined to be viable if the actual seed (endosperm and embryo) was visible within the glumes (Figure 2). The number of viable seeds produced per head varied from 6 to 642, with an average of 134 seeds per cross. The crosses can be grouped into two, dependent upon the parents, those that were outcrossed and those that were selfed. Crosses that were designated “out” were between genotypes that were geographically diverse, while crosses designated “self” were produced from members of the population collected at the same location. The average number of seeds produced from outcrossed individuals was 143, with a range of 12 to 250, while selfed individuals produced an average of 112 seeds per cross, with a range of 6 to 642 (Figure 3).Table 1
Summary of crosses performed amongst the core prairie cordgrass germplasm collection. The type of cross is designated “Out” for crosses between geographically diverse members and “Self” for members of the population collected at the same location.
Female parent
Male parent
Number of crosses
Type
Total no. of seeds
SP1
SP21
2
Out
126
SP1
SP71
1
Out
71
SP21
SP30
1
Out
51
SP21
SP31
6
Out
484
SP21
SP4
2
Out
248
SP21
SP42
1
Out
78
SP21
SP66
2
Out
233
SP21
SP7
1
Out
82
SP24
SP30
1
Out
180
SP29
SP22
1
Out
146
SP29
SP30
1
Out
42
SP3
SP77
1
Out
282
SP30
SP31
1
Out
164
SP30
SP52
1
Out
246
SP31
SP21
4
Out
408
SP31
SP30
1
Out
28
SP31
SP32
1
Out
38
SP31
SP46
3
Out
211
SP31
SP5
1
Out
81
SP31
SP66
1
Out
46
SP31
SP79
1
Out
198
SP32
SP21
1
Out
173
SP32
SP31
2
Out
310
SP32
SP45
1
Out
296
SP32
SP66
1
Out
240
SP32
SP67
1
Out
193
SP32
SP78
1
Out
94
SP35
SP78
1
Out
279
SP41
SP42
1
Out
6
SP41
SP45
1
Out
86
SP44
SP54
1
Out
312
SP44
SP59
1
Out
274
SP45
SP31
1
Out
282
SP45
SP49
1
Out
225
SP45
SP67
1
Out
138
SP49
SP40
1
Out
203
SP49
SP45
1
Out
176
SP49
SP66
2
Out
237
SP5
SP31
1
Out
189
SP5
SP78
1
Out
407
SP53
SP67
2
Out
43
SP54
SP44
1
Out
145
SP54
SP59
2
Out
81
SP56
SP58
1
Out
235
SP65
SP57
1
Out
70
SP66
SP31
1
Out
93
SP67
SP32
1
Out
99
SP67
SP45
2
Out
583
SP67
SP53
1
Out
185
SP7
SP46
1
Out
160
SP72
SP1
1
Out
125
SP72
SP30
2
Out
459
SP75
SP13
1
Out
59
SP77
SP72
1
Out
163
SP77
SP79
1
Out
184
SP78
SP35
1
Out
94
SP78
SP79
1
Out
113
SP82
SP14
1
Out
642
SP21
SP21
13
Self
1470
SP22
SP22
1
Self
110
SP31
SP31
13
Self
1377
SP40
SP40
1
Self
126
SP41
SP41
2
Self
393
SP53
SP53
1
Self
12
SP74
SP74
2
Self
229Prairie cordgrass seeds photographs taken over a light box. Illuminating prairie cordgrass with light box is used to distinguish the presence of a developed embryo in the seeds. (a) Non-viable seeds. (b) Viable seeds.
(a)
(b)Figure 3
Summary graph of results of crosses between members of the core germplasm collection of prairie cordgrass. White bars indicate self crosses and black bars indicate out crosses.


## 3.2. Microsatellite Characterization

Of the total 100 genomic clones sequenced, 70 contained microsatellite motifs, 26 from the (CA)n enriched library, 25 from the (GA)n enriched library, 3 from the (AAG)n enriched library, and 16 from the (CAG)n enriched library. All 70 loci were examined and 35 were amplified with the standardized conditions (Table 2). The repeat SSR structure that occurred most frequently was the pure repeat (27), followed by the compound repeat (7) and only one interrupted repeat sequence was found (Table 2).Table 2
Summary and description of primer sequences designed for SSR regions identified in prairie cordgrass and their amplification products.
Locus
Library
Primer sequence (5′3′)
SSR motif
Expectedsize (bp)
Sizerange  (bp)
T
a
(°C)
GenBank ID
SPSD001
(
CA
)
n
F: GCTGCTCCTCTTCTCTGTCT
(CA)13
180
212–386
55
GQ354531
R: ACGGCACACTTAGTTTTCTG
Perfect
SPSD002
(
CA
)
n
F: GCACTGTTTGGTGATGCTC
(CA)13
249
100–655
55
GQ354532
R: CTGACGCAAGGTTGATGAG
Perfect
SPSD009
(
CA
)
n
F: ATGGTTTCACAAGTCGGAAGT
(GT)15
166
215–432
55
GQ354533
R: CAGGGCTGCCTACAAGATG
Perfect
SPSD010
(
CA
)
n
F: AACCAAAAGGATAGACCCTA
(CA)28
168
127–310
53
GQ354534
R: ACGAAATATGTGACCGATAC
Perfect
SPSD011
(
CA
)
n
F: CTAACGTATGTCGTTCATGTGG
(GT)11
135
147–251
53
GQ354535
R: AAGGCGATTTTAAGAGGCTAAG
Perfect
SPSD013
(
CA
)
n
F: GGGATGCTTTGTAGATAAGAAA
(CA)41
157
85–173
55
GQ354536
R: TCTTCCTCTTTACTCTGTCACC
Perfect
SPSD016
(
CA
)
n
F: CCGACTACGAGCCACATT
(CA)14
155
156–268
53
GQ354537
R: GTTCCACACATACGAAGGAGA
Perfect
SPSD019
(
CA
)
n
F: CCTGCTTACTCTTACTCCGTC
(CA)13(GA)7
134
220–365
53
GQ354538
R: ACCCTTTTTTCTTTTGGTCTC
Compound
SPSD020
(
CA
)
n
F: ATGAGACGATAGCAGGATGAC
(CA)23
178
146–288
55
GQ354539
R: AGCAGATTACGATTCAGATGG
Perfect
SPSD025
(
CA
)
n
F: CAGTCCATGCAACTCAGAAGTA
(CT)10(CA)38
269
204–748
55
GQ354540
R: AACCTGATAGAAGTGGTCATGC
Compound
SPSD026
(
CA
)
n
F: GTGGAATCAACAACACCAGA
(GA)13(GT)20
209
196–539
55
GQ354541
R: GTCGCTTTAGCCCGTAAG
Compound
SPSD003
(
GA
)
n
F: ATGGAAACTGTCTGGAACTGAC
(CT)28
294
231–263
55
GQ354542
R: AGCAATAACCACAGAAGAGACC
Perfect
SPSD027
(
GA
)
n
F: TCAAACAATGGCGGAGAAG
(CT)17
214
188–224
55
GQ354543
R: CTGGCTCCACCTCTTTGG
Perfect
SPSD004
(
GA
)
n
F: GTTGCTCGGTTCCAGTTG
(GA)22
178
133 & 164
55
GQ354544
R: CGCCACACAAAAGTAGCC
Perfect
SPSD031
(
GA
)
n
F: TCGCACTTTTGATTCTCTTTAC
(CT)20
188
155–346
53
GQ354545
R: TGGATGGATTAGGTTACTGTTG
Perfect
SPSD032
(
GA
)
n
F: CTCTCGCCCATTGCTACTTA
(CT)13
196
146–193
53
GQ354546
R: CCATTGCTATGTTGTTTGAGC
Perfect
SPSD034
(
GA
)
n
F: CAGGTCTACGGAGGTCACTAC
(CT)12
160
144–306
53
GQ354564
R: TCAAAAGAAGAGCACATACACA
Perfect
SPSD036
(
GA
)
n
F: TTCACCACACCACTTTATCC
(CT)29
271
222–260
53
GQ354547
R: GGAAGCAACAAACATTGATG
Perfect
SPSD039
(
GA
)
n
F: CTTTCAGATAGCTCCACTGATC
(GA)12
206
193–448
55
GQ354548
R: AGCAATAACTGTGCATACCTCT
Perfect
SPSD040
(
GA
)
n
F: AATCGAAGTAGCAGACACCAAC
(GA)11
214
190–416
55
GQ354549
R: CATGCGTTTTTCACTCATGTAG
Perfect
SPSD041
(
GA
)
n
F: CCCAACGATGATTTCTCTTG
(GA)26
135
104–180
53
GQ354550
R: TCACGGTAACACGATTAGTCC
Perfect
SPSD042
(
GA
)
n
F: ACCTCCCACTCGTTGCTAC
(CT)19
211
131–257
53
GQ354551
R: GCCATTGCTCTGTTGTTTG
Perfect
SPSD043
(
GA
)
n
F: GTTCAAATGCGAACAAATCAG
(GA)30
277
231–252
53
GQ354552
R: ATTCGATCTCACATGCAACAC
Perfect
SPSD044
(
GA
)
n
F: AGCTATATGACCCGAGACTGTG
(GA)20
275
254–276
55
GQ354553
R: GGAATGGTCCCATACTTAATCC
Perfect
SPSD045
(
GA
)
n
F: AACGGAGGAAGTAATAAATAGC
(CT)16
241
214–426
53
GQ354554
R: AGCACACACTAGCAAGGAC
Perfect
SPSD046
(
GA
)
n
F: CAGGTTTATCAGTGAAGACATC
(TA)9(GA)12
278
57–426
53
GQ354555
R: GAGGTTCTTAAAGGAACATAGC
Compound
SPSD047
(
GA
)
n
F: CCACCTTCCTTGGATACAC
(CT)17
194
156–326
53
GQ354556
R: CCACAACTACCACCTC
Perfect
SPSD005
(
AAG
)
n
F: TGAACCAACATAACCTACCTG
(TTC)19
297
192–452
55
GQ354557
R: CCACACTAAACCGAAACTTG
Perfect
SPSD048
(
AAG
)
n
F: AAGGGCATAGTTTCAACCAAG
(AAG)10
197
88
55
GQ354565
R: CTTTTGCTTGTTCATCAACATG
Perfect
SPSD007
(
CAG
)
n
F: AATCCTTCGCCTATCCTACAC
(CTG)8(CT)9
168
146–519
55
GQ354558
R: TTCACACAGCAGACAGAACTG
Compound
SPSD008
(
CAG
)
n
F: GCAAGAACAGACTCAAGAGC
(CAG)9(CAA)4(CAG)4(CAA)2
276
144–309
55
GQ354559
R: CTGCTGCTGAAGTAAAAGTTG
Compound
SPSD049
(
CAG
)
n
F: TGGATTGTTTCCTGATACTCCA
(TTG)5(CTG)5
299
303–667
53
GQ354560
R: CCATAAATTGCTGCATTATTCC
Compound
SPSD050
(
CAG
)
n
F: GAAGCAGAAAACACAGTATTGC
(CTG)5
225
225–505
53
GQ354561
R: TTGCTGGAATTTAACCTATCTG
Perfect
SPSD053
(
CAG
)
n
F: ACGCCTTCTTCACTCCAAC
(CTG)8
208
166–316
53
GQ354562
R: GCCACCAGTTTTCATCACC
Perfect
SPSD056
(
CAG
)
n
F: GTTCTCCAAAGTCTCCTCCT
(TCC)6TTC(TCC)2
197
79–230
53
GQ354563
R: ATCTTTACCTTCCTTCTGGG
InterruptedOnly two primers produced monomorphic profiles from the analysis of the sixteen lines, SPSD004 from the (GA)n library and SPSD048 from the (AAG)n library. The remaining primers produced between 1 and 12 scorable bands. Of the examined primers, 11 were amplified between 1 and 7 bands in S. spartinae (Table 2).All sequences were examined using BLASTn [27] on the National Center for Biotechnology Information (NCBI) server for similarities to other recorded sequences. Only two of the prairie cordgrass sequences displayed homology (e≤e-20) to recorded sequences, SPSD003 to six sequences from rice, wheat, and field mustard (Brassica rapa) (accession numbers: AP008214.1, AP005495.3, NM_001067901.1, AK111788.1, EU660901.1, and AC189183.2) and SPSD050 to five sequences all from rice (accession numbers: CR855236.1, AP008210.1, AL606607.3, NM_001059819.1, and AK108706.1) [25].


## 3.3. Crossing Population

A total of 94 individuals from the RR2 × RR21 and the RR21 × RR2 crosses were examined with 35 SSR primers. The amplified bands were scored based on presence or absence, the resulting data was analyzed with Nei and Li’s coefficient producing a similarity matrix (data not shown), and the data was clustered with UPGMA producing a dendogram (Figure4). The results of the dendogram indicate that each individual was a cross between the two parents. When the population derived from the cross between the two plants (SP1.1B and SP1.2A) and determined to be clones was examined, bands present in both parents were found to segregate in the progeny (data not shown), indicative of sexual recombination; this result provides validation that successful crossing occurred.Figure 4
Unrooted dendogram clustered with UPGMA of the genetic association of the F1 progeny from a cross between prairie cordgrass lines RR21 and RR2, from the analysis of SSR regions with Nei and Li’s Coefficient of genetic similarity.


## 4. Discussion

The primary requirement of any breeding program is to ensure that accurate crosses are made; in many other members of the Poaceae this is achieved by physical or chemical emasculation. The prolific numbers of flowers per head in addition to the small size of the flowers make physical emasculation unfeasible. Furthermore, due to limited knowledge about the nature of the fertility of this species, chemical emasculation has not been developed for prairie cordgrass. The results from the SSR analysis indicate that utilizing the inherent protogyny of prairie cordgrass allows successful crossing between two individuals without the need for emasculation, confirming the validity of the breeding methodology used. The presence of individuals in the F1 mapping populations which show limited genetic dissimilarity from the parents could be evidence of selfing; further investigations are required. The presence of these potential selfed individuals indicates that future breeding and/or mapping populations should be examined with the molecular markers devised in this research to remove suspect individuals. Subsequently, the successful crossing between two clones is indicative that prairie cordgrass may not be self-incompatible and that it may be possible to develop in this species conventional mapping populations, such as recombinant inbred lines. Further studies into self-compatibility with investigations into potential apomictic prairie cordgrass plants are underway. The number of seeds observed in this research appears to be larger than what was previously described by Clayton et al. [7]. The variation between the two studies can be attributed to both environmental and genetic variations. Genetic variation in seed set in prairie cordgrass, although at this stage not quantified, is demonstrated by the range in viable seed set observed in this research.The amplification ofS. spartinae with the SSR primers developed in this research are indicative of the potential colinearity amongst the genomes of Spartina spp. and other grass species; this colinearity will allow easier identification and characterization of genes. The colinearity between prairie cordgrass and other Poaceae is currently utilized to examine genes identified in related species in prairie cordgrass, specifically genes utilized to examine phylogeny (i.e., the waxy gene for granule bound starch synthase).The prevalence and distribution of SSR regions across plant genomes are extremely variable. Variation in SSRs is not limited to their location, but also their motif, putative function, abundance, and repeat number [28]. The results of this analysis indicate that the two dinucleotide repeats (CA)n and (GA)n are more prevalent in prairie cordgrass (31% and 46%, resp.), than the two trinucleotide repeats (AAG)n and (CAG)n (6% and 17%, resp.). The prevalence of the dinucleotide motif in prairie cordgrass is similar to what was observed in the characterization of SSRs in other Spartina sp., where Blum et al. [16] found 82% of isolated SSRs contained dinucleotide repeats and Sloop et al. [17] found 71% containing similar motifs. In all three studies the di- and trinucleotide repeats occurred as perfect, compound, and interrupted motifs. Based on the results found in this research, the genomic libraries enriched with the dinucleotide repeats have been extensively sequenced; the resulting sequence information will be screened to isolate additional SSR regions, primers will be designed, and the resulting markers will be used to develop a molecular map of prairie cordgrass. The molecular map will then be used to find linkage between SSR markers and traits of interest allowing future MAS to be performed.



---

*Source: 313545-2012-11-26.xml*