Cytological observations
The W19513 line was derived from the cross between CS and SY159, followed by one backcross to CS and repeated selfing to generate the BC1F10 population. Cytological examination revealed that the number of mitotic metaphase chromosomes (44) in the root tip of indoor- and field-cultivated plants was identical (Fig. 1A). Twenty-two pairs of chromosomes were observed in the pollen mother cells at meiosis, the chromosomes were evenly paired, and no unpaired chromosomes were observed (Fig. 1B). At meiotic anaphase chromosome separation was uniform, the chromosomes moved to the opposite poles synchronously, and no laggard chromosomes were observed (Fig. 1C). Therefore, W19513 showed high cytological stability.
Genomic DNA of SY159 (as the probe DNA) and that of CS ( as the blocking DNA) were extracted using a modified cetyltrimethylammonium bromide (CTAB) method [33] for genomic in situ hybridization (GISH). Two chromosomes showed the probe signal (Fig. 2A). This result implied that a pair of alien chromosomes were added to the genome of W19513. To further verify this result, GISH was conducted using CS as the blocking DNA, and Ae. umbellulata (Fig. 2B), and Ae. comosa (Fig. 2C) as the probe DNA. These results indicated that W19513 contained a pair of additional chromosomes from the M genome.
Functional molecular markers analysis
To determine the homologous group relationship of the alien chromosomes pair, 156 pairs of expressed sequence tag–sequence-tagged site (EST-STS) primers and 173 pairs of PCR-based landmark unique gene (PLUG) primers distributed across the seven homologous groups were analyzed using molecular markers. The pair of alien chromosomes was then compared with the amplified bands of the parents SY159 and CS. Five primers (BM134465, CD343475, BQ169491, CD452844, and BF429203) amplified specific bands (Fig. 3H–L). Specific bands of Ae. geniculata were amplified by five PLUG primers (TNAC1300, TNAC1341, TNAC1383, TNAC1627, and TNAC1364) (Fig. 3A–G), all of which belonged to the third homologous group (Table 1). Thus, the alien chromosomes belong to the third homologous group of the M genome (3M).
Table 1
Expressed EST–STS and PLUG marker list W19513
Marker
|
Type
|
Primer (5’-3’)
|
Location
|
Geltype/Restrictionenzyme
|
Tm ◦C/t (h)
|
TNAC1300
|
PLUG
|
F: TCTGCAGGTTCGGTAGACAAT
|
3AS 3BS 3DS
|
2% agarose gel/TaqI
|
60
|
R: AGTACGGGAGGACGCATGT
|
TNAC1341
|
PLUG
|
F: GTTGAAGCCTACATGCCACAC
|
3AL 3BL 3DL
|
2% agarose gel/TaqI
|
54
|
R: TAGCATGGGCTCCTAACATTG
|
TNAC1383
|
PLUG
|
F: GCGGTCGATCTTCTTCAAGTC
|
3AS 3BS 3DS
|
2% agarose gel/TaqI/HaeIII
|
60
|
R: TCAGATGGACTATGGGAGCAC
|
TNAC1627
|
PLUG
|
F: CAGGAGGCCTACGAGACG
|
3AS 3BS 3DS
|
2% agarose gel/TaqI/HaeIII
|
60
|
R: TTCTTCAGCTCGGATATTTGG
|
TNAC1364
|
PLUG
|
F: CGTCAGGCTCAGGGTGTC
|
3AL 3BL 3DL
|
2% agarose gel/TaqI
|
60
|
R: AAAGAGCCTCTGTCTCTCAGG
|
BM134465
|
EST-SSR
|
F: CAATTGAACCCATCCGAAAG
|
3AL 3BL 3DL
|
8% non-denaturing
|
60
|
R: CTGCCCGAACTATCCACAAT
|
polyacrylamide gel 8% non-denaturing/-
|
CD343475
|
EST-SSR
|
F: CCGAAGACGAAGTCGAGAAC
|
3AS 3BS 3DS
|
8% non-denaturing
|
62
|
R: ACACATCCCGTCCTTCTTTG
|
polyacrylamide gel 8% non-denaturing/-
|
BQ169491
|
EST-SSR
|
F: TGGCCATCATTGAACTGAAA
|
3AL 3BL 3DL
|
8% non-denaturing
|
56
|
R: CAATCAGATTTTTCGGCCAT
|
polyacrylamide gel 8% non-denaturing/-
|
CD452844
|
EST-SSR
|
F: AAAAGTTGGCCACCAATCTG
|
3AL 3BL 3DL
|
8% non-denaturing
|
60
|
R: TGGCATATGTCGCTCTGAAG
|
polyacrylamide gel 8% non-denaturing/-
|
BF429203
|
EST-SSR
|
F: CTTCGTAGCCTCCTCACTGG
|
3AL 3BL 3DL
|
8% non-denaturing
|
60
|
R: AGATTATGTGCGTGCTGTGC
|
polyacrylamide gel 8% non-denaturing/-
|
In Situ Hybridization.
The oligonucleotide probes Oligo-PTa535 and Oligo-pSc119.2 were used for fluorescence in situ hybridization(FISH) analysis, and then the pair of alien chromosomes was compared with the CS standard karyotype map [34]. The karyotype of W19513 was essentially identical to that of CS, comprising 42 chromosomes with an additional pair of alien chromosomes (Fig. 4A). The results of sequential FISH-GISH analysis further confirmed that the line W19513 carried a pair of alien chromosomes from Ae.geniculata. Comparison of the extra pair of chromosomes on the FISH map showed signal from the Ae. geniculata probe (Fig. 4B). Interestingly, comparison of the FISH karyotype of W19513 with that of CS (Fig. 4C) revealed variations in chromosomes 4A and 6B (Fig. 4D). Collectively, the aforementioned results showed that W19513 carried an additional pair of chromosomes 3Mg derived from Ae. geniculata.
Development of specific molecular markers
The SLAF-seq data comprised a total of 4,733,453, 8,752,434, and 6,943,541 raw reads for CS, SY159, and W19513, respectively. The average Q30 score was 94.15% and the average GC content was 48.66%. After filtering out low-depth data, the final numbers of SLAF-seq reads were 314,571, 193,701, and 371,647 for CS, SY159, and W19513, respectively, and the average sequencing depth was 6.3850. Using the Burrows–Wheeler Alignment (BWA) tool, 2888 reads were observed to show 50% similarity to the CS reference genome (IWGSC RefSeq v1.0). Among these reads, 634 reads showed at least 90% similarity to SY159 and were considered to be specific fragments of chromosome 3Mg. To develop chromosome 3M-specific markers, 128 primers were designed based on 128 randomly selected fragments and were used to amplify the sequence of CS, SY159, Ae. comosa, Ae. umbellulata, and W19513. Among these markers, 61 markers amplified SY159-specific bands with a maximum success rate of 47.66% (Table 2). Ultimately, 61 sequences of chromosome 3M were obtained (data not shown). The 61 3M-specific markers were further divided into three categories, namely W19513, SY159, and Ae. comosa and Ae. umbellulata, of which four markers (Fig. 5) showed the same amplification patterns in W19513, SY159, Ae. comosa and Ae. umbellulata (Fig. 5 Type 1); 13 markers amplified the same bands in W19513 and SY159, but not the corresponding bands in Ae. comosa and Ae. umbellulata (Fig. 5 Type 2); and 44 markers amplified the same bands in W19513, SY159, and Ae. comosa, but not Ae. umbellulata (Fig. 5 Type 3).
Table 2
Detection of W19513 specific-locus amplified fragment sequencing (SLAF) markers
Type
|
SY159
|
Ae. Comosa
|
Ae.Umbellulata
|
W19513
|
No. of Specific
|
(UUMM)
|
(MM)
|
(UU)
|
Type1
|
+
|
+
|
+
|
+
|
4
|
Type2
|
+
|
+
|
-
|
+
|
44
|
Type3
|
+
|
-
|
-
|
+
|
13
|
Assessment of agronomic traits and disease resistance
The agronomic traits of W19513 and the parents CS and SY159 were analyzed. No significant differences in plant height, number of grains per spikelet, spikelet number, spikelet length, and 1000-grain weight were observed between W19513 and CS. However, notable differences in the lower spike nodes between W19513 and CS were noted. Significant differences were observed in plant height, spikelet number, tiller number, and 1000-grain weight between W19513 and SY159. These results inferred that the W19513 line showed overall morphological intermediacy between the parents and produced long panicle nodes and stable agronomic traits (Fig. 6A–D, Table 3).
Table 3
Analysis of the agronomic traits of W19513 and its parents (CS, SY159)
Materals
|
Plant
|
Tillering
|
Spikelets/
|
Kernels/
|
Spike
|
Subpanicle
|
Thousand Kenel
|
Awnedness
|
Height (cm)
|
Spike
|
Spikelet
|
Length(cm)
|
node(cm)
|
Weight(g)
|
CS
|
128
|
21
|
21
|
4
|
9
|
40
|
32
|
Awnless
|
SY159
|
59
|
68
|
19
|
2
|
5
|
23
|
23
|
Long awn
|
w19513
|
138
|
33
|
20
|
4
|
9
|
47
|
34
|
Awnless
|