Efficient mutation induction using carbon-ion beam in japonica rice in northeast China
Our results showed that seed germination was significantly repressed by 200 Gy carbon-ion beam treatment; seed germination vigor, germination rate, and survival rate decreased by 76.72%, 67.52%, and 96.58%, respectively (Fig. 1B). After irradiation, the rice seeds were planted from generation M1 to M5, and the field experiment data showed that the mutant population was more abundant from generation M3 to M5. Most of the mutant materials have good stability, of which a small number of progeny materials present good comprehensive properties than 'Tonghe899'. For example, some stable mutants have shortened maturation periods than the mutagenic parent, namely '9001', '9002', '9003', '9004', '9005', '9006', '9007', '9008', '9009', '9010' and '9011'. Table S3 describes the main maturation period traits of these mutants. In the present study, we used the above 11 mutants to further analyze DNA polymorphism patterns after irradiation.
RAPD and SSR markers analysis in ‘Tonghe 899’ and mutants
RAPD markers were used to confirm the polymorphism among ‘Tonghe 899’ and mutants (Fig. 2A and B). As shown in Table S4, RAPD identified a total of 202 bands with a molecular weight ranging from 200–2,200 bp, of which 100 bands were polymorphic (49.50%). The total number of bands for each primer varied from 2 to 15, with a mean number of 8.78. The number of polymorphic bands for each primer ranged from 1 to 11, with an average of 4.35. S22 primer amplified 15 bands, out of which seven bands showed polymorphism, S7, S155, and S350 primers produced fewer bands while they presented the highest polymorphism (100%). The PIC for each RAPD primer varied to a large extent between 0.718 in S350 and 0.924 in S22.
A total of 25 SSR primers were used to measure the genetic diversity of the ‘Tonghe 899’ and mutants (Fig. 2C). With a molecular weight ranging from 100–500 bp, SSR primers generated a total of 371 bands of which 285 bands were polymorphic (76.81%), and per pair of primers produced 14.84 bands. Primers RM19 obtained the highest number of amplicons (25 alleles) with 72% polymorphism, primers RM85 detected the lowest number of amplicons (8 alleles) with 100% polymorphism, and RM8277 and RM258 detected the minimum polymorphism (40.00%). The PIC for each primer pair varied from 0.836 in RM190 to 0.954 in RM19 (Table S4).
Combined with 23 RAPD and 25 SSR primer data, a total of 574 DNA fragments had been produced of which 385 were polymorphic (67.07%). Table S6 showed Jaccard’s similarity coefficient of the genotypes ranging between 0.69 and 0.89. And the maximum three similarity values were recorded between ‘9005’ and ‘9011’ (0.89), ‘Tonghe 899’ and ‘9009’ (0.87), as well as ‘Tonghe 899’ and ‘9007’ (0.84), while‘Tonghe 899’ and ‘9001’ (0.69), as well as ‘Tonghe 899’ and ‘9002’ (0.70) had the minimum two similarity values. The dendrogram divided 12 japonica cultivars into five clusters. There was only one japonica in the first three clusters, namely ‘9001’, ‘9002’ and ‘9003’, respectively, the fourth group included ‘9004’, ‘9005’, ‘9011’, ‘9008’, ‘9010’ and ‘9006’, besides, ‘9007’, ‘9009’, and ‘Tonghe 899’ separated from the other genotypes and tightly linked in the fifth cluster (Fig. 3A). PCA analysis indicated that the first three components explained 46.70% of the total variation, with 21.21%, 14.28%, and 11.21%, by PCA1, PCA2, and PCA3, respectively. Furthermore, the three-dimensional (3D) graph also tended to cluster the samples into five groups (Fig. 3B).
RAPD and SSR markers analysis in local cultivars and mutants
RAPD markers evaluated the level of polymorphism among mutants, ‘Tonghe 899’, and other local varieties (Fig. 2A and B). In RAPD, primers generated 250 clear and repeatable bands with molecular weight ranging from 200 to 2,000 bp, with 183 polymorphic bands (73.20%). The range of fragments number amplified by each primer varied from 2 to 19, and the number of polymorphic bands ranged from 2 to 16, the number of average amplified fragments and polymorphic bands per primer was 10.87 and 7.95, respectively. Additionally, the primers S7, S230, S310, and S350 presented 100% polymorphism, S22, S195, S218, S374 were identified as good primers for the amplification of more bands. Besides, the PIC values ranged from 0.500 in primer 155 to 0.925 in S22 with an average of 0.852 over all loci (Table S5).
SSR primer pairs generated a total of 408 bands of which 347 bands presented polymorphic (85.05%), with a molecular weight of 100–500 bp, and the number of bands amplified per pair of primer ranged from 7 to 33 with an average of 16.32. The RM71 and RM19 primers amplified more bands (26 alleles) with 96.15% and 76.92% polymorphism, respectively, however, RM190 presented the fewer amplified bands (7 alleles) and polymorphic (42.86%). The PIC values ranged from 0.835 in RM190 to 0.952 in RM19 with an average of 0.913 over all loci (Table S5).
Combined with RAPD and SSR data, primers produced 658 DNA fragments of which 530 bands were polymorphic (80.55%). The dendrogram shows that ‘9001’, ‘Jijing 809’, and ‘Jijing 816’ are distinctly formed in a single cluster, respectively. ‘9002’ and ‘ChangJing 616’ forms group B, and the remaining samples separates from the other genotypes and links in the same group (Fig. 4A). Besides, the first three Eigen values accounted for 25.67% of the total variation. Based on the 3D PCA plot, major genotypes inclined to cluster except ‘Jijing 809’, ‘Jijing 816’, ‘9002’, and ‘9001’ (Fig. 4B). Jaccard’s similarity coefficient of mutants and local japonica cultivars varied from 0.62 to 0.91 with an average similarity value of 0.67. And ‘Jijing 88’ and ‘Tonghe 66’ (0.91) presented the highest similarity values, yet ‘Jijing 809’ and ‘9001’ had the lowest similarities, only 0.62 (Table S7). However, the results of similarity coefficient among ‘Tonghe899’ and other 22 japonica varieties showed that the highest similar value was observed between ‘Jijing 88’ and ‘Tonghe 66’ (0.91), and the lowest value was found between ‘Changjing 616’ and ‘Jijing 816’ (0.65) (Table S8), the adding mutants reduced the lowest Jaccard’s similarity coefficient of local varieties from 0.65 to 0.62.