{"id":26344,"date":"2015-10-17T17:39:31","date_gmt":"2015-10-17T17:39:31","guid":{"rendered":"http:\/\/healthmedicinet.com\/news\/asymmetric-somatic-hybridization-induces-point-mutations-and-indels-in-wheat\/"},"modified":"2015-10-17T17:39:31","modified_gmt":"2015-10-17T17:39:31","slug":"asymmetric-somatic-hybridization-induces-point-mutations-and-indels-in-wheat","status":"publish","type":"post","link":"http:\/\/healthmedicinet.com\/news\/asymmetric-somatic-hybridization-induces-point-mutations-and-indels-in-wheat\/","title":{"rendered":"Asymmetric somatic hybridization induces point mutations and indels in wheat"},"content":{"rendered":"

cDNA sequencing<\/h4>\n

A total of 19,045 SR3 and 10,327 JN177 clones were sequenced, resulting in the acquisition
\n of, respectively, 18,192 and 9770 usable sequences (Additional file 1: Table S1). The sequences resolved into 9634 unigenes (2097 contigs and 7537 singletons)
\n from SR3, and 7107 unigenes (1207 contigs and 5900 singletons) from JN177, of which
\n full length cDNAs were 4825 and 2975, respectively (Additional file 1: Table S1). The length of most of the unigenes laid in the range 700\u00e2\u20ac\u201c1000\u00c2\u00a0nt (Additional
\n file 2: Figure S1), and their mean GC content was 53.85\u00c2\u00a0% (SR3) and 55.46\u00c2\u00a0% (JN177). The
\n BLASTn analysis of the unigenes revealed that 2581 were shared (96\u00c2\u00a0% identity) between
\n SR3 and JN177 (Table\u00c2\u00a01). In all, 5072 (71.4\u00c2\u00a0%) of the JN177 and 7284 (75.6\u00c2\u00a0%) of the SR3 unigenes shared
\n 96\u00c2\u00a0% identity with sequences represented in the wheat EST database (Table\u00c2\u00a01).<\/p>\n

Table 1.<\/strong> BLASTn-based homology comparisons of unigene sequences\n <\/p>\n

Frequency of single nucleotide polymorphisms (SNPs) in the unigene sequences<\/h4>\n

Based on the unigene sequences sharing 96\u00c2\u00a0% identity, 15,226 SNPs were identified
\n within the unigene sequence shared between SR3 and JN177, equivalent to a SNP frequency
\n of 11.33 per 1000\u00c2\u00a0nt of coding sequence (Table\u00c2\u00a02). The transition and transversion frequencies were, respectively, 6.70 and 4.63 per
\n 1000\u00c2\u00a0nt. The SNP frequency between JN177 and the sequences represented in the wheat
\n EST database (JN177 vs<\/em> Ta comparison) was only about one half of this level (5.77 per 1000\u00c2\u00a0nt) (Table\u00c2\u00a02), demonstrating that the somatic hybridization process was effective in inducing
\n point mutations. A comparison based on the sequences of the unigenes shared between
\n the BA progenitor tetraploid (T. turgidum<\/em>) and the A genome carrier T. monococcum<\/em> revealed a SNP frequency of 15.48 per 1000\u00c2\u00a0nt, while that between T. turgidum<\/em> and Ae. speltoides<\/em> (related to the B genome progenitor) was 18.51, indicating that a high frequency
\n of mutation was induced during the formation of allotetraploid wheat. Similarly, the
\n estimated SNP frequencies between bread wheat and T. monococcum<\/em>, Ae. speltoides<\/em>, T. turgidum<\/em> and Ae. tauschii<\/em> (D genome progenitor) were, respectively, 12.02, 16.24, 12.13 and 5.40 per 1000\u00c2\u00a0nt
\n (Table\u00c2\u00a03). Thus the mutation frequency induced by the somatic hybridization process appeared
\n to be similar in extent to that induced by allopolyploidization. The frequency of
\n SNPs between the unigene sequences of bread wheat and those of either T. monococcum<\/em> or Ae. speltoides<\/em> was less than that between T. turgidum<\/em> and either T. monococcum<\/em> or Ae. speltoides<\/em> unigenes (Table\u00c2\u00a03). This coincided with the finding that the SNP frequency of SR3 and wheat database
\n EST (SR3 vs<\/em> Ta alignment) was lower than those of the SR3 vs<\/em> JN177 alignment (Table\u00c2\u00a02). The SNP frequency between SR3 unigene sequences and those of the A, B, BA and D
\n genome species was similar to those between JN177 unigenes and those of the A, B,
\n BA and D genome species (Table\u00c2\u00a03).<\/p>\n

Table 2.<\/strong> The SNP frequencies in SR3 and JN177\n <\/p>\n

Table 3.<\/strong> SNP and indel frequencies among wheat and its ancestors\n <\/p>\n

The size distribution of indels in the unigene sequences<\/h4>\n

The indels ranged from 1\u00c2\u00a0nt to 574\u00c2\u00a0nt, and a majority of the indels involved only
\n 1\u00c2\u00a0nt. A significant number of indels was revealed by aligning the matched unigene
\n sequences, with the frequency of larger indels (23\u00c2\u00a0nt) being clearly less than that
\n of the small ones (1\u00e2\u20ac\u201c10\u00c2\u00a0nt) (Table\u00c2\u00a04). There had 82.14\u00c2\u00a0% unigenes possessing small indels when compared between SR3 and
\n JN177, higher than those from SR3 vs<\/em> Ta and JN177 vs<\/em> Ta comparisons. On the contrary, 6.70\u00c2\u00a0% unigenes had large indels in the comparison
\n between SR3 and JN177, lower than those of other two comparisons. There had more unigenes
\n with small insertions than those with small deletions, and the difference was stronger
\n in the SR3 vs<\/em> Ta and JN177 vs<\/em> Ta comparisons. Unigenes with large insertions were similar to those with large deletions
\n in the SR3 vs<\/em> JN177 comparison, but unigenes with large deletions were more abundant than those
\n with large insertions in the other two comparisons. The comparison between the JN177
\n (and similarly SR3) unigene sequences with those represented in the wheat EST database
\n showed that for small indels, the ratio of insertion to deletion frequency was negatively
\n correlated to indel length (R<\/em>2<\/sup>
\n ?=?0.62 and 0.72, respectively) (Fig.\u00c2\u00a01a); the ratio was 1 for indels shorter than 6\u00c2\u00a0nt, and 1 for indels longer than 6\u00c2\u00a0nt.
\n However, for the larger indels, the insertion to deletion ratio was positively correlated
\n to indel length (R<\/em>2<\/sup>
\n ?=?0.59 and 0.65, respectively) (Fig.\u00c2\u00a01b); in indels ranging in length from 20 to 70\u00c2\u00a0nt, the ratio was just 0.01\u00e2\u20ac\u201c0.06, rising
\n to 0.28\u00e2\u20ac\u201c0.86 for indels of length 71\u00e2\u20ac\u201c200\u00c2\u00a0nt, and to ~1.5 for indels longer than 200\u00c2\u00a0nt
\n (Fig.\u00c2\u00a01b). The SR3 vs<\/em> JN177 comparison revealed an insertion to deletion ratio of ~1 irrespective of indel
\n length (Fig.\u00c2\u00a01a, b).<\/p>\n

Table 4.<\/strong> Indel variation in SR3 and JN177 unigene sequences\n <\/p>\n

Fig. 1.<\/strong> The distribution of indel lengths. a<\/strong>: small indels. b<\/strong>: large indels. The insertion\/deletion ratio was obtained by dividing the number of
\n insertions by the number of deletions. SR3-JN177: JN177 unigene sequences queried
\n with those of SR3. SR3-Ta: SR3 unigene sequences queried with wheat ESTs housed in
\n GenBank. JN177-Ta: JN177 unigene sequences queried with wheat ESTs housed in GenBank.
\n The correlation between indel size and insertion\/deletion ratio was performed using
\n the Pearson correlation analysis\n <\/p>\n

The frequency of indels in the unigene sequences<\/h4>\n

Small indels were used to calculate the indel frequency because they were markedly
\n more abundant than large indels (indel numbers not shown). In all, the 2581 matched
\n unigenes derived from the SR3 vs<\/em> JN177 comparison revealed 2120 indels (1.58 per 1000\u00c2\u00a0nt). Based on the JN177 sequences,
\n these comprised 1331 insertions and 789 deletions in SR3, equivalent to frequencies
\n of, respectively, 0.99 and 0.59 per 1000\u00c2\u00a0nt (Table\u00c2\u00a05). In the comparison with the sequences represented in the wheat EST database, the
\n indel frequency in SR3 was 1.36 per 1000\u00c2\u00a0nt. The similar comparison between JN177
\n and the sequences represented in the wheat EST database revealed an indel frequency
\n of only 0.93 per 1000\u00c2\u00a0nt, implying that the asymmetric somatic hybridization process
\n was effective in inducing indels in coding sequence.<\/p>\n

Table 5.<\/strong> The frequency of small indels in SR3 and JN177 unigene sequences\n <\/p>\n

To compare the induction rates of indels caused by somatic hybridization and allopolyploidization,
\n equivalent calculations were made using the matched unigene sequences present in bread
\n wheat and its relatives\/progenitor species (Table\u00c2\u00a03). Comparing the unigenes of T. turgidum<\/em> with those of T. monococcum<\/em> and Ae. speltoides<\/em> revealed an indel frequency of, respectively, 1.90 and 1.28 per 1000; the equivalents
\n between bread wheat and its various related species ranged from 1.42 to 2.31 per 1000\u00c2\u00a0nt,
\n with the comparison involving Ae. tauschii<\/em> producing the highest estimate. The indel frequencies estimated using the unigenes
\n of JN177 and SR3 also lay in the range 1 to 2 per 1000\u00c2\u00a0nt, with the exception of the
\n comparison with Ae. tauschii<\/em>, where the frequencies were, respectively, 4.42 and 5.37 per 1000\u00c2\u00a0nt (Table\u00c2\u00a03).\n <\/p>\n

In the SR3 vs<\/em> JN177 comparison, the insertion frequency (0.99 per 1000\u00c2\u00a0nt) was 1.69 fold to the
\n deletion frequency (0.59 per 1000\u00c2\u00a0nt) (Table\u00c2\u00a05). Based on the sequences represented in the wheat EST database, the insertion frequencies
\n were higher by 3.41 and 2.58 fold than deletion frequencies in SR3 and JN177, respectively.
\n Especially, the insertion frequency in the SR3 vs<\/em> Ta comparison (1.05 per 1000\u00c2\u00a0nt) was similar to the SR3 vs<\/em> JN177 comparison (0.99 per 1000\u00c2\u00a0nt), but its indel frequency was lower than the latter.
\n Thus, the preference to small insertion was decreased in wheat asymmetric somatic
\n hybrids in comparison with allopolyploid wheat.\n <\/p>\n

The function of unigenes showing sequence polymorphism<\/h4>\n

To know whether the genetic variation is associated with their biological processes,
\n we selected sequences participating in gene expression regulation and other processes
\n for analysis. A selection of polymorphic unigenes represented in the JN177 and SR3
\n libraries (948 and 1519, respectively) as well as in the wheat EST database showed
\n that for gene expression regulation, the frequency of SNPs differed most notably in
\n genes involved in nucleosome assembly and chromatin assembly\/disassembly (Table\u00c2\u00a06). The least polymorphic comparison was between JN177 and the wheat EST database unigenes
\n (4.59 SNPs per 1000\u00c2\u00a0nt for the genes involved in the former category and 3.72 per
\n 100\u00c2\u00a0nt in the latter). The same comparison between SR3 and JN177 produced, respectively,
\n 18.17 and 19.21 SNPs per 1000\u00c2\u00a0nt. The next most variable genes were those encoding
\n products involved in translation and post-translational modification, where the SR3
vs<\/em> JN177 comparison revealed a SNP frequency of, respectively, 13.30 and 13.97 per 1000\u00c2\u00a0nt,
\n while the JN177 vs<\/em> Ta comparison showed 8.69 and 7.98, respectively. The range in SNP frequency for
\n genes associated with metabolic processes ranged from 5.45 to 6.29 per 1000\u00c2\u00a0nt in
\n the JN177 vs<\/em> Ta comparison and 9.16\u00e2\u20ac\u201c14.25 per 1000\u00c2\u00a0nt in the SR3 vs<\/em> JN177 comparison (Additional file 3: Table S2). SNP frequencies were also higher in the SR3 vs<\/em> Ta comparison than in the JN177 vs<\/em> Ta comparison for genes encoding proteins involved in various metabolic processes
\n except for glycolysis as well as nucleobase, nucleoside, nucleotide and nucleic acid
\n metabolic process (Additional file 3: Table S2). This difference in SNP frequencies was also found in ESTs of protein
\n fate, transport, cell redox homeostasis, and response to (oxidative) stress (Additional
\n file 4: Table S3). With respect to unigenes varying at the level of indels, the frequency
\n of polymorphism was lower in the JN177 vs<\/em> Ta than in the SR3 vs<\/em> JN177 comparison (Table\u00c2\u00a06; Additional files 3 and 4: Table S2 and S3). Indel events were noticeably rare in genes involved in nucleosome
\n assembly and chromatin assembly\/disassembly (Table\u00c2\u00a06). The respective frequencies were 0.43 and 0.38 per 1000\u00c2\u00a0nt in the JN177 vs<\/em> Ta comparison and 0.95 and 0.92 per 1000\u00c2\u00a0nt in the SR3 vs<\/em> JN177 comparison.<\/p>\n

Table 6.<\/strong> The function of unigenes affected by SNP and indels in SR3 and JN177\n <\/p>\n

Sequences flanking indels in the unigenes<\/h4>\n

The sequences flanking indels were characterized by calculating the GC content of
\n the ten nucleotides flanking either side of the indel. There was no obvious difference
\n in GC content between 5? and 3? terminal flanking sequence in any of the comparisons
\n (JN177 or SR3 vs<\/em> wheat database ESTs, SR3 vs<\/em> JN177) (data not shown). The trend of GC content was similar when the second to tenth
\n nucleotides of the flanking sequence were considered (Fig.\u00c2\u00a02). The GC content of the nucleotides positioned three, six and nine away from the
\n indels was higher than that of the ones positioned four, five, seven or eight away
\n in the 3? terminal flanking sequences, but the rule was not found in the 5? terminal
\n flanking sequences. The GC content of the flanking sequence was higher in the SR3
vs<\/em> JN177 comparison (53.47\u00e2\u20ac\u201c54.04\u00c2\u00a0%) than in the other two comparisons (51.88\u00e2\u20ac\u201c52.88\u00c2\u00a0%
\n in JN177 vs<\/em> Ta, 50.99\u00e2\u20ac\u201c52.63\u00c2\u00a0% in SR3 vs<\/em> Ta). In the SR3 vs<\/em> Ta and JN177 vs<\/em> Ta comparisons, the GC content of the flanking sequence of the deletions was higher
\n than that of the insertions, while the content of the indels was close to that of
\n the insertions. However, in the SR3 vs<\/em> JN177 comparison, the GC content of the flanking sequence of the deletions was generally
\n lower than that of the insertions, and the difference between the deletions and insertions
\n was weaker than that in the JN177 or SR3 vs<\/em> Ta comparisons; the content of the flanking sequence of the indels did not bias toward
\n either the deletions or insertions. In the JN177 or SR3 vs<\/em> Ta comparisons, with respect to the nucleotide lying on the 5? side of the indels,
\n the GC content of the nucleotide adjacent to the deletions was significantly lower
\n than that of other nucleotides in the flanking sequence, but the GC content of the
\n nucleotide adjacent to the insertions was significantly higher than that of other
\n nucleotides in the flanking sequence (Fig.\u00c2\u00a02b, c). On the other hand, for the nucleotides lying on the 3? side of the indels, the
\n GC content of the nucleotide adjacent to the deletions remained high and was comparable
\n to that of the second and third flanking nucleotides, but the GC content of the nucleotide
\n adjacent to the insertions was significantly lower than that of the other flanking
\n nucleotides (Fig.\u00c2\u00a02b, c). In contrast in the SR3 vs<\/em> JN177 comparison, for the nucleotides lying on the 5? side of the indel, the GC content
\n of the nucleotide adjacent to both the deletions and insertions was higher than that
\n of the second to tenth nucleotides of the 5? flanking sequence, while for the nucleotides
\n lying on the 3? side of the indels, the GC content of the nucleotide adjacent to both
\n the deletions and insertions was lower than the second and third nucleotides (Fig.\u00c2\u00a02a).<\/p>\n

Fig. 2.<\/strong> Variation in the GC content in the sequence immediately flanking indels. a<\/strong>: SR3-JN177, JN177 unigene sequences queried with those of SR3. b<\/strong>: SR3-Ta, SR3 unigene sequences queried with wheat ESTs housed in GenBank. c<\/strong>: JN177-Ta, JN177 unigene sequences queried with wheat ESTs housed in GenBank. -10?~??1:
\n The tenth to first nucleotides on the 5? side of the indel. 1?~?10: The first to tenth
\n nucleotides on the 3? side of the indel. In-mean, Del-mean and indel-mean: the GC
\n content of 5? and 3? flanking sequences of insertions, deletions and indels, respectively\n <\/p>\n

Indel classification<\/h4>\n

We further compared the characteristic of flanking sequences of large indels in the
\n SR3 vs<\/em> JN177 comparison. The flanking sequences of 45 large insertions and 39 large deletions
\n identified in the SR3 vs<\/em> JN177 comparison were identical (Fig.\u00c2\u00a03a, d). Two examples were SR3_2LCP226_E06 (474\u00c2\u00a0nt insertion) and SR3_firstas1573 (34\u00c2\u00a0nt
\n deletion) (Additional file 5: Figure S2A, B). A second group had 40 insertions and 36 deletions, whose two terminals
\n possessed repeated sequences in SR3 and JN177 (Fig.\u00c2\u00a03b, e). The flanking sequence of SR3_5V50 (179\u00c2\u00a0nt insertion) harbors a run of G\u00e2\u20ac\u2122s; the
\n 3? flanking sequence of SR3_firstas843 (55\u00c2\u00a0nt deletion) includes two copies of CATCCC
\n in JN177 but only one in SR3 (Additional file 5: Figure S2C, D). The repeat motifs present in the flanking sequence ranged in length
\n from 1 to 51\u00c2\u00a0nt (data not shown). A 1\u00c2\u00a0nt motif was present in 23 of the insertions
\n and 19 of the deletions, dominated by runs of G (data not shown). The third group,
\n in which the flanking sequence was modified (Fig.\u00c2\u00a03c, f), is exemplified by SR3_firstas1573 (141\u00c2\u00a0nt deletion), where SNPs were generated
\n at three positions adjacent to the deletion (Additional file 5: Figure S2E). A few of the unigenes experienced multiple indel events: SR3_firstas1573
\n carries two deletions, one belonging to the first group and the other to the third
\n group (Additional file 5: Figure S2B, E). Other variants included the induction of translocated and chimeric
\n sequences. In the homologs SR3_2LCP192_G10 and JN177_firstas231, the same sequence
\n was found in positions 1187\u00e2\u20ac\u201c1367 in the former allele, but at 1\u00e2\u20ac\u201c181 in the other.
\n SR3_2LCP192_G10 also harbors a large deletion with a repeat sequence CAAGAAGGA (Additional
\n file 6: Figure S3A). SR3_firstas716 nucleotides 88\u00e2\u20ac\u201c196 do not align with JN177_LCP139_D11
\n nucleotides 157\u00e2\u20ac\u201c278, but their terminal sequences are identical (Additional file 6: Figure S3B).<\/p>\n

Fig. 3.<\/strong> Hypothetical model for the formation of large indels induced during asymmetric somatic
\n hybridization. Blue block: unigenes shared by SR3 and JN177. Red block: insertion
\n and deletion fragments. Black block: repetitive sequences in the indel flanking sequence.
\n Gray blocks: small fragments adjacent to insertions and deletions, differing sequence
\n between SR3 and JN177. SR3-JN177: JN177 unigene sequences queried with those of SR3.
\n SR3-Ta: SR3 unigene sequences queried with wheat ESTs housed in GenBank. JN177-Ta:
\n JN177 unigene sequences queried with wheat ESTs housed in GenBank\n <\/p>\n","protected":false},"excerpt":{"rendered":"

cDNA sequencing A total of 19,045 SR3 and 10,327 JN177 clones were sequenced, resulting in the acquisition of, respectively, 18,192 and 9770 usable sequences (Additional file 1: Table S1). The sequences resolved into 9634 unigenes (2097 contigs and 7537 singletons) from SR3, and 7107 unigenes (1207 contigs and 5900 singletons) from JN177, of which full […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[],"tags":[],"class_list":["post-26344","post","type-post","status-publish","format-standard","hentry"],"_links":{"self":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/26344","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/comments?post=26344"}],"version-history":[{"count":0,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/posts\/26344\/revisions"}],"wp:attachment":[{"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/media?parent=26344"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/categories?post=26344"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/healthmedicinet.com\/news\/wp-json\/wp\/v2\/tags?post=26344"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}