甘蔗栽培种单倍体基因组SSR位点的发掘与应用

doi:10.3724/SP.J.1006.2020.94135

Abstract

Abstract:

Sugarcane is one of the most important sugar crops in the world. However, it is difficult to develop SSR on a large scale since the genome of cultivar has not been sequenced, which limits the genetic improvement of sugarcane. In this study, a template of monoploid sugarcane genome was assembled using a set of 4660 BAC library sequences (with a cumulative length of 382 Mb, predicting 25,316 genes) from cultivar ‘R570’. SSR loci were identified by using MISA (Microsatellite identification tool) software. The distribution characteristics of the monoploid genome ‘R570’ was comprehensively analyzed by comparing with the SSR loci of four Gramineae plants (Sorghum bicolor, Zea mays, Oyrza sativa, and Brachypodium distachyon). Fifty pairs of primers with TG and AG repeat motifs were designed to verify the amplification efficiency and polymorphism by PCR amplification in four Saccharum clones (R570, ROC1, LA purple, and SES208) and twenty four core parents of sugarcane. A total of 27,241 SSR loci were identified, with an average of 6.29 SSR loci per BAC clone and an average density of 71.33 SSR Mb ^-1 which was much lower than that of sorghum (350.00 SSR Mb ^-1). The mono-nucleotide (11,079) and tri-nucleotide repeat motifs (6447) accounted for 64.33% of the total SSR loci. The number and proportion of tri-nucleotide repeat motifs were the largest in the four Gramineae plants. In addition, A/T (accounting for 84.8%) motif had the highest proportion and C/G (accounting for 15.2%) motif the lowest proportion in the mono-nucleotide repeat motifs and TGT/ACA (accounting for 16.04%) motif had the highest proportion in the trinucleotide repeat motifs. In general, the genomes in Gramineae plants are rich in A/T repeat motifs. In the polymorphism validation of 50 pairs of primers (41 pairs of TG motif and 9 pairs of AG motif), 45 pairs of primers (90%) were found to be able to amplify successfully, of which 35 (70%) were polymorphic in 4 sugarcane clones. Furthermore, 20 pairs of polymorphic SSR primers were used to detect 24 core parents of sugarcane, a total of 95 alleles were amplified with an average of 4.75 alleles per primer, verifying the application feasibility of these primers for the genetic diversity analysis in sugarcane. The development of SSR markers from the monoploid genome of cultivars ‘R570’ not only enriches the number of SSR markers available in sugarcane genetic analysis, but also facilitates the genetic diversity analysis of sugarcane population and the genetic mechanism dissection of important agronomic traits, which provides a foundation for the in-depth research of molecular breeding in sugarcane.

Key words: sugarcane cultivars, bacterial artificial chromosome library, SSR, development, polymorphism

WANG Heng-Bo,QI Shu-Ting,CHEN Shu-Qi,GUO Jin-Long,QUE You-Xiong. Development and application of SSR loci in monoploid reference genome of sugarcane cultivar[J].Acta Agronomica Sinica, 2020, 46(4): 631-642.

Figures/Tables 9

Table 1

Table 2

Table 3

Table 4

Fig. 1

Table 5

Primers information of sugarcane SSR with polymorphic amplification"

引物名称 Primer name	重复基序 Motif	左引物序列 Left-primer (5°-3°)	退火温度 T_m(℃)	右引物序列 Right-primer (5°-3°)	退火温度 T_m (℃)	产物大小 Product size (bp)	PIC
FAFUR-S1	(TG)69	TCATACCCATTGGAAGAAGC	60.5	GTTATGTTGCCGTGCCAAGT	59.8	278	0.85
FAFUR-S3	(TG)39	TAGCCTTTGGTCGTTCTTGG	58.2	AATGCTTCATCCATAGGGGA	59.3	259	0.84
FAFUR-S7	(TG)32	GCCTGGGGAACTATGCTGTA	59.1	CAAGCATTGAAGTTGCCAAA	59.0	254	0.61
FAFUR-S12	(TG)24	CGTCAGTTGCTCAGCTCTTG	58.0	CCCTGGGAAGAAGAGGTAGG	58.6	223	0.69
FAFUR-S17	(TG)19	AATGATGTTTCGCCTGATCC	60.2	ACCAACACAACTCGCTACCC	60.1	166	0.77
FAFUR-S18	(TG)19	CCACATTCTTCGACCCTGTT	59.8	CCATCCTGCGAACTAACCAT	59.7	183	0.71
FAFUR-S22	(TG)18	AGGGCACGAGGTATTGCTTA	58.9	AACCGGTCAAATCACACACA	59.2	179	0.68
FAFUR-S24	(TG)17	ATCTTTCGGCATCAACTTGG	60.1	GCTTCAAGCCATCTGTCTCC	60.3	274	0.73
FAFUR-S32	(TG)13	CAACGAATTCCACTTGCACA	60.0	TCATGGCTATTGTGGTCTGG	60.4	207	0.61
FAFUR-S33	(TG)13	CTCCTCTGTCACCCAGCACT	58.8	GATCACCCCAGATCCAGAGA	59.6	179	0.75
FAFUR-S34	(TG)13	TGCTGATTATGTGCTGCCTC	58.5	CACGCCTAGGGCATAAGAGA	58.4	222	0.67
FAFUR-S36	(TG)12	AGGCATGGGAATTTCTCTCC	60.1	GGCCTCTCTTTAGTGCAGGA	59.8	265	0.77
FAFUR-S38	(TG)12	GACACCCACCACAGGACTTT	60.3	CCCTCCCCAATCCTATCAGT	60.1	198	0.66
FAFUR-S40	(TG)11	GCTGATGTTTGGTCATGTGG	61.0	TGCAGACTCAGAAGTAGCCG	60.5	246	0.86
FAFUR-S41	(TG)11	TGTTTCAGGCACTGTTTTGG	60.9	AGCAATGTGTTCTCCATCCA	60.5	261	0.73
FAFUR-S42	(AG)38	CGGCACAAGTAAATGCAAGA	59.7	AGTACTGCCAACAAGGCAGG	58.3	230	0.85
FAFUR-S43	(AG)34	CTTGAGCTCGTAGCCTCCTC	60.3	GCCTCTGCTGTCTGCTCTCT	59.6	267	0.92
FAFUR-S44	(AG)31	AGTGCAGGTTGGCTTTCTGT	60.2	GGGGATTCCAAGTCTCAACA	59.8	206	0.82
FAFUR-S47	(AG)25	GTACCAGCCCAAAAACTGGA	59.8	TTGTCACTGGGAACACGGTA	60.1	280	0.73
FAFUR-S49	(AG)23	TTCTCCGTCAACTGTCATGC	59.6	TCCTACGGAGGGAAATCAAA	60.2	273	0.81
FAFUR-S4^#	(TG)33	CGACTGGAAGAAGATCGAGG	58.2	GAGGTACTGCATGCCCAAAT	60.1	185	-
FAFUR-S5^#	(TG)33	CTTCCTCCCAGTAGCCGAGT	59.3	TCTCGAATTCGCAAGGAACT	57.9	257	-
FAFUR-S6^#	(TG)32	GGAAGGAGGAGATGGAAAGG	59.4	CGCAACACGTACACACACAC	59.6	245	-
FAFUR-S9^#	(TG)26	GTTTTCTTCTCGGAGGGGAG	57.9	AATGCTGGGATCGAAGTTTG	60.2	213	-
FAFUR-S15^#	(TG)20	TGCTATCTCCTGCTTGGACA	60.2	GCCTCACACACACACACACA	59.4	268	-
FAFUR-S16^#	(TG)19	TGCTTGCTAGCTTGGCACTA	60.4	ACAACTAGGCCATCAGTGGG	59.7	268	-
FAFUR-S19^#	(TG)19	AGCCCAACAGAAATACGCAC	60.6	GGGCTCACTCAAAAACCAAA	58.8	269	-
引物名称 Primer name	重复基序 Motif	左引物序列 Left-primer (5°-3°)	退火温度 T_m(℃)	右引物序列 Right-primer (5°-3°)	退火温度 T_m (℃)	产物大小 Product size (bp)	PIC
FAFUR-S20^#	(TG)18	TCGATTGGAGTCTTCAGCAA	59.9	CCCATGAGATTGTATTCGGC	60.3	269	-
FAFUR-S21^#	(TG)18	TGCACTGTTTAAATTCCCCC	60.3	AAATCTCCCTTCATGATGCC	58.8	229	-
FAFUR-S25^#	(TG)17	TCGTAGAAGCACTTCAGGGAG	58.8	CCAAAATAAGGCCATCGAAA	60.1	162	-
FAFUR-S26^#	(TG)17	CTTTGTCCCCTTCTCCATCC	57.9	TCTCGAAGTCGCAAGGAACT	60.5	185	-
FAFUR-S28^#	(TG)16	TGGCTCACTGAAAATCTCCC	61.1	TGTGTGGCAAGATAAGAAGGG	60.3	250	-
FAFUR-S29^#	(TG)15	TGCTGATTATGTGCTGCGTC	59.6	ATCGATCACACACCTAGGGC	59.5	234	-
FAFUR-S46^#	(AG)25	ATCGATCCTGGGGTAGCTTT	58.4	TTTCCTCTGCAAGACTGCAA	58.7	262	-
FAFUR-S48^#	(AG)23	TTCCAGATTCTTTTCCACGG	60.3	GTCACCTGGGAACTACCCCT	59.6	257	-

Table 5

Fig. 2

Fig. 3

Fig. 4

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3	CP72-1210	17	Saccharun hybrid	美国USA
4	NCo 310	13	Saccharun hybrid	印度India
5	F108	12	Saccharun hybrid	中国台湾Taiwan, China
6	华南56-12 Huanan 56-12	10	Saccharun hybrid	中国China
7	崖城71-374 Yacheng 71-374	9	Saccharun hybrid	中国China
8	粤农73-204 Yuenong 73-204	9	Saccharun hybrid	中国China
9	CP28-11	8	Saccharun hybrid	美国USA
10	Co 1001	6	Saccharun hybrid	印度India
11	桂糖11号 Guitang 11	6	Saccharun hybrid	中国China
12	云蔗65-225 Yunzhe 65-225	6	Saccharun hybrid	中国China
13	川73-219 Chuan 73-219	-	Saccharun hybrid	中国China
14	ROC 1	6	Saccharun hybrid	中国台湾Taiwan, China
15	科5 Ke 5	4	Saccharun hybrid	菲律宾Philippines
16	CP67-412	3	Saccharun hybrid	美国USA
17	POJ2878	3	Saccharun hybrid	印度尼西亚爪哇岛Java, Indonesia
18	华南56-21 Huanan 56-21	3	Saccharun hybrid	中国China
19	R570	-	Saccharun hybrid	法国France
20	LA purple	-	Saccharun officinarum	美国USA
21	SES208	-	Saccharun spontaneum	美国USA
22	LCP85-384		Saccharun hybrid	美国USA
23	ROC16	-	Saccharun hybrid	中国台湾Taiwan, China
24	ROC22	-	Saccharun hybrid	中国台湾Taiwan, China

重复次数 Repeat number	核苷酸重复基序 Nucleotide repeat motif						合计 Total
重复次数 Repeat number	Mono-	Di-	Tri-	Tetra-	Penta-	Hexa-	合计 Total
3					2044	2104	4148
4				941	313	355	1609
5			3718	225	112	72	4127
6		1210	1342	78	26	18	2674
7		527	636	29	9	6	1207
8		305	281	10	7	5	608
9		199	168	9	4	3	383
10	6297	128	77	8	4	0	6514
11	1997	84	71	5	2	4	2163
12	891	57	38	8	3	2	999
13	496	45	17	6	3	2	569
14	262	49	11	2	3	0	327
15	182	39	17	2	0	0	240
>15	954	619	71	18	8	3	1673
合计Total	11079	3262	6447	1341	2538	2574	27241
优势重复次数 Dominant repeat number (%)	56.84	37.09	57.67	70.17	80.54	81.74	78.08
比例 Proportion (%)	40.67	11.97	23.67	4.92	9.32	9.45	100
平均重复次数 Mean repeat number	11.68	11.23	6.1	4.78	3.38	3.3
设计引物的位点数 Number of loci primer designed	11079	3262	6447	1122	1995	1815
比例 Proportion (%)	100	100	100	83.67	78.61	70.51

物种 Species	项目 Item	甘蔗 S. spp.	高粱 S. bicolor	玉米 Z. mays	水稻 O. sativa	二穗短柄草 B. distachyon
单核苷酸	数量 Number	11079.00	14294.00	30700.00	15311.00	7991.00
Mono-nucleotide	相对丰度 Relative abundance	29.00	19.34	14.90	41.16	29.38
二核苷酸	数量 Number	3262.00	38090.00	64663.00	35315.00	9175.00
Di-nucleotide	相对丰度 Relative abundance	8.54	51.54	31.37	94.93	33.73
三核苷酸	数量 Number	6447.00	80299.00	185973.00	77566.00	37005.00
Tri-nucleotide	相对丰度 Relative abundance	16.88	108.66	90.23	208.51	136.05
四核苷酸	数量 Number	1341.00	47062.00	58806.00	26411.00	17428.00
Tetra-nucleotide	相对丰度 Relative abundance	3.51	63.68	28.53	71.00	64.07
五核苷酸	数量 Number	2538.00	16630.00	38408.00	17080.00	7972.00
Penta-nucleotide	相对丰度 Relative abundance	6.64	22.50	18.64	45.91	29.31
六核苷酸	数量 Number	2574.00	62227.00	119813.00	38940.00	18629.00
Hexa-nucleotide	相对丰度 Relative abundance	6.74	84.20	58.13	104.68	68.49
SSR 数量(丰度) SSR number (abundance)		27241.00	258602.00	498363.00	210623.00	98200.00
基因组大小 Genome size (Mb)		382.00	739.00	2061.00	372.00	272.00
总的相对丰度 Relative abundance		71.33	350.00	152.54	566.45	361.15
SSR频率SSR frequency (1 kb^-1)		14.02	2.86	6.56	1.77	2.77

项目 Item	甘蔗 S. spp	高粱 S. bicolor	玉米 Z. mays	水稻 O. sativa	二穗短柄草 B. distachyon
单核苷酸	(T)75	(A)71	(A)88	(C)51	(A)49
Mono-nucleotide	(T)63	(A)59	(A)85	(A)49	(C)45
	(G)49	(A)53	(A)83	(A)48	(A)43
二核苷酸	(TA)71	(AT)280	(AC)1366	(AC)170	(AT)312
Di-nucleotide	(TG)69	(AT)276	(AC)910	(AT)104	(AT)182
	(TA)55	(AT)270	(AT)178	(AT)100	(AT)158
三核苷酸	(TGT)123	(ACT)366	(ACC)291	(AAT)165	(AAT)225
Tri-nucleotide	(ATT)59	(AAT)327	(AAT)207	(AAT)147	(AAT)171
	(TTA)56	(AAT)318	(ACT)132	(AAT)126	(AAT)138
四核苷酸	(TTAT)23	(ACAT)524	(ACAT)196	(ACAT)132	(ACAT)196
Tetra-nucleotide	(ACAT)25	(AGAT)388	(ACAT)144	(ACAT)96	(ACAT)180
	(ATGT)26	(ACAT)260	(AAAG)100	(ACAT)96	(ACAT)180
五核苷酸	(CTTTT)29	(AATAT)740	(AATAT)115	(AATAT)55	(AGATC)100
Penta-nucleotide	(TTTTG)25	(AATAT)430	(ACTAT)115	(AATAT)55	(ACGCC)75
	(AATAT)24	(AATAT)315	(AATAT)85	(AATAT)55	(AGATG)65
六核苷酸	(ATTGTC)43	(AAATAT)390	(AATAGT)198	(ACCTAT)90	(AACAGC)90
Hexa-nucleotide	(TTTTTG)32	(AGATAT)366	(AATAGT)72	(ACATAT)78	(ACTGAT)78
	(TTATAT)16	(AAATAT)294	(AACCAT)66	(ACATAT)78	(AGAGAT)66

Development and application of SSR loci in monoploid reference genome of sugarcane cultivar

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