Artocarpus hypargyreus



Premise of the study:

Artocarpus hypargyreus (Moraceae), a threatened species endemic to China, to investigate the genetic diversity and structure of the species.


Methods and Results:

A. hypargyreusA. hypargyreus. The results showed the number of alleles per locus ranged from three to 10, and the observed heterozygosity and expected heterozygosity per locus varied from 0.000 to 0.706 and from 0.328 to 0.807, respectively.


Conclusions:

A. hypargyreusA. nitidusA. heterophyllus, indicating that they will be of great value for genetic study across this genus.




Keywords: Primer Note
Keywords: Artocarpus hypargyreus, microsatellite marker, Moraceae, transcriptome
Artocarpus hypargyreus Hance (Moraceae), a tall evergreen tree endemic to southern China, is valued for its milky latex for making stiff rubber and for its wood for making furniture (Zhou and Gilbert, 2003). Its natural populations have declined because of overexploitation and habitat loss, and it was listed as a vulnerable species in the IUCN Red List of Threatened Species in 1997 (IUCN, 2015). Therefore, genetic information, such as genetic diversity and population structure, will be important for the conservation of this species.

Here, we developed 34 novel simple sequence repeat (SSR) markers for A. hypargyreus, of which 10 were polymorphic in A. hypargyreus and the additional 24 successfully amplified loci were monomorphic. These 10 polymorphic markers were tested on 67 individuals from four populations of A. hypargyreus, and their transferability was tested in two other Artocarpus species.

METHODS AND RESULTS

The transcriptome of A. hypargyreus was sequenced with Illumina paired-end sequencing for the development of expressed sequence tag (EST)–SSR markers. The total RNA was extracted from the fresh leaves of A. hypargyreus (Appendix 1) using the modified cetyltrimethylammonium bromide (CTAB) method (Fu et al., 2004). Normalized cDNA libraries were constructed and sequenced using the HiSeq 2000 system (Illumina, San Diego, California, USA). The raw reads were cleaned by removing reads containing unknown “N” bases or more than 10% bases with a Q value < 20 using custom Perl scripts. A total of 25.34 million cleaned 100-bp paired-end reads were de novo assembled into 121,556 contigs (N50 = 906 bp) using Trinity version 2.1.1 (Grabherr et al., 2011) with default parameters.

The software QDD version 3.1 (Meglécz et al., 2014) was used to search SSR motifs containing two to six nucleotides with the minimum number of repeats as follows: six for dinucleotide and five for trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide. A total of 14,143 SSR loci were detected in 12,013 contigs. Among them, dinucleotide repeats account for the largest proportion for 54.7%, trinucleotide repeats account for 40.5%, and tetranucleotide repeats account for 1.1%. Subsequently, using Primer3 (Rozen and Skaletsky, 1999) implemented in the QDD program, primer pairs were successfully designed for 3693 SSR loci, which were further subjected to an “all-against-all” BLAST with an E-value of 1E−40 to remove redundancy. Finally, we obtained 2084 unique SSR loci based on which primer pairs were successfully designed.

Field investigations indicated that the individuals of A. hypargyreus showed a scattered distribution in their natural environments, causing difficulties in collecting large samples for each population. A total of 67 individuals were collected from four populations of A. hypargyreus (16–18 individuals for each population, see Appendix 1) to evaluate the polymorphisms of these SSR loci. In addition, five individuals of A. nitidus Trécul and nine individuals of A. heterophyllus Lam. were sampled to test the transferability of these primers. Genomic DNA was extracted from silica gel–dried leaves with the CTAB method (Doyle and Doyle, 1986).

Amplification and polymorphism tests were performed for 63 randomly selected primer pairs using two individuals from each population of A. hypargyreus. PCR amplification was performed according to Fan et al. (2013) with an appropriate annealing temperature, and PCR products were detected on 1% agarose gels. A total of 34 primer pairs were successfully amplified, generating legible products of the expected fragment size. Sequences of these SSR loci have been deposited in GenBank (Table 1, Appendix 2). The products were inspected with the Fragment Analyzer Automated CE system (Advanced Analytical Technologies [AATI], Ames, Iowa, USA) with the Quant-iT PicoGreen dsDNA reagent kit, 35–500 bp (Invitrogen, Carlsbad, California, USA). The raw data were analyzed by using PROSize version 2.0 software (AATI). Ten loci were polymorphic among the populations, and 24 loci were monomorphic.


Table: Table 1.

Characteristics of the 10 polymorphic microsatellite loci developed for Artocarpus hypargyreus
Table: .

Locusa		Primer sequences (5′–3′)	Repeat motif	Product size (bp)	Allele size range (bp)A		GenBank accession no.	Putative function	OrganismE	-value
	 AH1	F: GCAGCCGCCGTTGTTTCTCTTC (GTG)5		 225	 204–231	 8	 KX495095	 Hypothetical protein L484_018850 Morus notabilis	 1.65E−70	
		R: ACGCACCGAAAACCCCACAAAC								
	 AH11	F: GGCTGAATCACCCACTTGAGTT (AT)8		 185	 170–182	 4	 KX495101 Ultraviolet-B receptor UVR8-like isoform X3 [Nicotiana sylvestris	]	  1E−85	
		R: TGAACCCTCGGCCACCAAAGAA								
	 AH14	F: GCTTGTGGGTTCTGGGATCTAT (AAG)5		 249	 240–261	 8	 KX495103	 Transcription factor MYB5 Fragaria ×ananassa	 1.21E−27	
		R: CAGACACTAGTTTGGATGTACT								
	 AH31	F: TCCTCTAACGTGCGCCCCTAAG (ACC)8		 204	 264–282	 7	 KX495107	 Hypothetical protein L484_012061 Morus notabilis	 9.80E−79	
		R: AAACCCAGCGTGCCACCATTG								
	 AH33	F: TCGTCTTTGCAGGGCGATAAGT (TTA)6		 242	 242–251	 4	 KX495109	 Xylosyltransferase 1 Morus notabilis	 5.72E−132	
		R: AGAATCGGAGCCATGTAGAAAT								
	 AH46	F: GGAGAGGGCGGTGCAGTAGAA (TTC)5		 249	 246–261	 5	 KX495114	 Polyribonucleotide nucleotidyltransferase Morus notabilis	 1.83E−36	
		R: GCAGAGCAGACACTACAGTAGC								
	 AH59	F: TCTCCTCCACCTCCTCCATTGT (GTG)6		 238	 220–244	 9	 KX495120	 E3 ubiquitin-protein ligase RING1 Morus notabilis	 4.19E−66	
		R: GACCTTGGGACCCGCACTTCTT								
	 AH76	F: GAACGGCAGATTTCACCATTTT (ATT)5		 207	 192–207	 5	 KX495125	 Beta-glucosidase 42 Morus notabilis		 0
		R: AGGATCAACTTAGCCCACTATA								
	 AH77	F: CGAGAAGGTTCCGAGCCAGATT (TTA)7		 237	 231–249	 7	 KX495126	 Hypothetical protein PRUPE_ppa010130mg Prunus persica	 1.52E−50	
		R: CCCGACCAAGACCCGGAGTATA								
	 AH80	F: GACGTTTGAGGTGCGCGAAAAG (TCA)6		 203	 200–206	 3	 KX495127	 PHD finger protein 3 Morus notabilis	 4.39E−134	
		R: GGCCTACCTCCTACGAACTCTA								
Note: A = number of alleles.

aAnnealing temperature for all loci was 55°C.


The allelic polymorphisms of the 10 loci were further tested in 67 individuals from four populations of A. hypargyreus, and the efficiency of these markers in cross-species amplification was detected in A. nitidus and A. heterophyllus. GenAlEx version 6.5 (Peakall and Smouse, 2012) was used to calculate the average number of alleles per locus, the observed heterozygosity, the expected heterozygosity, and deviation from Hardy–Weinberg equilibrium (HWE). The results showed that the number of alleles per locus ranged from three to 10 (Table 1). The observed and expected heterozygosity ranged from 0.00 to 0.706 and from 0.328 to 0.807, respectively, and all loci showed significant deviation from HWE (Table 2). The scattered distribution of A. hypargyreus may cause difficulties in the long-distance dispersal of pollen and eventually lead to a decrease in the observed heterozygosity values and the significant deviations from HWE. Of the 10 SSR markers tested, all successfully amplified in A. nitidus and nine successfully amplified in A. heterophyllus (Table 3).


Table: Table 2.

Polymorphism of the 10 EST-SSRs in four populations of Artocarpus hypargyreus.a
Table: 

	DGD (N	 = 16)NLD (N	 = 17)XG (N	 = 16)HSD (N	 = 18)
	LocusA	Ho	Heb	A	Ho	Heb	A	Ho	Heb	A	Ho	Heb	
	AH1	5	0.615	0.523	6	0.529	0.704**	5	0.375	0.635	2	0.167	0.674
	AH11	4	0.688	0.537	4	0.353	0.593	4	0.375	0.561**	3	0.111	0.623
	AH14	5	0.125	0.773**	5	0.235	0.633**	4	0.063	0.588**	4	0.333	0.656*
	AH31	5	0.188	0.717**	5	0.118	0.740**	5	0.000	0.758**	5	0.278	0.778*
	AH33	4	0.438	0.646*	4	0.235	0.670**	4	0.188	0.725**	2	0.056	0.495**
	AH46	4	0.125	0.328**	3	0.471	0.567**	4	0.438	0.717**	3	0.278	0.415*
	AH59	7	0.563	0.758	7	0.706	0.751	7	0.375	0.807**	5	0.333	0.765
	AH76	5	0.313	0.604**	4	0.353	0.471**	4	0.250	0.537*	4	0.222	0.634**
	AH77	5	0.500	0.684*	6	0.706	0.740	7	0.438	0.762**	6	0.611	0.759
	AH80	3	0.250	0.531	3	0.000	0.602**	2	0.063	0.404**	2	0.056	0.526**
Note: A = number of alleles; He = expected heterozygosity; Ho = observed heterozygosity; N = sampled individuals from each population.

aVoucher and locality information are provided in Appendix 1.

bSignificant deviations from Hardy–Weinberg equilibrium after sequential Bonferroni corrections: * represents significance at the 5% nominal level; ** represents significance at the 1% nominal level.



Table: Table 3.

Cross-amplification of the 10 polymorphic EST-SSR markers developed for Artocarpus hypargyreus in A. nitidus and A. heterophyllus
Table: .

	SpeciesN		AH1	AH11	AH14	AH31	AH33	AH46	AH59	AH76	AH77	AH80
A. nitidus		5+	+	+	+	+	+	+	+	+	+	
A. heterophyllus		9+	+	+	+	+	+	+	+		—+	
Note: + = primers could be successfully amplified in all individuals; — = primers could not be amplified in any individual; N = number of individuals.



CONCLUSIONS

Ten novel polymorphic SSR markers were developed for A. hypargyreus, which are likely to be useful for evaluating the genetic diversity and population structure of A. hypargyreus, and for facilitating the development of a conservation strategy for this species. The cross-amplification of these microsatellite loci in A. nitidus and A. heterophyllus suggests that they will also be useful in studies of other species within Artocarpus.


LITERATURE CITED

Development and characterization of microsatellite markers from the transcriptome of  Primer3 on the WWW for general users and for biologist programmers.  Moraceae Link. 

Table: Appendix 1.

Voucher specimen information for Artocarpus
Table:  populations used in this study. Specimens are deposited at the Herbarium of Sun Yat-sen University (SYSU), China.

	Species	Population	Voucher no.	Collection locality	Geographic coordinatesN	
A. hypargyreus Hancea			NLD20151219051	Neilingding Island, Shenzhen, China	22°24′29.85″N, 113°49′00.46″E	1
A. hypargyreusb		DGD	DGD20160112003	Zhuhai, Guangdong, China	22°02′28.51″N, 114°16′19.84″E	16
A. hypargyreusb		NLD	NLD20151219054	Shenzhen, Guangdong, China	22°24′36.23″N, 113°48′20.35″E	17
A. hypargyreusb		XG	JSGY20160119087	Hong Kong, China	22°21′19.29″N, 111°53′25.96″E	16
A. hypargyreusb		HSD	HSD20151223124	Fengkai, Zhaoqing, Guangdong, China	23°27′25.60″N, 113°48′20.35″E	18
A. nitidus Tréculc		Cultivated	SYSU2015060501	SYSU, Guanghzhou, China	23°5′51.88524″N, 113°18′4.34″E	5
A. heterophyllus Lam.c		Cultivated	SYSU2015060502	SYSU, Guanghzhou, China	23°5′51.88524″N, 113°18′4.34″E	9
Note: N = number of individuals sampled.

aSamples used for cDNA library construction.

bSamples used for initial PCR amplification trials and detailed evaluation for polymorphisms.

cSamples used for transferability test.





Table: Appendix 2.

Characteristics of 24 monomorphic EST-SSR markers in Artocarpus hypargyreus
Table: .

Locusa		Primer sequences (5′–3′)	Repeat motif	Expected allele size (bp)	GenBank accession no.
	Art_SSR2	F: CACACAAAATTCGTGCCCATTA(GGA)8		207	KX495096
		R: TCCTGAGGTTTTGGCTGCTGTT			
	Art_SSR3	F: CCAACAAACAGGGCCAACTCAA(CCA)5		164	KX495097
		R: ATGTCGCCAAGGGAGCTGTATC			
	Art_SSR4	F: TGGTGGTGGATGATGCACAATT(GTG)6		248	KX495098
		R: CGTCTCAATCTACCTTCGCATA			
	Art_SSR6	F: TTGAGGCAGGGTGGATGTAATC(AG)6		188	KX495099
		R: TGTTTCTTTTGCATCCTTCTTC			
	Art_SSR8	F: TGGCATCAACGCGGAAGGATAT(GAA)5		248	KX495100
		R: CCCCTCATCCTTCACCCTTCC			
	Art_SSR12	F: TGACAACCATGGGCACGATCAT(CAT)5		215	KX495102
		R: TGTCAGCCAGAACATGCAAGAA			
	Art_SSR24	F: AGGCTCAAAAGGGTGGCAATAA(GCA)5		204	KX495104
		R: GGGTGTTGGAGTTGGGCATCAT			
	Art_SSR25	F: GGTACACAGCCGCGAAGAATAA(TC)6		227	KX495105
		R: CACTTTTCACCACCACCAAACA			
	Art_SSR27	F: AAGGTGGCAGAGCAGAGGAGTC(AT)8		218	KX495106
		R: ACCAAGCAAGAACCAGGTCAGC			
	Art_SSR32	F: GGCACGGTTCTCAAGCCTGAA(GAA)5		234	KX495108
		R: TCATGATCAATCCAGGCACAAA			
	Art_SSR36	F: GGGCCTCGACGAGCACTCTATC(CCG)5		124	KX495110
		R: GGCAGCGTGGAACCGATCTG			
	Art_SSR39	F: CAACAGCCCATACGTCGGATCT(TAG)5		174	KX495111
		R: CTCTCTGAGGGTCCCACTATTC			
	Art_SSR40	F: TCGCCGCTGTCCTCGTCTTC(ACC)5		145	KX495112
		R: CCCTCTACCGTACGATCCTCAT			
	Art_SSR43	F: GCAAGCAGACAGTGGGGAGATA(AAG)6		232	KX495113
		R: GGTTGAGCCTTCTTCGCGTACA			
	Art_SSR49	F: AACAGCACCGTCAATGGAACTT(GA)6		202	KX495115
		R: CCTTGCAGCCTCCCGAGCTATC			
	Art_SSR52	F: TCCCCTGGAGCCTGATGAGTTT(TC)10		225	KX495116
		R: CGCGAACTGAAAAGGGGTTATG			
	Art_SSR55	F: GAGTTCAGCCCAGCCTGCA(TA)8		137	KX495117
		R: TTGCCAAAACACATGAAACAGT			
	Art_SSR56	F: AAGACCCGGAAGAAAGGAAAGA(GA)7		173	KX495118
		R: CCCTCTGTAACGACTGTGATTT			
	Art_SSR58	F: GCAAGGGGAAGCTGAGGGTATA(GA)6		239	KX495119
		R: AGGCCTTTTCTCGCTCCTCAAA			
	Art_SSR61	F: TTACCCTAATAGCCGCCGATTT(TTA)5		199	KX495121
		R: AGTAGCGCTCCAATGCCATTCA			
	Art_SSR62	F: GAAGGGACGGAGGGGAGAGTTT(GCG)5		208	KX495122
		R: CCGGCATGAGGGTCCAAATCAA			
	Art_SSR67	F: TCTTCTTCGGAGCTGGCATAGA(CGC)5		179	KX495123
		R: AGGAGGTTCGCTTGTCCTTGTT			
	Art_SSR72	F: ATGGGTGAGGAGAGCGTGATGA(AG)7		146	KX495124
		R: TTCTCCTTTCGCTTCCCCTTCT			
	 Art_SSR84	F: TGACCACCATCACCACCAAAAC (ACA)7		 165	 KX495128
		R: GCAGCCAAGAGACGGTGGTAAT			
aAnnealing temperature for all loci was 55°C.
Liu Haijun, Tan Weizheng, Sun Hongbin, Liu Yu, Meng Kaikai, Liao Wenbo