___________________________
Corresponding author: Li-jing Chen, E-mail: chenlijing1997@126.com, Phone: +86-24-88487163, Fax: +86-24-88492799.Tian-lai Li，E-mail: litianlai@126.com, Phone: +86-24-88487143, Fax: +86-24-88487143.
UDC 575:630 DOI: 10.2298/GENSR1502425C
Original scientific paper
CONSTRUCTION OF A GENETIC LINKAGE MAP IN LiliumUSING A RIL MAPPING POPULATION BASED ON SRAP MARKER
Li-jing CHEN#*, Hong-mei LI# , Shao-kun SUN#, Muhammad IRFAN, Jing-wei LIN, Ming ZHONG, Hui MA, Zhi-Fu GUO, Tian-lai LI*
Key Laboratory of Agricultural Biotechnology of Liaoning Province, Key Laboratory of Protected Horticulture (Ministry of Education), Bioscience and Biotechnology College, Shenyang
Agriculture University, Shenyang 110866 China
Chen L., H.Li , S. Sun, M. Irfan, J. Lin, M. Zhong, H. Ma, Z. Guo, T. Li (2015): Construction of a genetic linkage map in Lilium using a RIL mapping population based on SRAP marker- Genetika, Vol 47, No. 2, 425- 438.
A genetic linkage map of lily was constructed using RILs (recombinant inbred lines) population of 180 individuals. This mapping population was developed by crossing Raizan No.1 (Formolongo) and Gelria (Longiflomm) cultivars through single-seed descent (SSD). SRAPs were generated by using restriction enzymes EcoRI in combination with either MseI. The resulting products were separated by electrophoresis on 6% denaturing polyacrylamide gel and visualized by silver staining. The segregation of each marker and linkage analysis was done using the program Mapmaker3.0. With 50 primer pairs, a total of 189 parental polymorphic bands were detected and 78 were used for mapping. The total map length was 2,135.5 cM consisted of 16 linkage groups. The number of markers in the linkage groups varied from 1 to 12. The length of linkage groups was range from 11.2 cM to 425.9 cM and mean marker interval distance range from 9.4 cM to 345.4 cM individually. The mean marker interval distance between markers was 27.4 cM. The map developed in the present study was the first sequence-related amplified polymorphism markers map of lily constructed with recombinant inbred lines, it could be used for genetic mapping and molecular marker assisted breeding and quantitative trait locus mapping of Lilium.
Key words: genetic linkage map, genome length, Lily, map coverage, RIL, SRAP
INTRODUCTION
known as a large plant genome since Paris japonica discovered by Scientists at Kew's Jodrell Laboratory has the biggest genome (even larger than the previous record holder-the marbled lungfish) (http://www.sciencedaily.com/releases/2010/10/101007120641.htm). Molecular research in Liliumis not only useful to germplasm improvement, but also discover the regular pattern how big genome maintains itself steady normally under a complex environment. In Lilium, whole-genome sequencing needs a lot of work and research project grant, but constructing a molecular genetic map can conquer these disadvantages. Molecular genetic maps pavethe way for locating and cloning of some important agricultural traits and marker-assisted selection (MAS) breeding. The first molecular genetic map of lily was constructed by Heusden with 100 descendants of Asiatic lily hybrid based on AFLP markers (van HEUSDEN et al., 2002). In the same year, ISSR-RAPD based genetic map of 96 F1 progenies from Asiatic lily using a double pseudo-testcross
strategy was finished (ABEet al., 2002). After that, diversity array technology (DArT) markers were used to complete a genetic linkage map in a F1 population of Longiflorum ×Asiatic lily
hybrids (KHAN, 2009).
Sequence-related amplified polymorphism (SRAP) technique was created by Li and Quiros and then used to construct the genetic linkage map of Brassica (LI and QUIROS, 2002). Owing to many advantages, SRAP technique has been applied in construction of a genetic map in Chinese Hedychium (GAOet al., 2008), citrus (GULSENet al., 2010), Populus adenopoda Maxim (WANGet al., 2010), Kenaf (ZHANGet al., 2011), genetic relationship analysis in Ramie (LIUet al., 2008), porphyra (QIAOet al., 2007), cassava (QI et al., 2010) and chrysanthemum (ZHANG et al., 2011).
Recombinant inbred lines (RIL), as one of mapping populations, have two striking advantages: constant diversity among individuals can be kept forever with repeated trials, and bringing new molecular markers into genetic linkage maps which have been constructed to increase the density of molecular markers on the map. The genetic linkage maps of sunflower (YU et al.,, 2003), sorghum (PRASANTA and HENRY, 2000, MENZet al.,, 2002) have been completed with RILs based on AFLP, RFLP , and SSR markers. In the light of previous literature, little research has been done on on SRAP markers for genetic linkage map construction. So, this study was designed to construct the genetic linkage map of lily by SRAP markers using RIL population of 180 individuals.
MATERIALS AND METHODS Plant materials
The mapping population comprised of 180 RIL individuals, obtained from a cross between Raizan No.1 and Gelria. The process of RIL used in this study as follows: Raizan No.1 (L.longiflorum) as the male-parent originating from Lilium.formosanum × Lilium.japonicum, Gelria as female-parent, F1 which was bred and kept by embryo cultivated in vitro was obtained
from a cross of Raizan No.1 and Gelria. F2 obtained from a cross between F1, inbred continually 6
times by means of single seed heredity. Then these progenies were called recombinant inbred lines (RIL). Parents (from Tomita in Japan) and RIL are maintained in plant garden at Shenyang Agricultural University, China.
DNA extraction
placed into liquid nitrogen for 30 s and chopped with pestle. Then, 0.5 ml of 2 × CTAB buffer (pre-heat at 70 °C) was added to the tubes and incubated at 65 °C for 30min. After incubation, 0.6 ml of phenol-chloroform-isoamylol was added and mixed, then the supernatant was carefully collected after centrifuge at 16 ° C, 12 000 rpm for 10 min. Further extraction was done by repeating the previous operation using chloroform-isoamylol instead of phenol-chloroform-isoamylol. Finally, 10 uL RNase A (10 mg/m) was added into the sample which was later incubated at 37 °C for 30 min. The supernatant was transferred into a new tube and the DNA was precipitated in an equal volume of 2-propanol at -20 °C for more than 2 h. The DNA after centrifuging was then washed with 70% ethanol and dissolved in TE buffer.
PCR amplification
PCR was performed in 10 µl reaction volume consisting of 0.2 µl Taq DNA polymerase, 1 µl 10 × PCR buffer, 1 µl 10 µ M of each primer (table 1), 1.2 µl 3 mM Mg2+, 0.8 µ l 200 µ M dNTPs, and 1.0 µl of template DNA. The amplification reactions were programmed as follows: an initial denaturation at 94 °C for 5 min followed by 5 cycles at 94 °C for 1 min, 34 °C for 1 min and 72 °C for 1 min, subsequently followed by 35 cycles at 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min with a final extension step at 72 °C for 7 min. The resulting products were denatured in formamide at 95 °C and separated by electrophoresis on 6% polyacrylamide geland visualized by silver staining.
Table 1. The primer sequences used in SRAP analysis
Code forward primers Code reverse primers
ME1 5 TGAGTCCAAACCGGATA3 EM1 5 GACTGCGTACGAATTAAT3
ME2 5 TGAGTCCAAACCGGAGC3 EM2 5 GACTGCGTACGAATTTGC3
ME3 5 TGAGTCCAAACCGGAT3 EM3 5 GACTGCGTACGAATTGAC3
ME4 5 TGAGTCCAAACCGGACC3 EM4 5 GACTGCGTACGAATTTGA3
ME5 5 TGAGTCCAAACCGGAG3 EM5 5 GACTGCGTACGAATTAAC3
ME6 5 TGAGTCCAAACCGGTAA3 EM6 5 GACTGCGTACGAATTGCA3
ME7 5 TGAGTCCAAACCGGTCC3 EM7 5 GACTGCGTACGAATTCAA3
ME8 5 TGAGTCCAAACCGGTGC3 EM8 5 GACTGCGTACGAATTCTG3
ME9 5 TTCAGGGTGGCCGGATG3 EM9 5 GACTGCGTACGAATTCGA3
ME10 5 TGGGGACAACCCGGCTT3 EM10 5 GACTGCGTACGAATACGA3
EM11 5 GACTGCGTACGAATTCCA3
Data analysis
SRAPs were scored as dominant markers on the basis of the presence or absence of the band at a corresponding position among the segregating RILs population. Only clear and unambiguous bands were scored for genotyping. Segregating SRAP markers in the mapping population were named according to the primer combinations employed and the order in the gel image. Mapmaker3.0 was used for construction of linkage map and Mendel’s 1:1 segregation ratio was also measured (STEPHENet al.,, 1993). The Chi-Square Test was used for statistical analyses. The Kosambi mapping function was used for calculation of map distances.
The actual genome length contains two aspects, length of fragment map (Gof) is summation of all linkage groups and length of linked markers (Goa) includes triplet and linkage pairs. Here two methods (POSTLETHWAIT et al.,, 1994, ARAVINDA et al., 1991) were used to estimate the genome length (Gel),
Ge1=Gof+2·s·n
Ge2=Gof (m+1) (m-1) Gel= (Ge1+Ge2)/2
Where Ge1 equals to single linkage group length adds to twice mean interval of whole linkage map, remedying shortage of telomere and terminal markers on linkage map. S is mean interval among markers and n is number of linkage group, meanwhile m is marker accounts of linkage group. Mean value obtained from the two methods was estimated as genome length. Total map coverage (Ce) was calculated by the formula Ce=Gof/Ge
RESULTS
Among 110 screened primers, only 50 primers produced clear fragments and polymorphism, which were used to construct a genetic linkage map in lily (Figure1). These 50 pairs of primer combination produced total of 189 loci. From these 189 loci, 102 showed distinct polymorphism and exhibit segregation distortion. A 2 test (P < 0.05) was performed to authenticate alleles of each parent that deviated from Mendelian segregation ratios. It was shown that serious segregation distortion (53%) existed in this mapping population. A total of 87 polymorphisms loci gained from 2 test (P < 0.05), of which 78 loci were used to construct a map with Mapmaker3.0, which were assigned to 16 linkage groups (LOD > 3.0.) spanning 2,135.5 cM.
Table 2.The characters of 16 linkage groups constructed by SRAP markers in Lilium
Linkage Group
Marker numbers
Max map distance (cM)
Min map distance (cM)
Length (cM) Average
distance (cM)
LG1 4 34.7 15.3 67.6 22.5
LG2 2 11.2 11.2 11.2 11.2
LG3 2 17.6 17.6 17.6 17.6
LG4 2 17.6 17.6 17.6 17.6
LG5 3 17.6 17.6 35.2 17.6
LG6 3 17.6 13.1 30.7 15.4
LG7 2 17.6 17.6 17.6 17.6
LG8 4 30.3 13.1 58.7 19.6
LG9 12 39.7 11.2 283.9 23.7
LG10 9 34.7 15.3 224.1 24.9
LG11 4 345.4 34.7 425.9 106.5
LG12 4 75.2 15.3 113.7 28.4
LG13 10 62.6 9.4 291.7 29.2
LG14 9 75.2 15.3 279.0 31.0
LG15 3 53.2 39.7 138.7 46.2
The longest interval was 354.5 cM, while the shortest interval was 9.4 cM and mean interval of markers was 27.4 cM. The detailed information of 16 linkage groups was described in Table 2 and shown in figure 2.
The total genome length of this map was estimated to span 2,135.5 cM (Ge), and expected genome coverage was 81.6% for RIL from Raizan No.1 × Gelria.
LG1
LG2
LG3
LG4
ME2EM9_8
ME3EM6_5
17.6
ME2EM5_2
ME5EM11
17.6
ME2EM1_1
ME2EM1_2
11.2
ME1EM1_1
ME3EM3_4
15.3
ME3EM6_6
17.6
LG5
LG6
LG7
LG8
ME5EM6_5
ME5EM6_1
15.3
ME5EM2_1
13.1
ME5EM6_7
30.3
ME4EM5_6
ME4EM5_8
17.6
ME4EM5_4
ME4EM5_2
13.1
ME4EM8_2
17.6
ME2EM10_1
ME4EM8_4
17.6
LG9
LG10
ME3EM2_5
ME3EM2_8
17.6
ME3EM4_1
15.3
ME3EM6_7
34.7
ME3EM6_8
34.7
ME3EM7_2
30.3
ME3EM8
26.5
ME3EM11_3
34.7
ME4EM5_3
30.3
ME1EM1_3
20.3
ME1EM5
11.2
ME1EM8_1
20.3
ME1EM8_2
13.1
ME2EM9_1
30.3
ME2EM9_5
39.7
ME2EM9_7
20.3
ME2EM10_6
30.3
ME2EM11_2
13.1
ME3EM1_2
15.3
ME3EM2_4
39.7
LG11
LG12
ME5EM5_7
ME5EM7_3
15.3
ME10EM11
23.2
ME5EM5_4
75.2
ME4EM6_2
ME4EM11_1
45.8
ME4EM11_2
345.4
LG13
LG14
ME3EM2_9
ME3EM3_3
30.3
ME3EM3_6
30.3
ME3EM4_2
34.7
ME3EM4_4
30.3
ME3EM7_1
15.3
ME3EM7_3
23.2
ME3EM9_2
39.7
ME3EM11_6
75.2
ME1EM1_4
ME1EM4_
9.
4
ME1EM7_1
13.
1
ME1EM9_1
62.
6
ME2EM3_2
34.
ME2EM4_1
45.
8
ME2EM8
45.
8
34.
ME3EM2_3
15.
LG15
LG16
Figure 2. The genetic linkage group of SRAP markers in Lilium
DISSCUSSION
In this study, we reported the first SRAP genetic linkage map of lily based on 180 RILs from a cross between Raizan No.1 (Formolongo) and Gelria (Longiflomm) cultivars. Although 89.7% of polymorphic markers were used to construct the genetic linkage map and 10.3% of markers were not polymorphic. Two main possible reasons can interpret this phenomenon: first, the information of the genome of lily cannot be completely included in the genetic linkage map, the second, Partial markers (rejected) belong to the same chromosome with some markers on map, but far distance between markers rejected cannot be brought into the map due to lack of sufficient
ME5EM4_1
ME5EM7_2
45.8
ME5EM7_4
17.6
ME5EM5_8
45.8
ME5EM7_7
13.1
ME4EM1
ME4EM2_1
53.2
ME4EM5_7
45.8
linkage relationship on corresponding mapping requirement. Incompatible allele and continuous selfing of recombinant inbred lines possibly cause the distorted segregation.
The total genome length of L.longiflorum is bigger than Asiatic lily (HEUSDEN et al., 2002, ABE et al., 2002) and Longiflorum × Asiatic lily hybrids (KHAN 2009), but our average interval was so bigger that there was a little difficulty to locate some traits on RIL population. It was worth being satisfied that map constructed by RIL mapping population can accommodate more new follow-up molecular markers to integrate the present map. Although we gained a framework map of lily, RIL mapping population and SRAP marker were firstly used to construct a genetic linkage map of lily. As one of the plants with an enormous genome, the construction of high-density genetic map in Lilium can be applied to locate some important traits accurately. In fact, a high density genetic linkage map in F1 population of Longiflorum × Asiatic lily hybrids
using diversity array technology (DArT) markers was successfully completed (KHAN, 2009). Density of the DArT map was 3.5 cM, smallest mean interval in molecular genetic map of lily according to published papers till now. High-density genetic mappings of Potato /tomato, Rice, Italian ryegrass, lettuce, rye, eucalyptus have been completed with closest mean interval of 0.7 cM (TANKSLEY et al., 1992, HARUSHIMA et al., 1998, INOUEet al., 2004, TRUCOet al., 2007, HANNAet al., 2009, NEVESet al., 2011).Although its marker density was too low to locate some important agronomic traits, it will be impossible to come true the location after having constructed a higher density map of lily by adding new molecular markers to the primary map.
ACKNOWLEDGEMENTS
This study was financially supported by the Specialized Joint Research Fund for the Doctoral Program of Higher Education (20112103120005) and the China Postdoctoral Science Foundation funded project (20100471471), and National High Tech Research and Development Program of China (2006AA100109).
Received December 27th , 2014 Accepted March 25th
, 2015
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KOSTRUKCIJA GENETIŠKE MAPE UKOP ANOSTI KOD KRINA PRIMENOM RAIL MAPIRANJA POPULACIJE ZASNOVANA NA SRAP MARKERIMA
Li-jing CHEN#*, Hong-mei LI# , Shao-kun SUN#, Muhammad IRFAN, Jing-wei LIN, Ming ZHONG, Hui MA, Zhi-Fu GUO, Tian-lai LI*
Glavna Laboratorija poljoprivredne biotehnologije Liaoning provincije, Glavna Laboratorija u zaštiti hortikulturnih biljaka (Ministarstvo za obrazovanje), Fakultet bioloških nauka i
biotehnologije, Shenhjang Poljoprivredni Univerzitet, Shenjang 110866, Kina.
Izvod
Geneti ka mapa ukop anosti je konstruisana, koriš enjem rekombinovanih samooplodnih linija (RIL) u populaciji od 180 individua. Populacija je konstruisana ukrštanjem Raizan No.1 (Formolongo) i Gelria (Longiflomm) kultivara selekcijom pojedina nih zrna (SSD) SRAPs markeri su generisani koriš enjem restrikcionih EcoR1 u kombinaciji sa MseI. Segregacija svakog markera i analiza ukop anosti je vršena primenom Mapmaker3.0. Sa 50 parova prajmera detektovano je ukupno 189 roditeljskih polimorfnih traka a 78 je koriš eno u mapiranju. Ukupna dužina mape je bila 2,135.5 cM koja se sastoji od 16 grupa ukop anosti. Mapa konstruisana u ovim istraživanjima je prva bazirana na markerima zasnovanim na polimorfizmu markera amplikovanih skvenci rekombinovanih linija krina i može da se koristi u marker – asistiranom oplemenjivanju i mapiranju lokusa koji kontrolišu kvantativne osobine krina.
