Molecular mapping of flowering time genes in chickpea (Cicer arietinum L.)

Transcription

1 Molecular mapping of flowering time genes in chickpea (Cicer arietinum L.) Mallikarjuna B.P. Ph.D. Scholar University of Agricultural Sciences, Raichur, Karnataka Supervisors: Dr. Pooran Gaur, ICRISAT Dr. K. P. Viswanatha, UAS-Raichur

2 Chickpea (Cicer arietinum L.) A diploid (2n=2x=16), self-pollinated food legume with genome size of 740 Mbp World s second most important food legume, grown in 13 mha area, over 50 countries globally (FAOSTAT, 2015) India - largest chickpea producing country having cultivated in 9.6 mha with total production of 8.8 m tonnes (FAOSTAT, 2015) Major source of protein (22-28%), good source of Fe, Zn, β- carotene, dietary fiber Mostly grown as a post-rainy season crop in the semi-arid tropics and a spring/summer crop in the Mediterranean and Temperate regions 2

3 Constraints to chickpea production: Abiotic stress Drought stress Heat stress Salinity stress Low temperature stress Biotic stress Fusarium wilt (FW) Ascochyta blight dry root rot (DRR) botrytis grey mold (BGM) Pod borer Terminal drought/heat stress Moisture & high temperature stress that occurs at pod filling & seed development stage with increasing severity towards the end-of-season Major constraint to chickpea production in over 80% of global chickpea area Breeding strategies: Developing early maturing varieties Importance of early maturing varieties Escape end-of-season high/low temperature stress Can be cultivated in a short window of cropping season Enhance cropping intensity and crop diversification opportunities 3

4 Flowering time Number of days from sowing to appearance of first fully opened flower Can be recorded with high precision and provides good indication of days to podding and maturity Important component for adaption of chickpea to various environments Influenced by many factors (temperature, photoperiod, altitude, latitude, genotype) Four early flowering genes have been reported in chickpea - efl-1 (in ICCV 2, ICCV 96029) - elf-2 (in ICC 5810) - efl-3 (in BGD 132) and - efl-4 (in ICC 16641) 4

5 Objectives I. Genetics of flowering time genes II. Development of genetic linkage map III. QTL mapping for flowering time genes IV. Association of flowering time with maturity and yield related traits Desi Kabuli 5

6 Objective 1 Genetics of flowering time genes 6

7 Materials and methods Experimental site: ICRISAT, Patancheru Plant Material: Female parents Male parent Line Origin Early flowering gene reported Reference ICCV Cross-bred efl-1 Kumar and Rheenen (2000) ICC 5810 India ppd/efl-2 Or et al. (1999) BGD 132 Cross-bred efl-3 Hegde (2010) ICC Pakistan efl-4 Gaur et al. (2015) CDC Frontier Cross-bred (Canada) - Warkentin et al. (2005) All the four female lines were crossed to a common male parent (CDC Frontier) to develop F 1 s and the F 1 seeds were selfed to develop F 2 mapping populations 7

8 Evaluation for flowering time: Post rainy season, : Parental lines, F 1 s and F 2 populations were evaluated for flowering time and yield related traits Planting was done in 4m row at a wider spacing of 60 cm x 20 cm Post rainy season, : Parents and F 3 progenies (20 plants in each progeny row of 2m length) were evaluated for flowering time Sl. No. Cross Population size F 2 F 3 1 ICCV CDC Frontier ICC 5810 CDC Frontier BGD 132 CDC Frontier ICC CDC Frontier

9 Figure 1. General view of the experimental plot of evaluation of F 2 populations for flowering time conducted during post-rainy season of Desi Kabuli Early Figure 2. Segregation for flowering time and maturity in the F 2 population of the cross ICC 5810 CDC Frontier Late Medium 9

10 Results: Post rainy season, Table 1. Flowering time & maturity of parental lines Sl. No. Parents N Flowering time Days to maturity Mean ± SE Range Mean ± SE Range 1 ICCV ± ± ICC ± ± BGD ± ± ICC ± ± CDC Frontier ± ± Table 2. Flowering time & maturity of F 1 s Sl. No. Cross N Flowering time Days to maturity Mean ± SE Range Mean ± SE Range 1 ICCV CDC Frontier ± ± ICC 5810 CDC Frontier ± ± BGD 132 CDC Frontier ± ± ICC CDC Frontier ± ± N = Total no. of plants 10

11 Figure 3. Frequency distribution of flowering time in F 2 populations Desi Kabuli 11

12 Table 3. Segregation of flowering time in F 2 populations Sl. No Cross N Observed Expected Ratio Late Early Late Early tested χ 2 P-value* ICCV CDC Frontier : ICC 5810 Desi Kabuli CDC Frontier : BGD 132 CDC Frontier : ICC CDC Frontier : *Null hypothesis of the test is that progeny segregate in the ratios tested. If the p-value (probability) is less than or equal to 5, then reject the null hypothesis. Otherwise one fails to reject the null hypothesis. 12

13 Figure 4. Segregation of flowering time in F 3 progenies of different crosses Early Segregating Late Late Segregating Early ICCV CDC Frontier ICC 5810 CDC Frontier Late Segregating Early Late Early Segregating BGD 132 CDC Frontier ICC CDC Frontier 13

14 Post rainy season, Table 4. Segregation of flowering time in F 3 progenies of four crosses Sl. No. Cross Phenotypic class No. of progeny tested Segregating Observed Segregating Expected Nonsegregating Nonsegregating Ratio tested χ 2 P-value* 1 ICCV CDC Frontier 2 ICC 5810 CDC Frontier 3 BGD 132 CDC Frontier 4 ICC CDC Frontier Late : Early : Late : Early : Desi Kabuli Late : Early : Late : Early : *Null hypothesis of the test is that progeny segregate in the ratios tested. If the p-value (probability) is less than or equal to 5, then reject the null hypothesis. Otherwise one fails to reject the null hypothesis. 14

15 Objective 2 Development of genetic linkage map 15

16 Table 5. Markers used for parental polymorphism study Marker No. Reference KASPar SNPs 714 Hiremath et al. (2012); Jaganathan et al. (2013) SSRs CaM-series 146 Thudi et al. (2011) Winter-series 135 Winter et al. (1999) ICCM-series 124 Nayak et al. (2010) H-series 57 Lichtenzveig et al. (2005) NCPGR-series 10 Gaur et al. (2011); Sethy et al. (2006) Total

17 Sl. No Results: Table 6. Parental polymorphism status Cross ICCV CDC Frontier ICC 5810 CDC Frontier BGD 132 CDC Frontier ICC CDC Frontier Total No. of markers screened SNPs No. of polymorphic markers Total No. of markers screened SSRs No. of polymorphic markers (6.9 %) (21.4 %) (6.2 %) (20.1 %) (5 %) (19 %) (4 %) (19.7 %) Polymorphic markers were genotyped on respective mapping population Construction of Genetic linkage map: using JoinMap v. 4.0 software 17

18 Figure 5. Genetic linkage map of the cross ICCV CDC Frontier CaLG01 CaM TA8 1.2 TR ICCM TR42 CaLG02 CaLG03 CaLG04 TR19 CaM1122 TS H4D TR CaM TA H1B H1H TA TR CaM H5A CaM0886; H3F H4G H6D CaM CaM2049 ; NCPGR7 3.8 H3C ICCM TA H3F ICCM0263a 13.6 NCPGR10 CaLG TAA170 TA196 CaLG05 TA TR18 TR TA TA CaM TA CaM CaM TR TR01 CaM1402 TR40 CaM0753 TA176 TA22 TA80 TR44 Sfl/sfl 43.7 TA64 CaLG07 NCPGR GAA47 ICCM0192a B/b CaLG08 GA6 Total No. of markers -77 Total Map length cm GA34 NCPGR93 CaM0244 TA H1C22 TA21 CaM0661 TAA58 TA180 CaM0111 TA78 ICCM0160 ; CaM0443 TA28 TS46 Desi 19.3 TA TS45 Kabuli H1C TA144

19 Figure 6. Genetic linkage map of the cross ICC 5810 CDC Frontier CaLG01 CaLG02 CaLG03 CaLG04 TR56 TS5 TS72 TA122 GA TA TA TR ICCM H3F08 H3C TS36 H1F TS104 ; CaM CaM CaM H4G TR H3F CaM0507 TA30 ; H3D TR CaM TR H5A CaM ICCM0297 H6D11 H4D TR42 H4A TA TA TA TAA170 CaLG TA NCPGR21 TA GAA47 CaLG08 CaLG TR01 GA TA71 ICCM0243c TA116 ; CaM0038 CaM0805 TA11 Total No. of markers -76 Total Map length cm TR44 TA22 TR7 ; TA80 CaM1402 ; TA176 CaM0317 NCPGR93 CaM0244 TA CaLG TAA CaM0443 TA21 ICCM0160 ; TA78; CaM0661 ; TA180; H1C22 CaM0111 TA TS TA118 TS45 Desi NCPGR89 TA B/b TR33 Kabuli 19

20 Figure 7. Genetic linkage map of the cross BGD 132 CDC Frontier CaLG03 CaLG01 ICCM0263a CaLG04 TA H3C06 TS TR H4G11 ; CAM2049 CaLG CaM CaM0507 H3F08 ; H1B TS36 ; CaM0113 TR H3F H1H15 H4D08 ; TA CaM1515 H4A04 ; TA TR H5A CaM H6D NCPGR TA H3D ICCM TA TAA TR42 CaLG06 TA TA64 CaLG05 TR TA H3A CaM CaM TR TA TA TA196 TR01 TR44 TR7 TR40 CaM1402 TA22 CaLG07 NCPGR NCPGR12 TA ICCM0192a CaLG08 TA H1D GA6 4.5 TA144 Total No. of markers -68 Total Map length cm 65.4 TA H1C22 TAA58 ; CaM0111; TA21 ; CaM0443; TA78 ; H1N12; CaM0661 TS TA118 20

21 Figure 8. Genetic linkage map of the cross ICC CDC Frontier CaLG03 CaLG01 CaLG02 CaM1122 TA122 GA TS5 4.1 TR CaM TR13 ; H3F H1F CaM CaM CaM NCPGR TA142 CaLG04 TS TS CaM0507 ; H4G NCPGR7 ; CaM ICCM H1H TAA H3D05 TR43 TA8 ICCM H5A08 ; H4A04 TA200 TA110 CaLG TA NCPGR21 GAA47 ICCM0192a TA TR CaLG05 TA71 TA05 CaM1228 TA116 CaM0805 TA179 CaM0038 TA11 TR18 Total No. of markers -67 Total Map length cm TR44 TA22 CaM1402 TA176 NCPGR93 CaM0244 TA106 CaLG TR33 NCPGR19 CaLG08 GA6 5.7 ICCM0130a TA118 TS ICCM CaM0661 ; CaM TAA58 ; H1C TA TS H1D24

22 Table 9. Features of Consensus map based on four mapping populations Linkage group No. of markers common among four crosses No. of markers mapped Consensus map Map distance (cm) Inter marker distance (cm) CaLG CaLG CaLG CaLG CaLG CaLG CaLG CaLG Total

23 Figure 9. Consensus map based on four mapping populations CaLG01 CaLG02 CaLG03 CaLG04 CaLG05 TR42 ICCM0297 CaM1451 TA8 TR43 TA30 H3D05 TA122 TA71 ICCM0243c TR59 H3A10 CaM1228 TA05 CaM0805 TA116 CaM0038 TA179 TA11 TR29 TR18 Total No. of markers -111 Total Map length cm CaLG06 TA14 TA196 TR01 Sfl/sfl TR44 TR40 CaM1402 TR7 TA80 CaM0317 CaM0753 TA176 TA22 GA16 GA34 NCPGR93 CaM0244 TA106 TR19 TA200 H4D08 H5A08 H4A04 ; TA96 TA59 TR14 H6D11 TA CaLG07 NCPGR H1C22 TAA58 TA21 CaM0661 H1N12 TA78 CaM0111 TA180 CaM0443 ICCM0160 TA28 TS46 TA142 CaM1122 CaM1020 H3C06 TS5 TR56 H1B04 H3F09 CaM1515 CaM0886 H1F14 ; CaM0475 TA34 H3F08 TR13 CaM1358 ICCM0263a NCPGR10 TA64 NCPGR12 TR32 TA CaLG08 TA118 TS45 ICCM0130 GA6 TA127 H1D24 TA144 H1C092 NCPGR TS72 TA132 TS36 TS104 CaM0113 CaM0507 H4G11 CaM2049 NCPGR7 H1H15 ICCM0250 CaM1158 TAA170 NCPGR21 GAA47 ICCM0192a B/b TR33 23

24 Objective 3 QTL mapping for flowering time genes The linkage map data and phenotyping data was used for QTL analysis by using QTL-ICiMapping (ICIM) software version 4.0 (Wang et al. 2014). 24

25 Results: Table 8. QTLs identified for flowering time in four chickpea crosses Sl. No Cross QTL CaLG 1 ICCV CDC Frontier Position (cm) LOD PVE (%) Additive effect Flanking markers Left Right marker marker Closest marker Qefl CaM1122 TR13 CaM1122 Qefl GAA47 ICCM0192a GAA ICC 5810 CDC Frontier BGD 132 CDC Frontier ICC CDC Frontier Qefl TA122 TA30 TA30 Qefl CaM1358 TA142 TA142 Qefl NCPGR21 GAA47 GAA47 Qefl GA6 TA118 TA118 Qefl CaM1515 TR13 TR13 Qefl TA142 TA64 TA142 Qefl TA127 H1D24 H1D24 Qefl TA14 TR44 TR44 25

26 Figure 10. QTLs for flowering time in the cross ICCV CDC Frontier CaLG03 CaLG Qefl1-1 LOD=3.55 PVE=7.07 % B/b Qefl1-2 LOD=5.95 PVE=12.34 % 26

27 Figure 11. QTLs for flowering time in the cross ICC 5810 CDC Frontier CaLG01 B/b CaLG04 LOD Score Qefl2-1 LOD=12.77 PVE=20.13 % Qefl2-3 LOD=9.10 PVE=10.44 % CaLG03 CaLG08 LOD Score Qefl2-2 LOD=16.82 PVE=25.19 % Qefl2-4 LOD=17.67 PVE=25.57 % 27

28 Figure 12. Major QTL for flowering time in the cross BGD 132 CDC Frontier CaLG08 efl-3 GA6 TA144 H1D24 TA127 TA127 H1D24 GA6 TA144 LOD Score CaLG Qefl3-3 LOD=44.83 PVE=65.35 % TA118 TA118 (a) (b) Figure 13 (a) Mapping of major flowering time gene efl-3 on CaLG08 based on F 3 segregating data of the cross BGD 132 CDC Frontier (b) Identification of major QTL for flowering time Qefl3-3 on CaLG08 28

29 Figure 13. Major QTL for flowering time in the cross ICC CDC Frontier CaLG06 CaLG06 LOD Score TA14 TA efl TR44 TA22 CaM1402 TA176 TR44 TA22 CaM1402 TA176 Qefl4-1 LOD=55.6 PVE=88.19 % NCPGR93 55 CaM0244 NCPGR (a) TA106 CaM0244 TA106 (b) (b) Figure 14 (a) Mapping of major flowering time gene efl-4 on CaLG06 based on F 3 segregating data in the cross ICC CDC Frontier (b) Identification of major QTL 29 for flowering time Qefl4-1 on CaLG06

30 Figure 14. Genomic regions identified for flowering time genes TR42 ICCM CaM TA8 5.7 TR TA H3D TA GA16 TR TA H4D H5A H4A04 ; TA96 TA TR H6D TA TA142 CaM CaM H3C TS TR H1B H3F CaM CaM H1F14 ; CaM TA34 H3F TR CaM ICCM0263a 17.1 NCPGR TA NCPGR TR TA TS72 TA TS TS CaM CaM H4G CaM NCPGR H1H ICCM CaM TAA NCPGR GAA ICCM0192a 51.9 B/b 71.6 TR TA71 ICCM0243c 1.1 TR H3A CaM TA CaM TA CaM TA TA TR TR CaLG01 CaLG02 CaLG03 CaLG04 CaLG05 TA14 TA TR Sfl/sfl 16.2 TR TR CaM TR7 3 TA CaM CaM TA TA GA NCPGR CaM TA CaLG06 NCPGR19 H1C TAA TA CaM H1N TA CaM TA CaM ICCM TA TS46 CaLG07 TA118 TS ICCM GA TA H1D TA H1C NCPGR CaLG08 efl1 efl4 efl3 efl-1 efl-3 efl-4

31 Identification of candidate genes in flowering time QTL regions Primer sequences (of flanking markers) were blasted (BLASTN) against the CDC Frontier genome (Varshney et al. 2013) For each primer pair, the best hit was selected based on the E-value and percent identity Gene ontology was carried out by the BLAST of these genes against the SWISS-PROT and TrEMBL database. The resulted genes were assigned with Uniprot ID and the functions for these genes were retrieved from Uniprot KB database ( 31

32 Results: The results of BLAST search indicated two relatively narrow regions on Ca4 (0.7 Mb region between the markers GAA47-ICCM192a) and Ca08 (region of about 3.5 Mb within the markers GA6-TA118). Gene ontology analysis by the blast of these genes against the SWISS- PROT and TrEMBL database resulted in 30 flowering related genes (06 genes on CaLG04 and 24 genes on Ca LG08 region) assigned with Uniprot ID. Table 9. Important flowering related genes Ca Genes Function Reference 4 ELF6, JMJ11 Acts as Repressor of the photoperiodic flowering pathway 4 TEM1 Acts as Transcritptional repressor of flowering time in long day plants Noh et al. (2004) Castillejo and Pelaz (2008) 8 SUVR5, SET6 Regulators of flower timing in Arabidopsis Krichevsky et al. (2007) 8 HOS1 Controls flowering time in response to ambient temperatures Lee et al. (2012) 8 VRN2 Plays a central role in vernalization Gendall et al. (2001) 8 AP2 Early floral meristem identity and transition into floral meristem Drews et al. (1991) 32

33 Objective 4 Association of flowering time with maturity and yield related traits 33

34 Results: Table 10: Association of flowering time with maturity and yield related traits Cross Days to pod initiation Days to maturity Plant height Biomass Pods per plant Seeds per plant Yield per plant 100 Seed weight Harvest index ICCV CDC Frontier ICC 5810 CDC Frontier BGD 132 CDC Frontier ICC CDC Frontier 0.988** 0.882** 0.507** 0.264** ** ** 0.987** 0.888** 0.189** 0.181* ** ** 0.992** 0.934** 0.453** 0.331** ** ** 0.997** 0.950** 0.471** 0.280** ** 34

35 Conclusions: Major gene inheritance for flowering time - Selection in F 2 and subsequent generations is effective Four genetic maps & consensus map developed - Can be used to develop high density genetic map Ten QTLs (7 major & 3 minor) identified for flowering time - Major genes/qtls can be transferred to any desired genetic background through simple crossing - Genomic regions can be fine mapped to identify candidate genes for flowering time 35

36 Acknowledgements Chickpea Breeding Unit, ICRISAT Dr. Pooran Gaur (Co-Chairperson) Dr. S Srinivasan Dr. S Shoban Centre of Excellence in Genomics, ICRISAT Dr. Rajeev Varshney, Director (RPGL) Dr. Mahendar Thudi Advisory committee Dr. KP Viswanatha (Chairman) Dr. R Lokesh Dr. BS Janagoudar Dr. Ayyanagouda Patil Department of Genetics & Plant Breeding, UAS-Raichur, Karnataka CGIAR Research Programme (CRP) on Grain Legumes Learning System Unit, ICRISAT Library and information services, ICRISAT Housing and food services, ICRISAT 36

37 Thank you 37