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DA DANG DITRUYEN CAC GIONG Bl/OfI DONG BANG SONG CUTl LONG DUA TREN TRINH TU ADN MA VACH VA DAU PHAN TEf ISSR Do Ian Khang1, Tram Thi Thanh Tien1, Tran Gia Huy1, Nguyen Van Ay*2, Tran Thanh Men3 T6M TAT Cac giong bddi d Dong bang song Cdu Long dddc khao sat trinh td ADN ma vach vdi vbng trinh td ITS, ycflb, psbK-psbl ket hop vdi phan tich da dang di truyen bang dau phan td ISSR Ket qua khao sat cac viing trinh tv£ cho thay cac giong biidi nghien cdu tUdng doi dong nhat vdi ve mat di truyen qua phan tich cac vung trinh tu ITS, ycflb, psbK-l Ket qua PCR vdi moi ISSRK2 va ISSR22 da khuech dai dilqc 19 bang ADN co 11 bang da hinh chiem 57,89% va bang don hinh chiem 42,11% Dau phan td ISSRK2 phan biet diiqc biidi da xanh vdi budi nam roi va budi ruby Biidi dudng trang va budi kieu co he so tUcfng dong den 95% Nhu vay dua tren hai dau phan td ISSRK2 va ISSR22 da cho thay sd da hinh trinh td cac giong bddi nghien cdu Dieu cho thay tiem nang cda dau phan td ISSR phan tich da dang di truyen cac giong bddi, phuc vu cho cong tac chon giong Td khoa Bddi, da dang di truyen, ma vach ADN, dau phan td ISSR ! Vien Nghien cdu va Phat trien Cong nghe Sinh hoc, Trddng Dai hoc Can Tho 2Khoa Nong nghiep, Trddng Dai hoc Can Tho;3Khoa Khoa hoc Td nhien, Trddng Dai hoc Can Tho 19 Tap chf Khoa hoc va Cong nghe Nong nghiep Viit Nam - So 05(126)/202 i d At v a n d e Viet Nam thuoc khu vdc Dong Nam A, la mot nhdng trung tam phat sinh cua cac loai cay co mui (Rainer, 1975) Mot so cong trinh da cong bo cho thay Viet Nam co nguon gen cay co mui kha da dang vdi nhieu vung bddi truyen thong co nhieu giong bddi quy nhd bddi dien, bddi tra, biidi da xanh, biidi bien hoa, biidi nam roi, (Nguyen Thi Tuyet va ctv., 2018; Nguyen Phiiong Thuy va ctv., 2016) Trong nam 2017, niidc ta xuat khau hon 10.000 tan biidi, tang gap doi so vdi nam 2016 d hau het thi triidng ldn nhii: My, EU, Canada va cac niidc Trung Dong (Tap chi Doanh nhan Viet Nam, 2020) Ngay nay, de xac dinh va nhan dien cac giong loai khac hoac cac loai co dac diem hinh thai tddng til co rat nhieu phiiOng phap Phiiong phap dddc dung chu yeu bien dda tren phan tich hinh thai cua mau vat Tuy vay, phddng phap hinh thai hoc thiidng gap nhieu trd ngai Trong boi canh hien tai d midc ta, cac nghien cdu ve sd dung ADN ma vach de phan tich cac loai khac dang dan dddc quan tam va phat trien Biidi duac khap cac tinh dong bang song Cdu Long (DBSCL), m6i vung deu co nhflng dieu kien sinh thai nhat dinh anh hiidng den qua trinh song cung nhii bao ton nguon gen Viec nghien cdu da dang di truyen nguon gen biidi da xanh d khu viic DBSCL co y nghia viec bao ton tinh da dang sinh hoc va sd dung co hieu qua cac nguon gen quy phuc vu cho cong tac chon tao giong De tai diidc thiic hien nham phan tich diidc da dang trinh td ADN ma vach (ITS, ycflb, psbK-psbl) va danh gia da dang di truyen cac giong bddi d nam tinh khu vdc DBSCL bang dau phan td ISSR II VAT LIEU VA PHUCfNG PHAP NGHIEN CUU 2.1 Vat lieu nghien cdu Mau la bddi da xanh (Citrus maxima) dddc thu tai tinh gom: Tien Giang, Soc Trang, Hau Giang, Ben Tre, Vinh Long Bddi “Ruby”, bddi kieu (Thdi An - Mon - Can Thd), bddi dddng trang, bddi nam roi 2.2 Phddng phap nghien cdu 2.2.1 Thu mdu va tach chietAD N Tien hanh trich ADN td mau la, ADN dddc chiet tach theo quy trinh cua Doyle va Doyle (1990) Kiem tra chat lddng cua ADN ly trich dddc bang phddng phap dien di tren gel agarose 1% d hieu dien the 100 V 30 phut Ban gel dddc quan sat dddi anh sang td ngoai tren he thong may Bio-Rad UV 2.000 Nong ADN dddc xac dinh bang phddng phap quang d bddc song 260 va 280 nm 2.2.2 Khuech dai vung trinh tu ITS, ycflb, psbK-l Moi phan dng PCR 50 pi gom co 25 pL nddc khd ion; 20 pL master mix 2X (Buffer, MgCl2, dNTPs, Taq polymerase); pL moi xuoi va moi ngddc va pL ADN khuon Chu ky nhiet PCR khuech dai cua vung trinh td ITS, ycflb va psbK-l dddc the hien dbang Bang Trinh td va chu ky nhiet cua cac moi sd dung nghien cdu Trinh td Chu ky nhiet ycfl fo F: TCTCGACGAAAATCAGATTGTTGTGAAT W ib-R ACATGTCAAGTGATGGAAAA (Dong et al, 2015) 94°C -5 phut; 94°C - 30 giay, 55°C - 40 giay, 72°C -1 phut (35 chu ky); 72°C -10 phut psbK-I-F:TGCCTTTGTTTGGCAAG psbK-I-RiAGAGTTTGAGAGAGCAT (Reginato etal, 2010) 94°C - phut; 94°C - 30 giay, 55°C - 40 giay, 72°C - 40 giay (35 chu ky); 72°C - phut ITS1-F: TCCGGGAACCTGCGG ITS4-R; TCCTCCGCTTTGATGC (“White et al., 1990) 95°C - phut; 95°C - 30 giay, 58°C - 30 giay, 72°C - phut (35 chu ky); 72°C - phut ISSR22: TGTGTGTGTGTGTGTGCA ISSRK2: GTGGTGGTGGTGAC (Samriti et al., 2017) 94°C - phut; 94°C - phut, 50°C - 45 giay, 72°C - phut (35 chu ty); 72°C - phut 2 Pkmm tkh so lieu K£t qua giai trinh td ADN dddc kiem tra tren d n a a t o p a m bang chddng trinh Bioedit Td cac «■»*» H c m cac giong bddi da xanh, xac dinh trinh W d o n g (Consensus sequence) cho giong bddi m a a n i l t Ialignment) vdi cac trinh td cua cac giong riba kM e tnm g n^iien cdu de xac dinh cac SNPs tiem nang So sanh sd tddng dong gifla cac giong bddi nghien cdu vdi co sd dd lieu cua ngan hang gene NCBI San pham khuech dai dau phan td ISSR dddc phan tich bang phan mem NTSYSpc2 Bieu hinh cay ve moi quan he phat sinh loai dddc xay ddng theo phddng phap UPGMA Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 m KET QUA VA THAO LUAN ro ret Khi so sanh vdi thang chuan thitong mai co kich thudc kb cho ket qua khuech dai dupe chieu dai cac doan gen lan lupt la 900 bp, 500 bp, 700 bp tUdng ling vdi moi la ycflb (Hinh 1A), psbK-psbl (Hinh IB) va ITS (Hinh 1C) Tif nhQng ket qua dat dupe cho thay san pham PCR tren da du dieu kien de tien hanh budc giai trinh tU 3.1 Khuech dai va giai trin h tit Ket qua dien di san pham PCR khuech dai vimg trinh tU ITS, ycfl b va psbK-psbl dupe thilc hien tren gel agarose 2%, sau kiem tra ket qua cho thay vdi moi tren dieu cho hien cac bang ADN sang r 1500bp -800bp— 10 500bp 10 11 700bp - C Hinh Pho dien di san pham khuech dai geneycflb (A), trinh tilpsbK-1 (B) va ITS (C) cua cac giong budi nghien ciiu Ghi chu: (1) thangchuan 3kb; (2) biidi daxanh Tiengiang(BDTG) + doi chvingdudng; (3) budi daxanh Soc Trdng (ST);(4) budi da xanh Hau Giang (BDHG); (5) btidi da xanh Vinh Long (BVL); (6) budi da xanh Ben Tre (BDB); (7) budi ruby (BRB1); (8) budi kieu (BTK); (9) budi dudng trdng (BDT); (10) budi roi (STR); (11) Doi chUng dm Cac doan ADN ma vach cua giong biidi tren vung trinh til ycfl b sau giai trinh til co chieu dai 800 - 820 bp, ket qua dung nhii dil kien ban dau la khoang 800 - 900 bp Cac doan ADN ma vach cua giong biidi tren moi psbK-psbl sau giai trinh til co chieu dai 430 - 440 bp, ket qua cung dat dung nhil dil kien ban dau gbi trinh til la khoang 400 - 500 bp Cac doan ADN ma vach cua cac giong biidi tren vimg trinh til ITS sau giai trinh til co chieu dai khoang 600 - 700 bp Ket qua giai trinh til cho thay cac doan trinh til tren vimg trinh til ITS co chieu dai san pham khuech dai xap xi vdi nghien ciiu cua Kyndt va cong tac vien (2010) vimg ITS nghien ciiu tren co chieu dai la 703, cac trinh til thu dupe tot, khong bi nhieu, du co sd de phan tich 3.2 Phan tich trinh tU ADN Ket qua phan tich vung trinh til ycflb cua cac giong budi nghien ciiu cho thay khong co sil khac biet qua ldn ciing mot giong cung nhu gifla cac giong budi khac cua cac doi tupng dupe nghien ciiu Qua cho thay gene ycfl b co tinh bao ton cao giQa cac giong budi thu dupe khu vile DBSCL Khi phan tich vung trinh tU ITS cho thay mot so vi tri sai khac d giong budi ruby va dot bien diem mat nucleotide C d giong budi ruby, budi kieu va nucleotide C d budi dudng trang Co vi tri xuat hien dot bien mat nucleotide d vi tri 10 hien dien d giong budi ruby, budi kieu va vi tri thd 21 d giong budi dudng trang Ben canh do, nghien ciiu cung xac dinh dupe SNPs cua giong ruby so vdi cac giong lai Cac trinh tU gen ycfl b va psbK-psbl cua cac giong budi khong co nhieu tren ngan hang gen day la hai trinh tU mdi, chUa dupe nghien ciiu nhieu Chi Citrus chi co nghien ciiu cua Xu va cong tac vien (2016) tai Trung Quoc Nghien ciiu da giai ma toan bo genome he luc lap cua budi da xanh Vi vay, trinh tU dupe sh dung de lam cP sd so sanh, doi chieu vdi cac trinh til nghien ciiu Rieng vung ITS la trinh tU bien nen dupe tim thay nhieu tren cP sd dQ lieu 21 Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 Theo Dong va cong tac vien (2015), ycflb dddc xem nhd dau phan td co kha nang nhan dien cao hon ca matK va rbcL day la vung trinh til bien dddc s£i dung rang rai de lam dau phan td, ycfl b dddc xem la dau phan td hieu qua nhan dien phan td d cac cap phan loai thap Tuy nhien, nghien cdu dddc thdc hien d cap cao hdn tren cay co mui ycflb lai khong the hien sd da hinh trinh td Td cho thay rang viec sd dung ycfl b de phan loai d thdc vat noi chung va cay co mui noi rieng khong that sd hieu qua d vung trinh td ITS co xuat hien nhieu sai khac chiia the ket luan no co phai la ma vach tren cay co mui hay khong, nhdng day cung cho thay dddc sd da dang di truyen tren vimg trinh td ITS d bddi vi tri sai khac co the xuat hien cung giong hoac khac giong Dieu cung phu hop vdi nghien cdu 0.66 0.73 cua Tran Thanh Men va cong tac vien (2008) tien hanh nghien cdu da dang sinh hoc cac giong bddi d Viet Nam 3.3 Moi quan he di truyen gifia cac giong bddi bang dau phan td ISSR Ket qua dien di dau phan td ISSRK2 cho xuat hien 11 bang co bang da hinh chiem ty le 72,73% so vdi tong so Ket qua dien di dau phan td ISSR22 cho xuat hien bang co bang da hinh chiem ty le 37,5% so vdi tong so Theo ket qua d hinh vdi tddng dong dao dong khoang 0,66 - 0,95 vdi khoang dao dong la 0,07 ddpc chia nhom chinh: nhom I vdi Nhom III co he so tddng dong la 0,76, nhom I, II vdi nhom III co he so tiidng dong thap nhat la 0,66 0.81 C oefficient 0.88 0.95 Hinh Gian pha he the hien moi tUdng quan di truyen cua giong biidi Nhom I: Chi co nhat mau budi da xanh Tien Giang (BDTG) Nhom II: nhom lai dddc chia lam nhom nho la: (II.l) gom biidi da xanh Soc Trang (ST) va bddi da xanh Hau Giang (BDHG) vdi he so tddng dong la 0,89 II.2: Gom bddi da xanh VInh Long (BVL) va bddi da xanh Ben Tre (BDB) wji he so tddng dong la 0,94 II.3: Gom bddi kieu (BTK) va bddi dddng trang (BDT) vdi he so tifdng dong gan 0,95 Nhom III: chi co giong la bddi ruby (BRB) va bddi nam roi (STR) vdi he so U n f dong la 0,74 Tuy nhien ket qua tren cho thay cung mot giong bddi da xanh lai d nhom k k k nhan, la giong tai Tien Giang (BDTG) co khac I s x U n wdi he so tddng dong la 0,76 vdi cac giong bddi da xanh d cac tinh lai nghien cdu A Sdc Trang (ST), Hau Giang (BDHG), Vinh Long (PITJL B£b The (BDB), mau bddi da iH fe Ip cung nam nhom II vdi bddi Kiiu litXlk]) xa bddi dddng trang (BDT) nhdng van co tach biet vdi m lu bddi da xanh vdi he s6 tddng dong la 0,85 Co nhieu yeu to tao nen sd khac biet quan the nhd kha nang phat tan cua loai va sd anh hddng cua moi trddng song (Kerdelhue et al, 2002) hoac tac dong cua ngddi Quan diem cung phu hdp vdi nghien cdu cua Snkiewicz va cong tac vien (2001) nghien cdu ve moi lien he di truyen cua cac giong cay trong canh tac nong nghiep cho thay ngddi la yeu to quan trong viec di chuyen giong cay gida cac vung dia ly Tuy nhien, ket qua chi la tam thdi dda tren hai dau phan td nghien cdu De tang cao ket qua can phan tich them nhieu dau phan td hdn nda Dda vao hinh cho thay, vi tri bang phan biet bddi da xanh vdi bddi roi va bddi “Ruby” d vi tri khoang 450 bp (mui ten mau trang) Cac mau bddi da danh deu co bang bddi nam roi va bddi “Ruby” khong co Tap chi' Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 n 3000bp 1500bp lOOObp 500bp - SB \ \\ Hinh Ket qua dien di cac mau bddi vdi dau phan td ISSRK2 Ghi chu: gieng 1: thang chuan 100 bp (Omega Bio-tek); gieng2: Doi chiing dUdng; gieng 3: bildi da xanh Soc Trang; gieng 4: btidi da xanh Hau Giang; gieng 5: bUdi da xanh Vinh Long; gieng 6: budi da xanh Ben Tre; gieng 7: budi ruby; gieng 8: budi kieu; gieng 9: budi diidng trang; gieng 10: budi nam roi Khuat Hutu Trung va cong tac vien (2009) da tien hanh nghien cdu da dang di truyen tren 41 giong btidi ban dia Viet Nam bang chi thi SSR lad cho ket qua bddi da xanh nam ciing nhom vdi bddi nam roi vdi he so tiicfng dong la 0,5 nghien cutu dtipc thdc hien lai cho ket qua btidi da xanh va biidi roi thupc nhom khac Trong nghien cdu nay, vdi dau phan td ISSR thi cho tdcfng dong 0,66 N hd vay, co the thay cac chi thi phan td khac se cho tdong dong khac Trong nghien cdu co giong la Bddi Thanh Kieu (BTK) va bddi dddng trang (BDT) co tdong dong di truyen den 95% mac du rat khac biet ve hinh thai Do nddc ta co nhieu giong bddi co hinh thai trai kha giong nen de dda den nham lan ten giuta cac giong bddi d nhutng vung khac (Vu Cong Hau, 2000) IV KET LUAN Gen ycflb va trinh td psbK-psbl chda that sd la mot trinh td ma vach tiem nang de phan loai cac giong bddi nghien cdu Khong co sd khac biet trinh td d cung giong cung nhd d nhdng giong khac Nhd vay cac viing trinh td ADN ma vach nghien cdu chda phan biet ddpc cac giong bddi, do can chon cac trinh td ADN ma vach khac de tiep tuc khao sat Dau phan td ISSR co the phan biet ddpc cac giong bddi co sd da hinh, nhdng can nhieu dau phan td hdn nda de sd phan biet ddpc ro rang hon gida cac giong Nghien cdu tren se gop phan xay ddng nen tang cho cac nghien cdu tiep theo tren chi Citrus cung nhd d thdc vat ve dng dung ADN ma vach dinh danh TAI LIEU KHAM KHAO Vii Cong Hau, 2000 Trong cay an qua d Viet Nam Tai ban lan NXB Nong Nghiep TR Ho Chi Minh: 100-116 Tran Thanh Men, Nguyen Thi Pha, Tran Nhan Ddng, Ha Thanh Toan, Tina Kyndt, Godelieve Gheysen va Marcelle Holsters, 2008 Nghien cdu da dang sinh hoc cac giong bddi (Citrus maxima (Burm) Merr.) d Viet Nam bang phddng phap PCR-RFLP Hoi nghi khoa hoc “cay an trai quan d DBSCL 14-20 Khuat Hflu Trung, Ha Trong Huy, Nguyen Trddng Khoa, Ngo Hong Binh, Nguyen Thanh Binh, Dang Trong Ldofng va Le Huy Ham, 2009 Nghien cdu da dang di truyen mot so giong bddi ban dia Viet Nam (Citrus grandis) bang chi thi microsatellite Tap chi Cong nghe Sinh hoc, 7(4): 485-492 Nguyen Phddng Thuy, Nguyen Thi Nga, Le Thi Cam Tu, Dao Thi Be Bay va Tran Tlii Oanh Yen, 2016 Danh gia dac tinh di truyen cua cac ddng bddi tif nuoi cay hat nhd bang chi thi phan td microsatellites Tap chi Khoa hoc vd Cong nghe Viet Nam, 8: 30-35 Tap chi Doanh nhan Viet Nam, 2020 Tim co hoi cho trai bddi Viet Nam md rang thi trddng ldn, https://doanhnhanvn.vn/m o-rong-thi-truongcho-trai-buoi-viet-nam-22572.html, truy cap: 20/5/2021 Nguyen Thi Tuyet, Nguyln Thi Xuyen, Nguyen Thi Lan Hoa, Bui Thi Thu Giang va Tran Danh Situ, 2018 Danh gia da dang di truy£'n mot so nguon gen bddi (Citrus spp.) bang chi thi SSR Tap chi Khoa hoc Cong nghe Nong nghiep Viet Nam, 1(86): 14-18 Dong, W.P., Xu, C., Li, C., Sun, J., Zuo, Y., Shi, S., Cheng O., Guo J and Zhou S., 2015 ycfl, the most promising plastid DNA barcode of land planted Scientific reports, 5(1): 1-5 23 Tap chi Khoa hoc va Cong nghe Nong nghiep Viet Nam - So 05(126)/2021 Doyle, J.J and Doyle, J.L., 1990 Isolation of plant DNA from fresh tissue Focus, 12:13-15 Kerdelhue, C., Roux, G., Forichon, ]., Chambon, J., Robert, A and Lieutier, F., 2002 (Curculionidae: Scolytinae) on different pine species and validation of T destruens (woll.) Molecular Ecology, (11): 483-494 Kyndt, T., Dung, T.N., Goetghebeur, R, Toan, H.T and Gheysen, G., 2010 Analysis of ITS of the rDNA to infer phylogenetic relationships among Vietnamese Citrus accessions Genetic resources and crop evolution, 57(2): 183-192 Rainer, W S., 1975 On the history and origin of Citrus Bulletin of the Torrey Bonical Club, 102(6): 369-375 Snkiewicz, M., Gadamski, G and Gawronski, S.W., 2001 Genetic variation and phylogenetic relationships of triazine resisnt and triazine susceptible biotypes of Solanum nigrum analysis using RAPD markers Weed Res., 41: 287-300 Xu, S-R., Huang, C-Y., Deng, Y-T., Zhou, L-L., Pan, D-M., and Pan, H-L., 2016 The complete chloroplast genome sequence of Citrus maxima (Burm.) Merr ‘Guanximiyou’ Mitochondrial DNA, 5(1): 482-483 Genetic diversity o f pomelo varieties in the Mekong Delta based on DNA barcode and ISSR markers Do Tan Khang, Tram Thi Thanh Tien, Tran Gia Huy, Nguyen Van Ay, Tran Thanh Men Abstract Pomelo varieties in the Mekong Delta were examined by sequencing of three DNA barcode regions, including ITS, ycflb, psbK-psbl in combination with genetic diversity analysis by ISSR markers The results indicated that the pomelo varieties in the study were similar in term of genetic diversity based on analyzing the sequences of ITS, ycflb and psbK The two ISSRK2 and ISSRK22 markers had amplified 19 DNA bands, including 11 polymorphic bands accounting for 57.89% and monomorphic bands for 42.11%, respectively The marker ISSRK2 could distinguish Da xanh from Nam Roi and Ruby pomelo varieties Genetic similarity between Duong Trang and Thanh Kieu pomelo varieties was 0.95 Therefore, based on the ISSR markers K2 and K22 the polymorphisms of pomelo varieties were observed The finding showed the potential of ISSR markers in analyzing genetic diversity of pomelo and could be used in pomelo breeding Keywords: Pomelo variety, genetic diversity, DNA barcode, ISSR marker Ngay nhan bai: 07/5/2021 Ngay phan bien: 18/5/2021 Ngiicfi phan bien: TS Tran Ngoc Hung Ngay duyet dang: 04/6/2021 ... Nam co nguon gen cay co mui kha da dang vdi nhieu vung bddi truyen thong co nhieu giong bddi quy nhd bddi dien, bddi tra, biidi da xanh, biidi bien hoa, biidi nam roi, (Nguyen Thi Tuyet va ctv.,... giong bddi d Viet Nam 3.3 Moi quan he di truyen gifia cac giong bddi bang dau phan td ISSR Ket qua dien di dau phan td ISSRK2 cho xuat hien 11 bang co bang da hinh chiem ty le 72,73% so vdi tong... da xanh Hau Giang; gieng 5: bUdi da xanh Vinh Long; gieng 6: budi da xanh Ben Tre; gieng 7: budi ruby; gieng 8: budi kieu; gieng 9: budi diidng trang; gieng 10: budi nam roi Khuat Hutu Trung va
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