Молекулярная структура генома и селекция

ÑÅËÜÑÊÎÕÎÇßÉÑÒÂÅÍÍÀß ÁÈÎËÎÃÈß, 2015, òîì 50, ¹ 1, ñ. 30-36
Ìîëåêóëÿðíàÿ ñòðóêòóðà ãåíîìà è ñåëåêöèÿ
ÓÄÊ 633.367.2:632.4:575.174.015.3:577.21
doi: 10.15389/agrobiology.2015.1.30rus
ÈÄÅÍÒÈÔÈÊÀÖÈß ÃÅÍÀ ÓÑÒÎÉ×ÈÂÎÑÒÈ Ê ÀÍÒÐÀÊÍÎÇÓ Lanr1
Ó ËÞÏÈÍÀ ÓÇÊÎËÈÑÒÍÎÃÎ (Lupinus angustifolius L.) Ñ ÏÎÌÎÙÜÞ
ÄÍÊ-ÌÀÐÊÅÐÎÂ AnSeq3 È AnSeq4*
Ñ.Þ. ÃÐÈØÈÍ1, Â.Â. ÇÀßÊÈÍ1, È.ß. ÍÀÌ1, Ï.À. ÀÃÅÅÂÀ2,
Ì.È. ËÓÊÀØÅÂÈ×2, Í.Ñ. ÊÓÏÖÎÂ3
Àíòðàêíîç — çàáîëåâàíèå ëþïèíà óçêîëèñòíîãî (Lupinus angustifolius L.), âûçûâàåìîå
ãðèáîì Colletotrichum lupini. Óñòîé÷èâîñòü ê íåìó íîñèò íå àáñîëþòíûé, à îòíîñèòåëüíûé õàðàêòåð: âûñîêîóñòîé÷èâûå îáðàçöû ìîãóò ïîðàæàòüñÿ èíôåêöèåé, íî â ìåíüøåé ñòåïåíè, ÷åì íåóñòîé÷èâûå ðàñòåíèÿ. Îòíîñèòåëüíî îáùåãî ÷èñëà ãåíîâ, ñâÿçàííûõ ñ òîëåðàíòíîñòüþ ê àíòðàêíîçó, äî ñèõ ïîð íåò åäèíîãî ìíåíèÿ.  íàñòîÿùåå âðåìÿ ñåëåêöèîíåðû èç ðàçíûõ ñòðàí çàíÿòû
ñîçäàíèåì áîëåå óñòîé÷èâûõ ê àíòðàêíîçó ñîðòîâ ïîñðåäñòâîì îáúåäèíåíèÿ â îäíîì ãåíîòèïå íåàëëåëüíûõ ãåíîâ óñòîé÷èâîñòè ê áîëåçíè. Äëÿ áûñòðîãî è ýôôåêòèâíîãî îòáîðà óñòîé÷èâûõ ðàñòåíèé ðàçðàáàòûâàþòñÿ ÄÍÊ-ìàðêåðû, ñöåïëåííûå ñ ãåíàìè óñòîé÷èâîñòè ê çàáîëåâàíèþ. Ïðåæäå ÷åì èñïîëüçîâàòü ÄÍÊ-ìàðêåðû â ñåëåêöèè, íåîáõîäèì ïðåäâàðèòåëüíûé àíàëèç èõ ýôôåêòèâíîñòè äëÿ òåõ èëè èíûõ ñåëåêöèîííûõ îáðàçöîâ èç-çà âîçìîæíûõ «ëîæíîïîëîæèòåëüíûõ» ðåçóëüòàòîâ. Íàèáîëåå áëèçêèìè ê Lanr1 ïðåäñòàâëÿþòñÿ ÄÍÊ-ìàðêåðû AnSeq3 è AnSeq4 (îäíîíóêëåîòèäíûå ïîëèìîðôèçìû — single nucleotide polymorphisms, SNPs), êîòîðûå ôëàíêèðóþò ãåí íà ðàññòîÿíèè 0,9 ñÌ (H. Yang et al., 2012).  íàñòîÿùåé ðàáîòå ìû èññëåäîâàëè âîçìîæíîñòü èñïîëüçîâàíèÿ ÄÍÊ-ìàðêåðîâ AnSeq3 è AnSeq4 â ñåëåêöèè ðîññèéñêèõ è áåëîðóññêèõ
ñîðòîâ ëþïèíà óçêîëèñòíîãî íà óñòîé÷èâîñòü ê àíòðàêíîçó. Ïîëèìåðàçíóþ öåïíóþ ðåàêöèþ
(ÏÖÐ) ïðèìåíÿëè äëÿ âûÿâëåíèÿ àëëåëåé ÄÍÊ-ìàðêåðîâ, ñöåïëåííûõ ñ ãåíîì Lanr1 óñòîé÷èâîñòè ê àíòðàêíîçó ëþïèíà óçêîëèñòíîãî. Îáúåêòàìè äëÿ èçó÷åíèÿ ñëóæèëè 50 ñîðòîâ è îáðàçöîâ
ëþïèíà ðîññèéñêîé, áåëîðóññêîé, ïîëüñêîé è àâñòðàëèéñêîé ñåëåêöèè. ÄÍÊ âûäåëÿëè èíäèâèäóàëüíî èç ñåìÿí â 3-êðàòíîé ïîâòîðíîñòè. Ïðåäñòàâëåí ñïèñîê ñåëåêöèîííûõ îáðàçöîâ, à òàêæå
ñîñòàâ ðåàêöèîííîé ñìåñè äëÿ ÏÖÐ è óñëîâèÿ àìïëèôèêàöèè ÄÍÊ. Äëÿ ÏÖÐ èñïîëüçîâàëè
ôåðìåíòû è ðåàêòèâû ôèðìû «ÑèáÝíçèì» (Ðîññèÿ), ïðàéìåðû ê ìàðêåðàì AnSeq3 è AnSeq4
ñàéòñïåöèôè÷íû è ñèíòåçèðîâàíû ôèðìîé «Ñèíòîë» (Ðîññèÿ). Ýëåêòðîôîðåç â ïîëèàêðèëàìèäíîì ãåëå ïðèìåíÿëè äëÿ ðàçäåëåíèÿ ìàðêåðíûõ àëëåëåé. Óñòîé÷èâûé è âîñïðèèì÷èâûé ê àíòðàêíîçó àâñòðàëèéñêèé ñåëåêöèîííûé ìàòåðèàë èñïîëüçîâàëñÿ â êà÷åñòâå êîíòðîëåé äëÿ àëëåëåé AnSeq3 è AnSeq4. ÄÍÊ-ôðàãìåíòû äëèíîé 92 è 87 ï.í., ñîîòâåòñòâóþùèå ìàðêåðàì
AnSeq3 è AnSeq4, áûëè ïîëó÷åíû äëÿ âñåõ 50 ñåëåêöèîííûõ îáðàçöîâ, âêëþ÷åííûõ â èññëåäîâàíèå. Äëÿ 13 ñîðòîâ ðîññèéñêîé è 10 ñîðòîâ ïîëüñêîé ñåëåêöèè áûëè îáíàðóæåíû ìàðêåðíûå
àëëåëè, ñîîòâåòñòâóþùèå âîñïðèèì÷èâîñòè ê àíòðàêíîçó. Äëÿ áåëîðóññêîãî ñåëåêöèîííîãî îáðàçöà ÁÃÁ-6 àëëåëè óñòîé÷èâîñòè áûëè âûÿâëåíû ïî ìàðêåðàì AnSeq3 è AnSeq4, äëÿ ñîðòà
Ìèðòàí — òîëüêî ïî ìàðêåðó AnSeq3. Îñòàëüíûì 21 îáðàçöàì áåëîðóññêîé ñåëåêöèè ñîîòâåòñòâîâàëè àëëåëè âîñïðèèì÷èâîñòè ê àíòðàêíîçó. Ðàíåå ñ ïîìîùüþ äðóãèõ ÄÍÊ-ìàðêåðîâ —
AntjM1 è AntjM2 ìû ïîêàçàëè, ÷òî ñîâðåìåííûå ñîðòà Âñåðîññèéñêîãî ÍÈÈ ëþïèíà íå ñîäåðæàò óñòîé÷èâûõ ìàðêåðíûõ àëëåëåé, ñöåïëåííûõ ñ ãåíîì Lanr1. Ïðîâåäåííûå èññëåäîâàíèÿ ïîêàçàëè, ÷òî ÄÍÊ-ìàðêåðû AnSeq3 è AnSeq4, ñöåïëåííûå ñ ãåíîì Lanr1, ìîãóò áûòü ïîëåçíû â
ñåëåêöèè ðîññèéñêèõ è áåëîðóññêèõ ñîðòîâ íà óñòîé÷èâîñòü ê àíòðàêíîçó.
Êëþ÷åâûå ñëîâà: Lupinus angustifolius L., àíòðàêíîç, ïîëèìåðàçíàÿ öåïíàÿ ðåàêöèÿ
(ÏÖÐ), ÄÍÊ-ìàðêåðû AnSeq3, AnSeq4.
Àíòðàêíîç — èíôåêöèîííîå çàáîëåâàíèå ìíîãèõ ñåëüñêîõîçÿéñòâåííûõ êóëüòóð, â òîì ÷èñëå ëþïèíà óçêîëèñòíîãî Lupinus angustifolius L.
Áîëåçíü ó ðàñòåíèé ëþïèíà âûçûâàåò ãðèá Colletotrichum lupini (1). Â ãîäû
ðàçâèòèÿ ýïèôèòîòèé àíòðàêíîçà ïðîèñõîäèò çíà÷èòåëüíîå ñíèæåíèå óðîæàéíîñòè ó âîñïðèèì÷èâûõ ê ïàòîãåíó ñîðòîâ. Ïîñåâû ëþïèíà, ñèëüíî
ïîðàæåííûå çàáîëåâàíèåì, íå ðåêîìåíäóåòñÿ èñïîëüçîâàòü íà ñåìåííûå
öåëè, çåðíîôóðàæ è ñèëîñîâàíèå (2). Ïî äàííûì ëèòåðàòóðû, âïåðâûå â
*
Ðàáîòà âûïîëíåíà ïðè ôèíàíñîâîé ïîääåðæêå ãðàíòà Ìèíèñòåðñòâà îáðàçîâàíèÿ è íàóêè Ðîññèéñêîé
Ôåäåðàöèè, ñîãëàøåíèå ¹ 14.132.21.1768 «Ïðîâåäåíèå ïîèñêîâûõ è ïðèêëàäíûõ èññëåäîâàíèé ïî ðàçðàáîòêå è âíåäðåíèþ â ñåëåêöèîííûé ïðîöåññ ãåíîìíûõ òåõíîëîãèé ÄÍÊ-ìàðêèðîâàíèÿ õîçÿéñòâåííî
öåííûõ ïðèçíàêîâ ëþïèíà».
30
ÑÑÑÐ àíòðàêíîç áûë îáíàðóæåí èìåííî íà ëþïèíå óçêîëèñòíîì, êîòîðûé ïîðàæàëñÿ â íàèáîëüøåé ñòåïåíè ïî ñðàâíåíèþ ñ äðóãèìè âèäàìè
(3). Îäíàêî áëàãîäàðÿ ìíîãîëåòíåé ñåëåêöèè íà óñòîé÷èâîñòü ê àíòðàêíîçó íåêîòîðûå ñîâðåìåííûå ñîðòà L. angustifolius L. îáëàäàþò òîëåðàíòíîñòüþ ê áîëåçíè.  òî æå âðåìÿ íåîáõîäèìî îòìåòèòü, ÷òî óñòîé÷èâîñòü íîñèò íå àáñîëþòíûé, à îòíîñèòåëüíûé õàðàêòåð, ïîòîìó ÷òî òàê íàçûâàåìûå âûñîêîóñòîé÷èâûå îáðàçöû ìîãóò ïîðàæàòüñÿ èíôåêöèåé, íî â ìåíüøåé ñòåïåíè, ÷åì ðàñòåíèÿ, êîòîðûå ñ÷èòàþòñÿ íåóñòîé÷èâûìè.
Äî ñèõ ïîð ñðåäè èññëåäîâàòåëåé íåò åäèíîãî ìíåíèÿ ïî ïîâîäó
îáùåãî ÷èñëà ãåíîâ, ñâÿçàííûõ ñ òîëåðàíòíîñòüþ ê àíòðàêíîçó (4). Àìåðèêàíñêèå ñîðòà ëþïèíà óçêîëèñòíîãî Rancher, Frost è àâñòðàëèéñêèå ñîðòà
Marri, Illyarrie, Yandee, Danja íåñóò äîìèíàíòíûé ãåí óñòîé÷èâîñòè ê àíòðàêíîçó An (5). Àâñòðàëèéñêèé ñîðò Kalya îáëàäàåò äîìèíàíòíûì ãåíîì
Lanr2, îáóñëîâëèâàþùèì ñðåäíþþ óñòîé÷èâîñòü ê àíòðàêíîçó (6). Âûñîêàÿ óñòîé÷èâîñòü ê àíòðàêíîçó ó àâñòðàëèéñêèõ ñîðòîâ Wonga è Tanjil êîíòðîëèðóåòñÿ äîìèíàíòíûì ãåíîì Lanr1 (7). Ïî äàííûì ãèáðèäîëîãè÷åñêîãî àíàëèçà, â ãåíîòèïå áåëîðóññêèõ ñîðòîâ Ìèðòàí è Ïåðøàöâåò, ïîêàçûâàþùèõ óñòîé÷èâîñòü ê àíòðàêíîçó íà óðîâíå ñîðòîâ Wonga è Tanjil, ñîäåðæàòñÿ áëîêè èç òðåõ íåàëëåëüíûõ äîìèíàíòíûõ ãåíîâ Rcl1 Rcl2 Rcl3.
Ñëåäóåò ïîä÷åðêíóòü, ÷òî â íàñòîÿùåå âðåìÿ ñåëåêöèîíåðû èç ðàçíûõ ñòðàí ðàçâåðíóëè èññëåäîâàíèÿ ïî ñîçäàíèþ áîëåå óñòîé÷èâûõ ê àíòðàêíîçó ñîðòîâ ïîñðåäñòâîì îáúåäèíåíèÿ â îäíîì ãåíîòèïå íåàëëåëüíûõ
ãåíîâ óñòîé÷èâîñòè ê áîëåçíè (4). Ñ öåëüþ áûñòðîãî è ýôôåêòèâíîãî îòáîðà óñòîé÷èâûõ ê àíòðàêíîçó ðàñòåíèé ðàçðàáàòûâàþòñÿ ÄÍÊ-ìàðêåðû,
ñöåïëåííûå ñ ãåíàìè óñòîé÷èâîñòè ê çàáîëåâàíèþ. Òàê, àâñòðàëèéñêèìè
èññëåäîâàòåëÿìè áûëè ïðåäëîæåíû ÄÍÊ-ìàðêåðû AntjM1 (7), AntjM2 (8),
AnSeq3 è AnSeq4 (9), ñöåïëåííûå ñ ãåíîì Lanr1. Ñ ïîìîùüþ ïðàéìåðîâ
äëÿ ñîîòâåòñòâóþùèõ ìàðêåðîâ óñòîé÷èâîñòè ê àíòðàêíîçó ó ëþïèíà óçêîëèñòíîãî ìîæíî îïðåäåëèòü íàëè÷èå ãåíà Lanr1 â èçó÷àåìûõ îáðàçöàõ ïðè
èñïîëüçîâàíèè ïîëèìåðàçíîé öåïíîé ðåàêöèè (ÏÖÐ) ñ ïîñëåäóþùèì àíàëèçîì ÄÍÊ-ôðàãìåíòîâ.
Ñêðèíèíã ñåëåêöèîííîãî ìàòåðèàëà ñ ïðèìåíåíèåì ÄÍÊ-ìàðêåðîâ
ïîçâîëÿåò âûÿâèòü è ðåêîìåíäîâàòü äëÿ âêëþ÷åíèÿ â ñåëåêöèîííûé ïðîöåññ óñòîé÷èâûå ê àíòðàêíîçó ðàñòåíèÿ.  òî æå âðåìÿ, êàê ïîêàçàëè ðåçóëüòàòû ðàáîòû àâñòðàëèéñêèõ èññëåäîâàòåëåé, ïðåæäå ÷åì èñïîëüçîâàòü
ÄÍÊ-ìàðêåðû â ñåëåêöèè, íåîáõîäèì ïðåäâàðèòåëüíûé àíàëèç èõ ýôôåêòèâíîñòè äëÿ òåõ èëè èíûõ ñåëåêöèîííûõ îáðàçöîâ (10). Ðàíåå ïðè èçó÷åíèè óñòîé÷èâîñòè ê àíòðàêíîçó, êîíòðîëèðóåìîé ãåíîì Lanr1, ìû ïðèìåíèëè ìàðêåðû AntjM1, AntjM2 äëÿ àíàëèçà ÄÍÊ ó 14 ñîðòîâ ëþïèíà óçêîëèñòíîãî ðîññèéñêîé è áåëîðóññêîé ñåëåêöèè (11). Ê íàñòîÿùåìó âðåìåíè
íàèáîëåå áëèçêèìè ê Lanr1 ïðåäñòàâëÿþòñÿ ÄÍÊ-ìàðêåðû AnSeq3 è
AnSeq4, êîòîðûå ôëàíêèðóþò ãåí íà ðàññòîÿíèè 0,9 ñÌ (9).
Öåëüþ íàñòîÿùåé ðàáîòû áûëî èññëåäîâàíèå âîçìîæíîñòè èñïîëüçîâàíèÿ ÄÍÊ-ìàðêåðîâ AnSeq3 è AnSeq4, ñöåïëåííûõ ñ ãåíîì Lanr1, â
ñåëåêöèè ðîññèéñêèõ è áåëîðóññêèõ îáðàçöîâ ëþïèíà óçêîëèñòíîãî, â ñâÿçè ñ ÷åì ìû îöåíèëè íàëè÷èå ãåíà óñòîé÷èâîñòè ê àíòðàêíîçó Lanr1 ó
ñîðòîâ è îáðàçöîâ ðàçëè÷íîãî ñåëåêöèîííîãî ïðîèñõîæäåíèÿ ñ ïîìîùüþ
äâóõ óêàçàííûõ ìîëåêóëÿðíûõ ìàðêåðîâ.
Ìåòîäèêà. Ìàòåðèàëîì äëÿ èññëåäîâàíèÿ ñëóæèëè 50 ñîðòîâ è îáðàçöîâ ëþïèíà óçêîëèñòíîãî (Lupinus angustifolius L.) ðîññèéñêîé, áåëîðóññêîé, ïîëüñêîé è àâñòðàëèéñêîé ñåëåêöèè.
ÄÍÊ âûäåëÿëè èíäèâèäóàëüíî èç ñåìÿí â 3-êðàòíîé ïîâòîðíîñòè
ïî ìåòîäèêå, èñïîëüçîâàííîé ðàíåå (11). ÏÖÐ ïðîâîäèëè â àìïëèôèêàòî31
ðå Òåðöèê («ÄÍÊ-Òåõíîëîãèÿ», Ðîññèÿ).  ñîñòàâ ðåàêöèîííîé ñìåñè îáúåìîì 10 ìêë âõîäèëà ãåíîìíàÿ ÄÍÊ (10-20 íã), ïðÿìîé è îáðàòíûé ïðàéìåðû (0,3 ìêÌ), Taq-ïîëèìåðàçà Å 338 (0,25 åä.), MgCl2 (1,5 ìÌ), dNTPs
(0,15 ìÌ), Â321 AS áóôåð (1 ìêë). Èñïîëüçîâàëè ôåðìåíòû è ðåàêòèâû
ôèðìû «ÑèáÝíçèì» (Ðîññèÿ). Óñëîâèÿ àìïëèôèêàöèè — íà÷àëüíàÿ äåíàòóðàöèÿ 1 ìèí ïðè 95 °Ñ; äàëåå 30 öèêëîâ: äåíàòóðàöèÿ 30 ñ ïðè 95 °Ñ,
îòæèã 20 ñ ïðè 51 °Ñ äëÿ ïðàéìåðîâ â ñëó÷àå ìàðêåðà AnSeq3 è ïðè 53 °Ñ
äëÿ ïðàéìåðîâ ê ìàðêåðó AnSeq4, ýëîíãàöèÿ 10 ñ ïðè 72 °Ñ; çàêëþ÷èòåëüíàÿ ýëîíãàöèÿ 2 ìèí ïðè 72 °Ñ. Èñïîëüçîâàííûå ïðàéìåðû ê ìàðêåðàì AnSeq3 è AnSeq4 ñàéòñïåöèôè÷íû (9) è ñèíòåçèðîâàíû ôèðìîé
«Ñèíòîë» (Ðîññèÿ). Ðàçäåëåíèå ôðàãìåíòîâ ÄÍÊ ïðîâîäèëè â 11-13 % ïîëèàêðèëàìèäíûõ ãåëÿõ ñ èñïîëüçîâàíèåì êàìåðû äëÿ âåðòèêàëüíîãî ýëåêòðîôîðåçà VE-20 («Õåëèêîí», Ðîññèÿ). Ïðîäóêòû ÏÖÐ ðàçäåëÿëè â òå÷åíèå 5-6 ÷ ïðè íàïðÿæåíèè 270-300 Â. Ãåëü ïîìåùàëè â ðàñòâîð áðîìèñòîãî ýòèäèÿ (êîíöåíòðàöèÿ 0,5 ìã/ë) íà 20-30 ìèí. Ïîñëå îêðàøèâàíèÿ åãî
âèçóàëèçèðîâàëè íà ñêàíåðå ãåëåé GelDocXR («Bio-Rad», ÑØÀ).  êà÷åñòâå êîíòðîëåé äëÿ îïðåäåëåíèÿ äëèíû ìàðêåðíûõ àëëåëåé èñïîëüçîâàëè
ïðîäóêòû ÏÖÐ ñ ÄÍÊ ðàñòåíèé, ïðåäñòàâëÿþùèõ ñîðòà àâñòðàëèéñêîé ñåëåêöèè Tanjil, Wonga, Kalya.
Ðåçóëüòàòû. Íàìè áûëè èññëåäîâàíû 50 îáðàçöîâ ëþïèíà óçêîëèñòíîãî ðàçíîãî ïðîèñõîæäåíèÿ (òàáë.).
Îáðàçöû ëþïèíà óçêîëèñòíîãî, âêëþ÷åííûå â èññëåäîâàíèå ïî èäåíòèôèêàöèè àëëåëåé ÄÍÊ-ìàðêåðîâ AnSeq3 è AnSeq4 ãåíà óñòîé÷èâîñòè ê àíòðàêíîçó Lanr1
Ïðåäîñòàâëåíî Ðîññèåé
Ïðåäîñòàâëåíî Áåëîðóññèåé
Ñîðòà è îáðàçöû ðîññèéñêîé ñåëåêöèè (âñåãî 13):
Ðàäóæíûé, Ñèäåðàò 38, Êðèñòàëë, Ñíåæåòü, Áåëîçåðíûé 110, Ñìåíà, Íàäåæäà, Áðÿíñêèé 123, Âèòÿçü, Óçêîëèñòíûé 53-02, Âåêòîð, Áåëîçåðíûé 121, ÑÍ 78-07
Ñîðòà è îáðàçöû áåëîðóññêîé ñåëåêöèè (âñåãî 23):
Ïðèâàáíû, Ïåðøàöâåò, Ìèòàí, Ãåëåíà, Ìèðòàí,
Õâàëüêî, ßí, ÁÃÁ-6, Ãåðêóëåñ, Æîäèíñêèé, Ðàííèé,
ÁÃÁ-3, Ìèðòàí 312, ÌÊ-ÂÄÑ-Áåèñ, ÏÝË-ÌÐË, Ðàìîíîê, 4Á-129, ÁÃÁ-1, ÁÃÁ-2, ÁÃÁ-4, Âàäà 40, Âàñèëåê, Äèâíûé
Ñîðòà è îáðàçöû ïîëüñêîé ñåëåêöèè (âñåãî 10):
Áàðîí, Áîðóòà, Áîÿð, Ãðàô, Çåâñ, Êàëèô, Íåïòóí,
Ðåãåíò, Öåçàðü, Ýëüô
Ñîðòà è îáðàçöû àâñòðàëèéñêîé ñåëåêöèè (âñåãî 4):
Tanjil, Wonga, Kalya, W2248
Ï ð è ì å ÷ à í è å. Ðîññèéñêèé è áåëîðóññêèé ñåëåêöèîííûé ìàòåðèàë áûë ïðåäîñòàâëåí ñîîòâåòñòâåííî
Âñåðîññèéñêèì ÍÈÈ ëþïèíà (ã. Áðÿíñê) è ÍÏÖ ÍÀÍ Áåëàðóñè ïî çåìëåäåëèþ (ã. Æîäèíî ).
ÄÍÊ-ôðàãìåíòû äëèíîé 92 è 87 ï.í., ñîîòâåòñòâóþùèå ìàðêåðàì
AnSeq3 è AnSeq4, áûëè ïîëó÷åíû äëÿ âñåõ 50 ñåëåêöèîííûõ îáðàçöîâ,
âêëþ÷åííûõ â èññëåäîâàíèå.  õîäå ñîïîñòàâëåíèÿ ìàðêåðíûõ àëëåëåé,
âûÿâëåííûõ äëÿ àâñòðàëèéñêèõ êîíòðîëüíûõ ñîðòîâ è äðóãèõ ñåëåêöèîííûõ îáðàçöîâ, îïðåäåëèëè àëëåëè âîñïðèèì÷èâîñòè (S) è óñòîé÷èâîñòè
(R) ê àíòðàêíîçó. Àëëåëè AnSeq3 è AnSeq4 ïðåäñòàâëÿþò ñîáîé îäíîíóêëåîòèäíûå ïîëèìîðôèçìû (single nucleotide polymorphism — SNP), èìåþùèå îäèíàêîâóþ äëèíó äëÿ êàæäîãî ìàðêåðà. Ïðè ýëåêòðîôîðåòè÷åñêîì
ðàçäåëåíèè â ïîëèàêðèëàìèäíîì ãåëå ôðàãìåíòû, ñîîòâåòñòâóþùèå ìàðêåðíûì àëëåëÿì óñòîé÷èâîñòè äëÿ AnSeq3 èç-çà íàëè÷èÿ îäíîé çàìåíû
íóêëåîòèäà C íà T ðàñïîëàãàëèñü íèæå àìïëèêîíîâ àëëåëåé âîñïðèèì÷èâîñòè, òîãäà êàê äëÿ AnSeq4 íàáëþäàëîñü îáðàòíîå ðàñïðåäåëåíèå âñëåäñòâèå åäèíè÷íîé çàìåíû G íà A â íóêëåîòèäíîé ïîñëåäîâàòåëüíîñòè (7).
Òàê, ó ñîðòîâ Tanjil è Wonga (ïîëîæèòåëüíûå êîíòðîëè óñòîé÷èâîñòè ïî
ãåíó Lanr1) ÄÍÊ-ôðàãìåíòû äëÿ àëëåëåé AnSeq3 è AnSeq4 ðàñïîëàãàëèñü
ñîîòâåòñòâåííî íèæå è âûøå àìïëèôèöèðóåìûõ ó ñîðòà Kalya (îòðèöàòåëüíûé êîíòðîëü óñòîé÷èâîñòè ïî ãåíó Lanr1). Ïðîäóêòû ÏÖÐ, ïîëó÷åííûå äëÿ ÄÍÊ êàæäîãî èç ñîðòîîáðàçöîâ ëþïèíà óçêîëèñòíîãî, âêëþ÷åí32
íûõ â èññëåäîâàíèå, âîñïðîèçâîäèëèñü âî âñåõ òðåõ ïîâòîðíîñòÿõ. Ìàðêåðíûå àëëåëè AnSeq3, AnSeq4 äëÿ ðàçëè÷íûõ ñîðòîâ è îáðàçöîâ ðîññèéñêîé,
áåëîðóññêîé è àâñòðàëèéñêîé ñåëåêöèè ïðåäñòàâëåíû íà ðèñóíêå.
Ðåçóëüòàòû ýëåêòðîôîðåòè÷åñêîãî ðàçäåëåíèÿ ÏÖÐ-ïðîäóêòîâ, ïîëó÷åííûõ äëÿ àëëåëåé ÄÍÊìàðêåðîâ AnSeq3 (1-34, ââåðõó) è AnSeq4 (1-34, âíèçó) âîñïðèèì÷èâîñòè è óñòîé÷èâîñòè ê àíòðàêíîçó, ó èçó÷åííûõ ñîðòîâ è ñåëåêöèîííûõ îáðàçöîâ ðàçíîãî ïðîèñõîæäåíèÿ: À — ôîòîäîêóìåíòèðîâàíèå, Á — ñõåìàòè÷åñêèå èçîáðàæåíèÿ; 1, 17, 18, 34 — Tanjil, 2 — Ðàäóæíûé, 3 —
Ñèäåðàò 38, 4 — Êðèñòàëë, 5 — Ñíåæåòü, 6 — Áåëîçåðíûé 110, 7 — Ñìåíà, 8 — Íàäåæäà, 9,
26 — Wonga, 10, 27 — Kalya, 11 — Áðÿíñêèé 123, 12 — Âèòÿçü, 13 — Óçêîëèñòíûé 53-02, 14 —
Âåêòîð, 15 — Áåëîçåðíûé 121, 16 — ÑÍ 78-07, 19 — Ïðèâàáíû, 20 — Ïåðøàöâåò, 21 —
Ìèòàí, 22 — Ãåëåíà, 23 — Ìèðòàí, 24 — Õâàëüêî, 25 — ßí, 28 — ÁÃÁ-6, 29 — Ãåðêóëåñ, 30 —
Æîäèíñêèé, 31 — Ðàííèé, 32 — W 2248, 33 — ÁÃÁ-3; S è R — ñîîòâåòñòâåííî ìàðêåðíûå
àëëåëè âîñïðèèì÷èâîñòè è óñòîé÷èâîñòè, ñöåïëåííûå ñ ãåíîì Lanr1 (12 % ïîëèàêðèëàìèäíûé
ãåëü; îïèñàíèå ñîðòîâ è ñåëåêöèîííûõ îáðàçöîâ ñì. â òåêñòå ñòàòüè).
 ðåçóëüòàòå ïðè ÏÖÐ-àíàëèçå äëÿ îáðàçöîâ ðîññèéñêîé ñåëåêöèè
íå áûëè âûÿâëåíû R-àëëåëè ÄÍÊ-ìàðêåðîâ AnSeq3 è AnSeq4, ñöåïëåííûõ
ñ ãåíîì Lanr1, êîòîðûé êîíòðîëèðóåò óñòîé÷èâîñòü ê àíòðàêíîçó ó àâñòðàëèéñêèõ ñîðòîâ Wonga è Tanjil. Ðàíåå èíôîðìàöèÿ î òîì, ÷òî ñîâðåìåííûå
âíåñåííûå â Ãîñóäàðñòâåííûé ðååñòð ñåëåêöèîííûõ äîñòèæåíèé ÐÔ ñîðòà
Âñåðîññèéñêîãî ÍÈÈ ëþïèíà íå ñîäåðæàò óñòîé÷èâûõ ìàðêåðíûõ àëëåëåé, ñöåïëåííûõ ñ ãåíîì Lanr1, íàìè áûëà ïîëó÷åíà òàêæå ñ ïîìîùüþ
äðóãèõ ÄÍÊ-ìàðêåðîâ — AntjM1 è AntjM2 (11). Èçâåñòíî, ÷òî ãåíåòè÷åñêèé ìàòåðèàë, êîòîðûé íàõîäèòñÿ â îñíîâå óñòîé÷èâîñòè ê àíòðàêíîçó ó
àâñòðàëèéñêèõ ñîðòîâ Wonga è Tanjil, â ñåëåêöèè èññëåäîâàííûõ â íàñòîÿùåé ðàáîòå ðîññèéñêèõ ñîðòîâ èñïîëüçîâàí íå áûë, ÷òî ïîäòâåðäèëè
ðåçóëüòàòû ìîëåêóëÿðíî-ãåíåòè÷åñêîãî àíàëèçà àëëåëåé ÄÍÊ-ìàðêåðîâ ãåíà
Lanr1. Ñîðòàì ïîëüñêîé ñåëåêöèè, êàê è ðîññèéñêèì îáðàçöàì, ñîîòâåòñòâîâàëè ìàðêåðíûå àëëåëè âîñïðèèì÷èâîñòè ê àíòðàêíîçó ïî ãåíó Lanr1.
Ïî ôåíîòèïè÷åñêèì ïðîÿâëåíèÿì ó ñîðòîâ ïîëüñêîé ñåëåêöèè, âêëþ÷åííûõ â íàñòîÿùåå èññëåäîâàíèå, âûðàæåííàÿ óñòîé÷èâîñòü ê àíòðàêíîçó íà
óðîâíå àâñòðàëèéñêèõ ñîðòîâ Tanjil è Wonga îòñóòñòâîâàëà, ÷òî ñîãëàñóåòñÿ
ñ äàííûìè ïî ìàðêåðàì AnSeq3 è AnSeq4.
Äëÿ áîëüøèíñòâà áåëîðóññêèõ îáðàçöîâ áûëè õàðàêòåðíû àëëåëè
âîñïðèèì÷èâîñòè ê àíòðàêíîçó. Àëëåëè óñòîé÷èâîñòè ïî äâóì ìàðêåðàì
AnSeq3, AnSeq4 îáíàðóæèëè òîëüêî ó êîìïîíåíòà áèîëîãè÷åñêîãî áàíêà
ãåíîâ óçêîëèñòíîãî ëþïèíà ÁÃÁ-6. Ýòîò ñåëåêöèîííûé îáðàçåö èìååò áëèçêîðîäñòâåííûå ñâÿçè ñ ñîðòàìè Wonga è Tanjil è òàêæå ïðîÿâëÿåò âûñîêóþ
óñòîé÷èâîñòü ê àíòðàêíîçó â ïîëåâûõ è ëàáîðàòîðíûõ èñïûòàíèÿõ (4).
Äëÿ ñîðòà Ìèðòàí áûëè âûÿâëåíû ìàðêåðíûå àëëåëè óñòîé÷èâîñòè
òîëüêî ïî AnSeq3. Â ïðåäûäóùåé ðàáîòå ñ ìàðêåðàìè AntjM1 è AntjM2 ó
ýòîãî ñîðòà ìû îáíàðóæèëè âîñïðèèì÷èâûå àëëåëè, ïðè÷åì ñ ïîìîùüþ
ïðàéìåðîâ äëÿ ìàðêåðà AntjM2 áûë àìïëèôèöèðîâàí àëëåëü, êîòîðûé íå
33
èäåíòèôèöèðîâàëñÿ ó äðóãèõ îáðàçöîâ, ÷òî õàðàêòåðèçóåò ãåíåòè÷åñêîå
ñâîåîáðàçèå íàçâàííîãî ñîðòà (11). Âàðèàöèþ àëëåëåé óñòîé÷èâîñòè/âîñïðèèì÷èâîñòè ê àíòðàêíîçó ïî ìàðêåðàì AnSeq3 è AnSeq4 ìîæíî îáúÿñíèòü ðàçëè÷íîé ãåíåòè÷åñêîé îñíîâîé áåëîðóññêîãî ñîðòà Ìèðòàí è àâñòðàëèéñêîãî ñîðòà Tanjil. Íåñìîòðÿ íà òî, ÷òî ñîðò Ìèðòàí õàðàêòåðèçóåòñÿ
îòíîñèòåëüíî âûñîêîé óñòîé÷èâîñòüþ ê àíòðàêíîçó, ïðè åãî ñåëåêöèè íå
èñïîëüçîâàëñÿ àâñòðàëèéñêèé óñòîé÷èâûé ê çàáîëåâàíèþ ìàòåðèàë. Òàêèì
îáðàçîì, ïðåäñòàâëÿåòñÿ âåðîÿòíûì òî, ÷òî ó ñîðòà Ìèðòàí ìàðêåð AnSeq3
äàåò «ëîæíîïîëîæèòåëüíûå» ðåçóëüòàòû ïî ñðàâíåíèþ ñ ìàðêåðîì AnSeq4
è âûÿâëåííûé ïðè ýòîì àëëåëü óñòîé÷èâîñòè íå ñâÿçàí ñ ãåíîì Lanr1.
Êðîìå ñîðòà Ìèðòàí è îáðàçöà ÁÃÁ-6, îòíîñèòåëüíî âûñîêîé óñòîé÷èâîñòüþ ê àíòðàêíîçó, îáóñëîâëåííîé íàëè÷èåì â ãåíîòèïàõ ðàñòåíèé
áëîêà èç íåñêîëüêèõ íåàëëåëüíûõ ãåíîâ Rcl, îáëàäàåò ñîðò Ïåðøàöâåò. Ãåíû óñòîé÷èâîñòè ê àíòðàêíîçó, õàðàêòåðíûå äëÿ ýòîãî ñîðòà, ïî âñåé âèäèìîñòè, íåàëëåëüíû Lanr1, ÷òî ñîãëàñóåòñÿ ñ ðåçóëüòàòàìè íàñòîÿùèõ èññëåäîâàíèé, â êîòîðûõ äëÿ ñîðòà Ïåðøàöâåò íå íàáëþäàëè óñòîé÷èâîñòü ê
àíòðàêíîçó ïî ìàðêåðíûì àëëåëÿì, ñöåïëåííûì ñ ãåíîì Lanr1. Ñëåäóåò
îòìåòèòü, ÷òî àëëåëè âîñïðèèì÷èâîñòè âûÿâèëè ó îñòàëüíûõ áåëîðóññêèõ
îáðàçöîâ ëþïèíà óçêîëèñòíîãî, êîòîðûå íå èìåëè áëèçêîðîäñòâåííûõ
ñâÿçåé ñ àâñòðàëèéñêèìè ñîðòàìè Wonga è Tanjil. R-àëëåëè ìàðêåðîâ AnSeq3 è AnSeq4 áûëè òàêæå îáíàðóæåíû ó àâñòðàëèéñêîé ñåëåêöèîííîé
ëèíèè Walan 2248. Ýòîò ôàêò îòðàæàåò ðåçóëüòàòû ïðîãðàììû ñåëåêöèè íà
óñòîé÷èâîñòü ê àíòðàêíîçó, ðåàëèçóåìîé â Àâñòðàëèè ïðè ñîçäàíèè ñîðòîâ
óçêîëèñòíîãî ëþïèíà.
 öåëîì îêàçàëîñü, ÷òî ÄÍÊ-ìàðêåðû AnSeq3 è AnSeq4 ïîçâîëÿþò
äîñòàòî÷íî ýôôåêòèâíî âûÿâëÿòü óñòîé÷èâûå è âîñïðèèì÷èâûå ê àíòðàêíîçó ãåíîòèïû ïî ãåíó Lanr1. Òàê, äëÿ 50 ñîðòîâ è ñåëåêöèîííûõ îáðàçöîâ
ëþïèíà óçêîëèñòíîãî íåîäíîçíà÷íàÿ èíôîðìàöèÿ ïî àëëåëÿì èçó÷åííûõ
ìàðêåðîâ áûëà ïîëó÷åíà òîëüêî ó ñîðòà Ìèðòàí. Ýòè ðåçóëüòàòû ïîäòâåðäèëè íåîáõîäèìîñòü ïðåäâàðèòåëüíîãî àíàëèçà ïðèìåíèìîñòè ÄÍÊ-ìàðêåðîâ ïðè øèðîêîìàñøòàáíûõ ñêðåùèâàíèÿõ, òàê êàê èñïîëüçîâàíèå «ëîæíîïîëîæèòåëüíûõ» ìàðêåðíûõ àëëåëåé ìîæåò ïðèâåñòè ê îøèáêàì ïðè âûáîðå óñòîé÷èâûõ ê àíòðàêíîçó ðàñòåíèé.
Ñòàáèëüíî âûñîêàÿ óñòîé÷èâîñòü ñîðòîâ Tanjil, Wonga, íåñóùèõ ãåí
Lanr1, ïîäòâåðæäåíà â ðàçëè÷íûõ óñëîâèÿõ èñïûòàíèé (4, 5, 12). Ïîýòîìó
âêëþ÷åíèå ãåíà Lanr1 â ãåíîòèïû íîâûõ ñîðòîâ, ïîëó÷åííûõ ïîñëå ñêðåùèâàíèÿ áåëîðóññêîãî èëè ðîññèéñêîãî ñåëåêöèîííîãî ìàòåðèàëà ñ óñòîé÷èâûìè ê àíòðàêíîçó àâñòðàëèéñêèìè îáðàçöàìè, ñïîñîáíî ïîâûñèòü îáùóþ óñòîé÷èâîñòü ê çàáîëåâàíèþ. Äëÿ èíäèâèäóàëüíîãî, âûñîêîòî÷íîãî è
áûñòðîãî îòáîðà ãåíîòèïîâ, ñîäåðæàùèõ ãåí Lanr1, ìîãóò áûòü ïðèìåíåíû
ñîîòâåòñòâóþùèå ÄÍÊ-ìàðêåðû.
Òàêèì îáðàçîì, ïðè èçó÷åíèè îáðàçöîâ ëþïèíà óçêîëèñòíîãî (Lupinus angustifolius L.) ðàçíîãî ïðîèñõîæäåíèÿ ó ñîðòîâ ðîññèéñêîé è ïîëüñêîé ñåëåêöèè íå áûëè îáíàðóæåíû àëëåëè óñòîé÷èâîñòè ê àíòðàêíîçó ïî
ìàðêåðàì AnSeq3 è AnSeq4, ñöåïëåííûì ñ ãåíîì Lanr1. Ó áåëîðóññêîãî ñåëåêöèîííîãî îáðàçöà ÁÃÁ-6 àëëåëè óñòîé÷èâîñòè áûëè âûÿâëåíû ïî îáîèì èçó÷åííûì ìàðêåðàì AnSeq3 è AnSeq4, äëÿ ñîðòà Ìèðòàí — òîëüêî ïî
ìàðêåðó AnSeq3. Óñòîé÷èâûé è âîñïðèèì÷èâûé ê àíòðàêíîçó àâñòðàëèéñêèé ñåëåêöèîííûé ìàòåðèàë èñïîëüçîâàëñÿ â êà÷åñòâå êîíòðîëåé ñîîòâåòñòâóþùèõ àëëåëåé AnSeq3 è AnSeq4. Ïðîâåäåííûå èññëåäîâàíèÿ ïîêàçàëè, ÷òî èñïîëüçîâàíèå ÄÍÊ-ìàðêåðîâ AnSeq3 è AnSeq4, ñöåïëåííûõ ñ
ãåíîì Lanr1, ìîæåò áûòü ïîëåçíî â ñåëåêöèè ðîññèéñêèõ è áåëîðóññêèõ
ñîðòîâ, íåñóùèõ ãåí Lanr1 óñòîé÷èâîñòè ê àíòðàêíîçó.  òî æå âðåìÿ ïðè
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øèðîêîìàñøòàáíûõ ñêðåùèâàíèÿõ íåîáõîäèìî ïðåäâàðèòåëüíî îöåíèâàòü
ïðèìåíèìîñòü âûáðàííûõ ÄÍÊ-ìàðêåðîâ, òàê êàê èñïîëüçîâàíèå «ëîæíîïîëîæèòåëüíûõ» ìàðêåðíûõ àëëåëåé ìîæåò ïðèâåñòè ê îøèáêàì ïðè îòáîðå
óñòîé÷èâûõ ê àíòðàêíîçó ðàñòåíèé.
1ÔÃÁÎÓ ÂÏÎ Áðÿíñêèé ãîñóäàðñòâåííûé
óíèâåðñèòåò èì. àêàäåìèêà È.Ã. Ïåòðîâñêîãî,
Ïîñòóïèëà â ðåäàêöèþ
14 íîÿáðÿ 2013 ãîäà
241036 Ðîññèÿ, ã. Áðÿíñê, óë. Áåæèöêàÿ, 14,
e-mail: syugrishin@gmail.com;
2ÃÍÓ Âñåðîññèéñêèé ÍÈÈ ëþïèíà
Ðîññåëüõîçàêàäåìèè,
241524 Ðîññèÿ, ã. Áðÿíñê, ïîñ. Ìè÷óðèíñêèé, óë. Áåðåçîâàÿ, 2,
e-mail: lupin_mail@mail.ru;
3Íàó÷íî-ïðàêòè÷åñêèé öåíòð ÍÀÍ Áåëàðóñè
ïî çåìëåäåëèþ,
222160 Ðåñïóáëèêà Áåëàðóñü, ã. Æîäèíî, óë. Òèìèðÿçåâà, 1,
e-mail: tinck@inbox.ru
Sel’skokhozyaistvennaya biologiya [Agricultural Biology], 2015, V. 50, ¹ 1, pp. 30-36
IDENTIFICATION OF THE Lanr1 GENE
OF RESISTANCE TO ANTHRACNOSE OF NARROW-LEAFED LUPINE
(Lupinus angustifolius L.) USING DNA-MARKERS AnSeq3 AND AnSeq4
S.Yu. Grishin1, V.V. Zayakin1, I.Ya. Nam1, P.A. Ageeva2, M.I. Lukashevich2,
N.S. Kuptsov3
1Bryansk
State University, 14, ul. Bezhitskaya, Bryansk, 241036 Russia, e-mail syugrishin@gmail.com;
Research Institute of Lupine, 2, ul. Berezovaya, pos. Michurinskii, Bryansk, 241524 Russia, e-mail lupin_mail@mail.ru;
3Scientific and Practical Center on Agriculture of NAS of Belarus, 1, ul. Timiryazeva, Zhodino, 222160 Republic of
Belarus, e-mail tinck@inbox.ru
Supported by the Ministry of Education and Science of the Russian Federation
Received November 14, 2013
doi: 10.15389/agrobiology.2015.1.30eng
2All-Russian
Abstract
Anthracnose is one of the fungal diseases of the narrow-leafed lupine (Lupinus angustifolius L.) caused by Colletotrichum lupini. Resistance to anthracnose is not absolute in character, as
the plants with high resistance can be affected by the pathogen but in less extent than those nonresistant. Recent suggestions of the total number of genes involved in control of anthracnose tolerance are discrepant. Current approach in breeding anthracnose-tolerant lupine is based on combination of non-allele genes of resistance in a single genotype. Specific DNA markers are being developed which are linked to the genes of resistance and can be used for rapid and effective selection of
resistant plants, but prior to their application the efficacy of DNA marker should be specifically
tested with the breeding material of interest to avoid false positive responces. The AnSeq3 and AnSeq4 DAN markers flanking Lanr1 gene at 0.9 cM distance are considered the closest to it (H.
Yang et al., 2012). In this article we report the possibility of using DNA markers AnSeq3 and AnSeq4, the single nucleotide polymorphisms (SNPs), in selecting forms resistant to anthracnose
among varieties of narrow-leafed lupine. A total of 50 Russian, Belarusian, Polish and Australian varieties and samples were tested to detect the allele DNA markers of susceptibility or resistance to anthracnose. DNA was individually isolated from seeds in three replicates. Polymerase chain reaction
(PCR) was used to detect alleles of DNA markers linked to the Lanr1 gene. The list of tested plants,
PCR mix composition and protocol are specified. PCR enzymes and reagents of the SibEnzyme
company (Russia) were used. The praimers for AnSeq3 and AnSeq4 markers were site-specific and
synthesized by the Syntol company (Russia). Polyacrylamide gel electrophoresis was used for visualization of the allele markers. The Australian varieties resistant and susceptible to anthracnose were
used as a control for AnSeq3 and AnSeq4 alleles. DNA fragments of 92 and 87 base pairs corresponding to the markers AnSeq3 and AnSeq4, respectively, were obtained for all 50 breeding samples
included in the study. For 13 Russian and 10 Polish varieties the marker alleles of susceptibility to
anthracnose were detected. For BGB-6 Belarusian sample the resistance alleles were identified by
AnSeq3 and AnSeq4 markers, and for Myrtan variety only the AnSeq3-specific pattern was shown.
The rest of 21 Belarusian samples possessed the alleles of susceptibility to anthracnose. Earlier by
means of other DNA markers, AntjM1 and AntjM2, we showed the absence of alleles linked to
Lanr1 in currently registered varieties originated from All-Russian Research Institute of Lupine.
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Thus, the DNA markers AnSeq3 and AnSeq4 linked to gene Lanr1 may be useful in breeding Russian and Belarusian anthracnose-resistant lupine varieties.
Keywords: Lupinus angustifolius L., anthracnose, polymerase chain reaction (PCR), DNA
markers AnSeq3, AnSeq4.
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