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Evaluation and identification of stem rust resistance genes Sr2, Sr24, Sr25, Sr26, Sr31 and Sr38 in wheat lines from Gansu Province in China

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Evaluation and identification of stem rust resistance genes Sr2, Sr24, Sr25, Sr26, Sr31 and Sr38 in wheat lines from Gansu Province in China https://t.co/RutoE0ICO6 https://t.co/oqv4VRcaCC
RT @thePeerJ: Evaluation and identification of stem rust resistance genes Sr2, Sr24, Sr25, Sr26, Sr31 and Sr38 in wheat lines from Gansu Pr…
RT @thePeerJ: Evaluation and identification of stem rust resistance genes Sr2, Sr24, Sr25, Sr26, Sr31 and Sr38 in wheat lines from Gansu Pr…
Evaluation and identification of stem rust resistance genes Sr2, Sr24, Sr25, Sr26, Sr31 and Sr38 in wheat lines from Gansu Province in China https://t.co/Q1XxSPfeWT https://t.co/FM6JojvhX6
PEER-REVIEWED Plant Biology section

Introduction

Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn (Pgt) causes one of the most potentially destructive wheat diseases, seriously threatening world grain production (Pardey et al., 2013). Disease-resistance breeding to control wheat stem rust is economic, effective, and protective of the environment, and has been proved to be the best control method by repeated practice (Goutam et al., 2015). Wheat stem rust has been effectively controlled with the wide use of resistance gene Sr31 from a 1BL/1RS wheat–rye chromosome arm translocation (Rouse et al., 2012). However, a new race Ug99 virulent to Sr31 was identified in Uganda and classifed as TTKS by the North American Nomenclature System of Pgt in 1999 (Pretorius et al., 2000). Ug99 has broad virulence, and mutates and spreads quickly. Since 1999, 13 variants of Ug99 have been found in 13 countries (FAO, 2017). Recently, Ug99 has been monitored in Egypt, which is the main wheat production area of the Middle East, revealing that its mode of spread is similar to that of a virulent stripe rust pathogen race to Yr9 predicted by Geographic Information System of CIMMYT (Singh et al., 2006). Following the identification and spread of the Ug99 race group, a new race TKTTF caused a wheat stem rust epidemic with an estimated 20,000 to 40,000 ha likely planted to ‘Digalu’ (with resistance to Ug99 race group) in Southern Ethiopia during 2013–2014 (Olivera et al., 2015). Currently it has been confirmed in 11 countries, and given the rapid and destructive nature of race TKTTF, close monitoring of this race is advised—especially in countries which have cultivars carrying the SrTmp resistance gene.

A new race TTTTF with virulence to Sr9e and Sr13 attacked thousands of hectares of durum wheat in Sicily, Italy in 2016, resulting in the largest burst of wheat stem rust in Europe since the 1950s (Bhattacharya, 2017). The large number of spores produced by TTTTF may continue the epidemic in 2017. Moreover, the researchers from the Global Rust Research Center shared a major concern in the warning report that TTTTF could infect not only durum wheat and bread wheat but also dozens of laboratory-grown strains of wheat (FAO, 2017). In view of this, in February 2017, Nature highlighted the potential threat to European wheat production of this race (Bhattacharya, 2017). Therefore, the spread of Ug99, TKTTF and TTTTF, and their variants, threaten the wheat production safety in China.

Gansu Province, located in the northwest of China, plays a significant role in the epidemic and spread of wheat stem rust in China (Cao, 1994). Resistance breeding for this disease has not been a primary objective because it has been effectively controlled in China since the 1970s (Wu et al., 2014). However, durable resistance to stem rust has been re-emphasized with the occurrence and spread of new races of Pgt. It is necessary to analyze the resistance genes in wheat cultivars (lines) from Gansu Province, and the information provided here will be important for developing potentially durable combinations of stem rust resistance genes in cultivars.

Materials and Methods

Wheat cultivars and near-isogenic lines

A total of 75 tested wheat cultivars in Gansu Province were provided by Dr. Fangping Yang from the Wheat Research Institute, Gansu Academy of Agricultural Sciences.

Molecular markers linked to six Sr genes were tested: Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38. Near-isogenic lines carrying 45 Sr genes were used to confirm the validity of these molecular markers. The near-isogenic lines carrying these resistance genes were provided by Dr. Yue Jin from USDA-ARS, Cereal Disease Laboratory, University of Minnesota, USA.

The tested Pgt races included the 21C3CTHTM, 21C3CFHQC, 34MKGQM, 34MKGSM, 34C3MTGQM and 34C3RTGQM (race 34C3MTGQM and 34C3RTGQM identified from the alternative host, Berberis). These races were named according to the methods described in a published study (Li et al., 2016b). The full names of the races and their virulence/avirulence patterns are shown in Table 1. They were isolated and identified by the Plant Immunity Institute, Shenyang Agricultural University, China.

Table 1:
Virulence/avirulence patterns of six races of P. graminis f. sp. tritici.
Race Ineffective Sr genes Effective Sr genes
21C3CTHTM 6, 7b, 8a, 9a, 9b, 9d, 9f, 9g, 10, 11, 12, 13, 14, 15, 16, 17, 18, 24, 28, 29, 34, 35, Tmp, McN 5, 9e, 19, 20, 21, 22, 23, 25, 26, 27, 30, 31, 32, 33, 36, 37, 38, 47
21C3CFHQC 7b, 8a, 9a, 9b, 9d, 9f, 9g, 12, 13, 14, 15, 16, 17, 18, 28, 29, 34, 35, McN 5, 6, 9e, 10, 11, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 36, 37, 38, 47, Tmp
34MKGQM 5, 6, 7b, 8a, 9a, 9b, 9d, 9f, 9g, 12, 15, 16, 20, 24, 27, 28,29, McN 9e, 10, 11, 13, 14, 17, 18, 19, 21, 22, 23, 25, 26, 30, 31, 32, 33, 34, 35, 36, 37, 38, 47, Tmp
34MKGSM 5, 6, 7b, 8a, 9a, 9b, 9d, 9f, 9g, 10, 12, 15, 16, 20, 24, 27, 28, 29, McN 9e, 11, 13, 14, 17, 18, 19, 21, 22, 23, 25, 26, 30, 31, 32, 33, 34, 35, 36, 37, 38, 47, Tmp
34C3RKGQM 5, 6, 7b, 8a, 9a, 9b, 9d, 9f, 9g, 12, 16, 19, 21, 23, 24, 27, 28, 29, McN 9e, 10, 11, 13, 14, 15, 17, 18, 20, 22, 25, 26, 30, 31, 32, 33, 34, 35, 36, 37, 38, 47, Tmp
34C3MTGQM 7b, 8a, 9a, 9b, 9d, 9f, 9g, 11, 12, 13, 14, 15, 16, 17, 18, 28, 29, 34, 35, McN 5, 6, 9e, 10, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 36, 37, 38, 47, Tmp
DOI: 10.7717/peerj.4146/table-1

Seedling resistance evaluation

The cultivars were planted in porcelain pots with a 12-cm-diameter. Seven days later, the leaves were moistened by water with 0.1% Tween 20 using an atomizer and then sprayed with 1 g of fresh urediniospores and dried talc in a ratio of 1:20 (v:v). The inoculated seedlings were transferred to a greenhouse with the temperature in a range of 18 to 22 ± 1 °C. Three biological replicates of the seedling assays were performed for each Pgt race. After 14 days of inoculation, the infection types (ITs) were recorded using the 0–4 IT scale (Stakman, Stewart & Loegering, 1962). ITs were then grouped into low (‘0’, ‘;’, ‘1’, ‘1+, ‘2′, ‘2+, and X) and high (‘3–’, ‘3’, ‘3+’, and ‘4’) infection types. The ITs used in this study are shown in Fig. 1.

Infection types (ITs) used in this study.

Figure 1: Infection types (ITs) used in this study.

A–H indicate ITs: 0, ;, ;1, 1, 2, 3-, 3, 4.

DNA extraction

DNA was extracted from young leaves of 10-day-old seedlings using a genomic DNA extraction kit (http://www.sangon.com/, China). The DNA quality was examined by 1.2% (w/v) agarose gels and DNA quantification was performed using the NanoDrop-1000 version 3.3.1 spectrophotometer.

Polymerase chain reaction (PCR)-specific primers were synthesized by Shanghai Biotech Biotech Co., Ltd, China (Table 2). PCR amplifications were carried out in 25 µL volume, including 0.5 µL of 10 mmol L−1 deoxyribonucleoside triphosphates, 2.5 µL of 10× buffer (Mg2+), 0.2 µL of 5 U µL−1 Taq polymerase, 1 µL of 10 µmol L−1 of each primer, and 2 µL of 30 ng µL−1 DNA. De-ionized water was used to achieve 25 µL volume. Condition of PCR amplification were as follows: 94 °C for 4 min, 30 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1 min, followed by the final extension at 72 °C for 8 min; other specific conditions were as described in previous studies (Table 1).

Table 2:
The markers linked to resistance genes Sr2, Sr24, Sr26, Sr31 and Sr38 with their forward and backward primers.
Genes Marker Forward primer Reverse primer References
Sr2 Xgwm533 5′-GTTGCTTTAGGGGAAAAGCC 5′-AAGGCGAATCAAACGGAATA Hayden, Kuchel & Chalmers (2004)
csSr2 5′-CAAGGGTTGCTAGGATTGGAAAAC 5′-AGATAACTCTTATGATCTTACATTTTTCTG Mago et al. (2011)
Sr24 Sr24#12 5′-CACCCGTGACATGCTCGTA 5′-AACAGGAAATGAGCAACGATGT Mago et al. (2005)
Sr24#50 5′-CCCAGCATCGGTGAAAGAA 5′-ATGCGGAGCCTTCACATTTT Mago et al. (2005)
Sr25 Gb 5′-CATCCTTGGGGACCTC 5′-CCAGCTCGCATACATCCA Liu et al. (2010)
Sr26 Sr26#43 5′-AATCGTCCACATTGGCTTCT 5′-CGCAACAAAATCATGCACTA Mago et al. (2005)
Sr31 SCSS30.2576 5′-GTCCGACAATACGAACGATT 5′-CCGACAATACGAACGCCTTG Das et al. (2006)
Iag95 5′-CTCTGTGGATAGTTACTTGATCGA 5′-CCTAGAACATGCATGGCTGTTACA Mago et al. (2002)
Sr38 VENTRIUP-LN2 5′-AGGGGCTACTGACCAAGGCT 5′-TGCAGCTACAGCAGTATGTACACAAAA Helguera et al. (2003)
URIC-LN2 5′-GGTCGCCCTGGCTTGCACCT 5′-TGCAGCTACAGCAGTATGTACACAAAA Helguera et al. (2003)
DOI: 10.7717/peerj.4146/table-2

Results

Wheat seedling resistance

The resistance test results of 75 main wheat cultivars in Gansu to the races 21C3CTHTM, 21C3CFHQC, 34MKGQM, 34MKGSM, 34C3MTGQM, and 34C3RTGQM are shown in Table 3. Thirty-eight (50.7%) of the 75 tested wheat cultivars showed different resistance levels (ITs 0, ;, ;1, 1+, and 2) to the six races at the seedling stage (Table 4). The remaining 38 (50.7%) wheat cultivars showed varying levels of susceptibility (ITs 3, 3−, 3+, and 4) (Table 3).

Table 3:
Resistant proportion of 75 wheat cultivars to six races of P. graminis f. sp. tritici.
Races Susceptible Resistance
Number of cultivars Percentage/% Number of cultivars Percentage/%
21C3CTHTM 28 37.3 47 62.7
21C3CFHQC 25 33.3 50 66.7
34MKGQM 30 40.0 45 60.0
34MKGSM 26 34.7 49 65.3
34C3RKGQM 26 34.7 49 65.3
34C3MTGQM 25 33.3 50 66.7
All tested races 37 49.3 38 50.7
DOI: 10.7717/peerj.4146/table-3
Table 4:
Seedling infection types produced by six races of P. graminis f. sp. tritici on 75 wheat cultivars (lines).
Cultivars (lines) Pedigree Infection typesa
21C3CTHQM 21C3CFHQC 34MKGQM 34MKGSM 34C3RTGQM 34C3MTGQM
Ningchun 39 Yong 833/Ningchu 4 0 1 0 1 0 0
Dingfeng 10 Tal 73-3/Mota 0 0 0 1 0 ;
Linmai 32 Ganfu 92-310/Xianyang-dasui 4 4 4 3− 3 4
Wuchun 8 Shi 1269/Shi 1269 1+ 0 3− 1 0 0
Wuchun 7 Yong 434/Jian 94-114 4 3− 4 1 4 3−
Dingxi 41 8124-10/Dongxiang 77-011 ; 0 0 ; 0 0
Longchun 31 Genic male sterility of Taigu 0 ;1 0 1 0 ;
Longchun 22 CHIL/BUC 0 0 0 3 0 2
Ganchun 25 M34IBWSN-262/M34IBWSN-252//
Zhangchun 11/Yongliang 4
0 0 0 0 0 0
Longchun 25 Yong 1265/Corydon ; 1 2 0 2 0
Longchun 23 Introduced from CIMMYT 0 1 0 1+ 0 ;
Longchun 26 Yong 3263/Gaoyuan 448 0 0 0 1 0 ;
Ganchun 24 Zhangchun11/93-7-31//23416-8-1//Aibai/
Kavkaz
0 2 0 ; 0 ;
Yinchun 9 Dingxi 35/Xihan 1//Dingxi 37/9208 0 0 0 0 0 2
Longchun 28 8858-2/Longchun 8 ; 1 0 3 ; 3
Wuchun 5 7906/ROBLIN//21-27 1+ 1 3 4 ; 3−
Ganchun 20 88-862/630 4 4 4 4 4 3+
Ningchun 4 Sonora 64/Hongtu 4 4 3− 4 4 4
Linmai 35 Yong 2H15//Gui 86101/79531-1 4 4 4 4 4 1
Xihan 2 8917C/Qinmai 3/72114 4 4 4 3 2 ;
Dingxi 38 RFMIII-101-A/Dingxi 32 ;1 0 1 0 0 0
Ganchun 21 Aibai/Zhangchun 11//2014/82166-1-2//Zhangchun 17 4 4 1 4 4 ;
Dingxi 40 8152-8/Yong 257 4 3 4 1 4 4
Wuchun 4 80-62- 3/7586//Rye//India Aisheng/Liaochun 10/Paulin 0 0 0 1 0 0
Wuchun 3 Yi 5/Shi 857 4 4 4 3+ 4 3
Jinchun 5 Shanqianhong/Funo ; ; 0 2 0 1
Gansu 26 Unknown 1+ ;1 1 1 1 2
Linmai 33 92 Yuan 11/Guinong 20 1 1 ; 0 1 ;
Longchun 33 Longchun 19/Longchun 23 4 1 3 1 4 0
Jiuchun 6 Jiu 96159/Jiu 9061 ; 0 0 0 1+ 0
Longchun 27 8858-2/Longchun 8 1 1+ 1 1 ;1 1
Linmai 34 94 Xuan 4149/Guinong 20//82316/Linmai 26 0 0 0 0 2 ;
Dingfeng 12 Tal 73-3/Mota//Dingfeng 1 0 1+ 2 2 1 ;1
Dingfeng 16 8447/CMS420 4 3 2 1 4 ;
Zhangchun 21 Gaoyuan 602/I 97-2//Gaoyuan 602 1 1 0 ; 1+ 0
Wuchun 6 80-62-3/Ningchun 4//Rye/India Aisheng/Liaochun 10//Paulin 0 0 ; 2 0 1−
Lantian 23 SXAF4-7/87-121 3+ ; 4 1 0 ;
Lantian 19 Mega/Lantian 10 4 4 4 4 4 4
Lantian 25 95-173-4/Baofeng 6 3+ 0 4 4 0 4
Lantian 13 A21//832809/872121-7 ; 4 3 4 0 4
Xifeng 27 83183-1-3-1/CA837 ; 2 1 1+ ; 1+
Lantian 26 Flansers/Lantian 10 0 2 1 1+ 1 1
Longjian 101 85(1)F3 Xuan (2)-4/Shanhan 8968//85-173-12-2 4 1 4 4 4 4
Hangxuan 1 Unknown 0 0 0 0 0 0
Lantian 14 Qingshang 895/Zhongliang 17 0 1+ 0 0 0 ;
Lantian 31 Long Bow/Lantian 10 0 1 3− 3 2 3
Pingliang 42 tal Changwu 131/Pingliang 38/82(51) ;1 3− 3− 2 4 3
Xifeng 20 Xifeng 18/CA8055 1 3− 2 2 1 1
Longyu 4 Xifeng 20/Zhong 210 0 1 2 2 1 1
Changwu 131 7014-5/Zhongsu 68//F16-71 4 4 4 4 4 4
Zhongliang 18 Kangyin 655/Elytrigia trichophora//Jingai 21 4 3 0 1 4 4
Zhongliang 22 Zhong5/S394//Xiannong 4 0 0 ;1 1 0 0
Lantian 10 Xifeng 16/Predgornajia/68286-0-1-1 ; 2 0 1 1 1
Tianxuan 39 Unknown 1 1+ ;1 0 0 1
Huandong 6 Unknown 4 0 4 0 4 3
Longjian 196 64035/Taiyuan 89/Qinnong 4 4 4 4 4 4 4
Lantian 30 95-111-3/Shan167 1 2 2 3 1 2
Longnan 2000-8-2-1 Unknown 0 1 0 ;1 1 2
Longjian 301 DW803/7992 1+ 1 ; 1+ 1 2
Longyu 2 Longdong 3 //82(348)/9002-1-1 0 1 1 1 1 1
Longjian P430 Unknown 0 1 ; 1 1 0
Longjian 103 Longjian 127/Mo(W)697 4 4 4 2 4 2
Lantian 29 82F-37/83-44-20//8380 4 3 4 4 4 3
Lan 092 Unknown 0 2 1− 4 1 0
Qingnong 1 7084/2037 4 4 4 3 4 3+
Pingyuan 50 Local cultivar 3+ 4 4 4 4 4
Longyuan 034 Unknown 0 2 0 1+ 0 1
Lan 05-9-1-4 Unknown 4 4 4 2 4 3+
Gandong 017 Unknown 0 2 2 ;1 0 1
Longjian 19 Jinan 2/Qinnong 4 4 3 4 3 4 4
Lantian 24 92R137/87-121-2 4 0 4 4 4 2
863-13 Xiannong 4/Tianxuan 42 0 0 0 0 0 0
01-426e-1 Unknown 3+ 4 3 4 4 3
Tian 01-29 Unknown ; 2 2 2 ; 2
Tian 01-104 Unknown 4 4 4 3− 4 4
DOI: 10.7717/peerj.4146/table-4

Notes:

Infection types (ITs): are based on a 0-to-4 scale where ITs of 0, ;, 1, and 2 are indicative of a resistant (low) response and ITs of 3 or 4 of a susceptible (high) response; Symbols + and − indicate slightly larger and smaller pustule sizes, respectively (Stakman, Stewart & Loegering, 1962).

Validity of the markers

Six specific PCR markers closely linked with resistance genes Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were validated using 45 single differentials carrying known resistance genes. Table 5 shows that these ten markers amplified only specific bands in the expected wheat genetic stocks. For example, primer SCSS30.2576 amplified only 576-bp specific bands in Siouxland, Sisson, Sr31/6*LMPG, and Federation*4/Kavl, while in other wheat lines without Sr31, no bands were amplified, indicating that these markers are able to be well applied for the molecular detection of the six resistance genes.

Table 5:
Amplification results for the known Sr genes by markers.
Line Sr gene Source Sr2 Sr2 Sr24 Sr24 Sr25 Sr26 Sr31 Sr31 Sr38 Sr38
Xgwm533 csSr2 Sr24#12 Sr24#50 Gb Sr26#43 SCSS30.2576 Iag95 VENTRIUP-LN2 URIC-LN2
ISr5-Ra 5 11Aberdeen a
CnS_T_mono_der 21 11Aberdeen
Vernstine 9e 11Aberdeen
ISr7b-Ra 7b 11Aberdeen
IS11-Ra 11 11GH
ISr-Ra 6 11GH
ISr8a-Ra 8a 11Aberdeen
CnSr9g 9g 10Aberdeen
W2691SrTt-1 36 11GH
W2691Sr9b 9b 11Aberdeen
BtS30Wst 30 11Aberdeen
Combination VII 17+13 11Aberdeen
ISr9a-Ra 9a 11Aberdeen
ISr9d-Ra 9d 11Aberdeen
W2691Sr10 10 11Aberdeen
CnsSrTmp Tmp 11Aberdeen
LcSr24Ag 24 11Aberdeen + +
Sr31/6*LMPG 31 11Aberdeen + +
Trident 38 11Aberdeen + +
McNair 701 McN Griffey 2010
Line E 09AB
Acme 9g 09AB
Siouxland 24+31 2011 Baenzinger + + + +
Sisson 31+36 Griffey 2010 + +
SwSr22T.B. 22 12GH
Agatha/9*LMPG 25 08AB +
Eagle 26 10AB +
73,214,3-1/9*LMH? 27 08AB
Federation*4/Kavl 31 10AB + +
ER 5155 32 10AB
Tetra Canthatch/A? 33 09AB
Mq(2)5XG2919 35 10AB
W3563 37 09Aberd
L6082 39 10AB
L6088 40 10AB
TAF 2 44 10AB
DAS15 47 10AB
Satu Satu 09Aberd
TAM 107-1 1A.1R 12GH
Fed*3/Gabo*21BI R 10AB
Iumillo 9g,12,+ 09GH
Leeds 9e,13,+
Hope 2 +
ST464 13 08GH
Q21861 Rpg1,4,5 04NewZealand
DOI: 10.7717/peerj.4146/table-5

Notes:

Symbol ‘+’ indicates the cultivar (line) carry the tested genes; ‘–’ indicates that the cultivar (line) does not carry the tested genes.

Sr2 screening

A DNA marker was developed to accurately predict Sr2 in diverse wheat germplasm for the partial resistance of Sr2 is very difficult to screen under field conditions (Mago et al., 2011). Two markers, Xgwm533 and csSr2, were used to detect Sr2 in wheat cultivars of Gansu Province. A specific PCR band with 120-bp in size was amplified with marker Xgwm533, but no PCR product was amplified using marker csSr2 in Hope with Sr2. In this study, a similar 120-bp band was detected in the 13 cultivars, indicating that these cultivars carried Sr2 (Table 6).

Table 6:
Molecular detection of resistance genes Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 in the 75 wheat cultivars (lines).
Cultivars (lines) Sr2 Sr2 Sr24 Sr24 Sr25 Sr26 Sr31 Sr31 Sr38 Sr38
Xgwm533 csSr2 Sr24#12 Sr24#50 Gb Sr26#43 SCSS30.2576 Iag95 URIC-LN2 VENTRIUP-LN2
Ningchun 39 a
Dingfeng 10
Linmai 32
Wuchun 8 +
Wuchun 7
Dingxi 41 + +
Longchun 31 + +
Longchun 22
Ganchun 25 + +
Longchun 25 + +
Longchun 23 + +
Longchun 26 + + +
Ganchun 24 + + +
Yinchun 9 + + +
Longchun 28
Wuchun 5
Ganchun 20
Ningchun 4
Linmai 35
Xihan 2
Dingxi 38 + +
Ganchun 21 +
Dingxi 40
Wuchun 4
Wuchun 3
Jinchun 5 + + + +
Gansu 26 + + + +
Linmai 33 + +
Longchun 33 +
Jiuchun 6 + + +
Longchun 27 + + +
Linmai 34
Dingfeng 12 +
Dingfeng 16
Zhangchun 21 + +
Wuchun 6 +
Lantian 23
Lantian 19
Lantian 25
Lantian 13
Xifeng 27 + +
Lantian 26 + +
Longjian 101
Hangxuan 1 + +
Lantian 14 + + + + +
Lantian 31
Pingliang 42
Xifeng 20
Longyu 4
Changwu 131
Zhongliang 18 +
Zhongliang 22 + + + +
Lantian 10 + + + +
Tianxuan 39 + +
Huandong 6
Longjian 196
Lantian 30
Longnan-2000-8-2-1
Longjian 301 + +
Longyu 2 + +
Longjian P430 + +
Longjian 103
Lantian 29
Lan 092
Qingnong 1
Pingyuan 50
Longyuan 034 + +
Lan 05-9-1-4
Gandong 017 + +
Longjian 19
Lantian 24
863-13 + +
01-426e-1 +
Tian 01-29 + +
Tian 01-104
DOI: 10.7717/peerj.4146/table-6

Notes:

Symbol ‘+’ indicates the cultivar (line) carry the tested genes; ‘–’ indicates the cultivar (line) don’t carry the tested genes.

Sr24 screening

Two markers, Sr24#12 and Sr24#50, were developed to detect Sr24, located on chromosome 3DL in Agent- or 1BS in Amigo-derived lines (Mago et al., 2005). These two markers were applied to detect Sr24 existence in the 75 major wheat cultivars (lines) of Gansu Province in this study. The results showed that marker Sr24#12 amplified a 500-bp specific band and marker Sr24#50 amplified an approximately 200-bp specific band in the Sr24 control LcSr24Ag. No PCR fragment was amplified in Little Club (LC) and the tested cultivars, indicating that these cultivars lacked Sr24.

Sr25 screening

Because of the resistance of Sr25 to the new race Ug99 and related strains, a dominant marker Gb was developed for haplotyping Sr25, (FAO, 2017; Liu et al., 2010; Pretorius et al., 2000). The presence of the marker was confirmed by detection of a 130-bp fragment. The PCR results indicated that the 130-bp band was only amplified using the Sr25-positive line Agatha/9*LMPG (monogenic Sr25) genomic DNA (Liu et al., 2010; Yu et al., 2010), but not with other cultivar DNA samples, indicating that all 75 lines from Gansu Province examined lack Sr25.

Sr26 screening

Stem rust resistance gene Sr26 was transferred into the long arm of wheat chromosome 6A from Thinopyrum ponticum (Mago et al., 2005). Although the cultivars carrying Sr26 displayed resistance to all the dominant Pgt races in China, it is not utilized in wheat breeding. A dominant STS marker Sr26#43 was developed for detecting this wheat stem rust resistance gene and a 207-bp band was amplified in wheat lines with Sr26 (Mago et al., 2005). Marker Sr26#43 was used to detect this fragment in tested wheat cultivars. No any visible band was detected, suggesting that these varieties do not carry Sr26, as expected.

Sr31 screening

Two markers, SCSS30.2576 and Iag95, linked to resistance gene Sr31 were used for detecting these locus. SCSS30.2576 amplified a 576-bp fragment and marker Iag95 amplified an 1,100-bp PCR fragment in Sr31-carrying lines such as Sr31/6*LMPG and Siouxland (Fig. 2). No fragment was amplified in the negative control LC. These two markers were used to detect Sr31 in the tested cultivars. The result showed that these two fragments were detected in the 25 tested cultivars (Table 6).

Amplification result for parts of wheat varieties with markers SCSS30.2576 and Iag95.

Figure 2: Amplification result for parts of wheat varieties with markers SCSS30.2576 and Iag95.

(A) Iag95. (B) SCSS30.2576. Lane 1–11, Monogenic Sr31, Little Club, Wuchun 7, Dingxi 41, Longchun 31, Longchun 22, Ganchun 25, Longchun 25, Longchun 23, Longchun 26, Ganchun 24, Yinchun 9, ‘M’ indicates 2,000 bp DNA ladder and black arrow indicates the position of the specific band.

Sr38 screening

The Lr37-Sr38-Yr17 rust resistance gene cluster was transferred to the short arm of bread wheat chromosome 2AS from a segment of Triticum ventricosum (Tausch) Cess. chromosome 2NS (Helguera et al., 2003). The 2NS-specific primer VENTRIUP-LN2 and 2AS-specific primer URIC-LN2 were developed to detect this rust resistance gene cluster in commercial wheat cultivars and 262-bp and 285-bp PCR products were amplified in wheat line carrying Lr37-Sr38-Yr17, whereas none of these amplification products were found in negative control LC (without Lr37-Sr38-Yr17). In this study, both 262-bp and 285-bp PCR fragments were amplified in nine wheat cultivars, suggesting that these wheat cultivars carried Sr38 (Table 6).

Discussion

The broad-spectrum wheat stem rust resistance gene Sr2 confers adult plant resistance to stem rust and is located on chromosome arm 3BS. It originated in tetraploid Yaroslav emmer (T. dicoccum) and later was transferred to the susceptible bread wheat ‘Marquis’ in the 1920s (McFadden, 1930). Several varieties with Sr2 were cultivated worldwide (Singh et al., 2011). Markers Xgwm533 and csSr2 were used to detect Sr2 in wheat cultivars from Gansu. However, marker csSr2 failed to predict Sr2. Only marker Xgwm533 amplified a 120-bp band in the positive control and 13 tested cultivars, but the 120-bp band also occurred in many North American and CIMMYT lines which are considered not to have Sr2. Therefore, it is difficult to conclude that all the accessions that showed a 120-bp fragment size for this marker carry Sr2.

The stem rust resistance gene Sr24 is completely associated with leaf rust resistance gene Lr24. It has been widely used in wheat breeding programs worldwide, since it was introgressed into wheat lines (McIntosh, Wellings & Park, 1995). Gene Sr24 was ineffective to some variants of Ug99 but is effective to the new races TKTTF, TTTTF, and many Pgt races in China (Bhattacharya, 2017; Han, Cao & Sun, 2010). Therefore, two markers, Sr24#12 and Sr24#50, developed by Mago et al. (2005) were used to detect the gene in Gansu wheat cultivars in this study. Surprisingly, no wheat cultivars carried this gene. However, it is reported that Chinese wheat cultivars in other provinces carry Sr24 (Cao et al., 2007; Li et al., 2016b).

Wheat plants carrying stem rust resistance gene Sr25 were susceptible to several strains of Chinese Pgt races (Cao et al., 2007). Sr25 and its linked leaf rust resistance gene Lr19, were transferred into wheat from Thinopyrum ponticum to wheat chromosomes 7D and 7A (Friebe et al., 1994; Zhang et al., 2005). The use of Sr25-Lr19 was initially limited because of linkage with another Th. ponticum derived gene producing undesirably yellow flour. It has been further backcrossed into the Australian and CIMMYT wheat backgrounds with the mutant line (which contains Sr25-Lr19), but with white flour (Bariana et al., 2007; Knott, 1980). The use of this gene in wheat programs is increasing for its resistance to new races TTTTF and Ug99 race group, having potential yield increases under irrigated conditions (FAO, 2017; Liu et al., 2010; Monneveux et al., 2003; Singh et al., 1998). In this study, 75 wheat varieties from Gansu Province were examined for presence of marker Gb. The result showed that all 75 wheat varieties lack Sr25.

In Australia, Sr26 has been released in the cultivar Eagle since 1971 (Martin, 1971). Later, other major cultivars including Flinders, Harrier, Kite, Takari, and Sunelg, were cultivated. Lines containing the Sr26 fragment are resistant to new stem rust pathogen races such as Ug99 and its associated strains. None of the cultivars had Sr26 in the present study, as expected, and similar results were observed in our previous study (Li et al., 2016a).

The stem rust resistance gene Sr31 on 1BL/1RS was transferred into the bread wheat from ‘Petkus’ rye (Graybosch, 2001). Since then a higher number of wheat cultivars carrying Sr31 have been released in global wheat breeding (Das et al., 2006). It is reported that more than 60% (1.3 ×107 hm2) of the total wheat planting areas carried this translocation in China (Jiang et al., 2007). Although the gene is ineffective to Ug99 and related variants, it is also an effective gene against all Pgt races in China and the new races TKTTF and TTTTF. Molecular marker detection showed that 25 wheat cultivars carried Sr31. All these cultivars (lines) produced resistance ITs (0, ;, ;1, 1+, and 2) to all tested Pgt races, as expected. Moreover, pedigree tracking indicated that resistant materials carrying the 1BL/1RS translocation such as ‘Kavkaz’ and ‘Rye’ were widely used in wheat breeding in Gansu Province (Cao et al., 2011), revealing the origin of Sr31 in these wheat varieties.

Rust resistance gene cluster Yr17-Lr37-Sr38 was initially transferred into the winter bread wheat line ‘VPM1’ from T. ventricosum and was located in a 2NS/2AS translocation (Bariana & McIntosh, 1993; Cao et al., 2007; Maia, 1967). PCR assays using restriction fragment length marker cMWG682 were developed for selecting the 2NS/2AS translocation in wheat cultivars (Helguera et al., 2003). Sr38 became susceptible to new races related to Ug99 but no virulent Pgt race to Sr38 has been found in China. The results showed that nine wheat cultivars carried the gene cluster. The resistance of these cultivars against the tested Pgt races might be attributed to this gene.

Conclusion

Breeding resistant cultivars is an economic and effective way to protect wheat from disease. The development of molecular technology facilitated the identification and utilization of molecular markers for durable resistance breeding, leading to increased crop production. The molecular markers associated with Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were used to detect the occurrence of these genes in 75 major wheat cultivars (lines) in Gansu Province in this study. The results showed that 35 tested cultivars might carry one of these genes. This information can be used in breeding for stem rust resistance in the future.

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