Germplasm and Genetic Stocks

Gene isolation and its identification of salinity stress on G.hirsutum L. YE Wu-wei*, WANG Jun-juan，WANG De-long，YIN Zu-jun， FAN Wei-li，WANG Shuai，Song Liyan, Zhang Lina, Zhou Kai, LU Xu-ke (State Key Laboratory of Cotton Biology / Institute of Cotton Research of CAAS, Anyang455000, Henan, China. Emai:yew15@hotmail.com;Tel:86-372-2562283;0086-13803724991) Soil salinization has become a serious global problem affecting the agricultural development and the ecological environment. Salinity，as one of the most important abiotic stresses in the world, severely limits the production of crop. Saline-alkali land in our country is widely distributed with the character of multi types and serious salt-deposition. In order to carry out the utilization of saline-alkali land efficiently, it is necessary to develop the agriculture on the saline-alkali land.Cotton, as a pioneer crop in saline-alkali land, should be paid more efforts to conduct the mechanism research of salt-tolerace and to breed new varieties of salt-tolerant. Cotton, the major cash crop in China, is playing a crucial role in national economic development. China, with less cultivated lands and more people, faces the contradiction between food and cotton, which seriously affects the cultivation and production of cotton. Therefore, It is an effective way to farm saline land and to enhance the sustainable agricultural development by develop the salinity-tolerant varieties of cotton. Identification of salinity-tolerance also plays a vital role on cotton breeding. The abiotic-tolerant identification methods used before, mainly based on morphological characters, were usually restricted for time-wasting and labor-costing, environment influence, and seasonal restrictions. A new set of preliminary methods system, called SSR multi-markers salinity-identification method, was initially established to identify salinity tolerance of cotton by the standardization of the whole process of seedling nursing, DNA extraction, PCR amplification, amplification products detecting, and marker-combination. Another 11 materials were used to testify this method, which showed the coincidence of 90.91% in consistence with the identification result of 0.4%NaCl identification method. This study showed that the multi-markers identification method was proved to be used to assist identify the salinity tolerance of cotton germplasm. Seven salt-tolerance related genes, H+-pyrophosphatase gene and S-adenosylmethionine synthetase gene and others, were cloned from the salt-tolerance material on Gossypium hirsutum, which were named GhVP and GhSAMS, respectively. The bioinformatics analysis and their transformed accessions were tested and identified. Key word: Cotton germplasm；identification；salinity;

MNH-886(Bt.) is a upland cotton cultivar (Gossypium hirsutum L.) developed through hybridization of three parents [(FH - 207 × MNH- 770 ) × Bollgard-1] at Cotton Research Station Multan, Pakistan. It is resistant to CLCuVD with 16.25 % disease incidence (60 DAS, March sowing) whereas moderately susceptible to CLCuVD when planted in June with disease incidence 34 % (60 DAS). This disease reaction was lowest among 25 cotton advanced lines/varieties tested at hot spots of CLCuVD. Its performance was tested during 2009 to 2012 in various indigenous, provincial, and national varietal trials in comparison with the commercial variety IR-3701 and AA-802 & CIM-496. In PCCT trial during 2009-10; 2011-12, MNH-886 surpassed all the existing Bt. strains along with commercial varieties across the Punjab province with seed cotton yield production 2658 kg ha-1 and 2848 kg ha-1 which was 81.31 and 13% higher than checks, respectively. In National Coordinated Bt. Trial, MNH-886(Bt.) produced 3347 kg ha-1 seed cotton at CCRI, Multan; the hot spot of CLCuVD, in comparison to IR-3701 which gave 2556 kg ha-1. It possesses higher lint percentage (41.01%), along with the most desirable fibre traits (staple length 28.210mm, micronaire value 4.95 µg inch-1 and fibre strength 99.5 tppsi, and uniformity ratio 82.0%). The quantification of toxicity level of crystal protein was found positive for Cry1Ab/Ac protein with toxicity level 2.76µg g-1 and Mon 531 event was confirmed. Having tremendous yield potential, good fibre traits, and great tolerance to CLCuVD we can recommended this variety for cultivation in CLCuVD hotspots of Pakistan. Keywords: Cotton, Cultivar, Cotton leaf curl virus, CLCuVD hit districts.

Simple sequence repeat (SSR) techniques were used to identify the genetic diversity of 102 G. arboreum accessions which collected from India, Vietnam and China (Guizhou, Guangxi and Yunnan). Twenty-six pairs of SSR primers produced a total of 103 polymorphic loci among these materials and average 3.96 polymorphic loci per primer were detected. The mean effective number of alleles, the mean Nei's gene diversity and the mean Shannon's information index were 0.59, 0.2835 and 0.4361. The diversity varied among different geographic regions. The result of principal components analysis was almost consistent with that of UPGMA clustering analysis. The 102 G. arboreum accessions were clustered into 3 groups. Dianya 16 was far from others and belonged to a group with one accession.

This study examines trends in United States upland cotton yield productivity since 1980. The overall objective of the study was to document changes in productivity over this time period with a special emphasis to measure and examine changes in genetic gain. To meet this objective, on-farm and replicated variety trial yield datasets were analyzed and interpreted. Clearly, these analyses suggest that yield productivity and genetic gain have occurred since 1980. Trends and factors will be discussed that are associated with increased yield over this time period.

The aim of our study is development of molecular genetic fingerprints of commercial cotton varieties cultivated in Uzbekistan using SSR markers. Thirty seven samples from Upland cotton germplasm collection of the Centre of genomics and bioinformatics were genotyped using 185 primer pairs. From 185 SSR of 38 yielded polymorphic loci suitable to create molecular profiles of the studying samples. The minimum and maximum number of loci varied from 2 to 7 respectively, with average of 2,81 loci per marker. Having assessed the content of polymorphic information content (PIC) of the markers, we found that the minimum and maximum value was 0,051 and 0,609, respectively with a mean value of 0,358. The lowest and highest value expected heterozygosity value (He) ranged from 0,052 to 0,678 with a mean value of 0,421. Further phylogenetic analysis using microsatellite data and UPGMA (unweighted pair group method with arithmetic mean) grouped 37 cotton varieties to 6 clasters. The most distant from others samples were 6 and 12. Varieties Namangan 102, Namangan 34, AN 16 also formed a separate cluster, as well as the line C 8271. Results of the study revealed narrow genetic base of selected commercial cotton cultivars. Identified 38 SSR markers are highly polymorphic, and able to discriminate all tested cotton cultivars. These markesr could be used as a DNA fingerprinting set for local cotton cultivars.

Gossypium australe is a diploid wild cotton species (2n=26, GG) native to Australia that possesses valuable characteristics unavailable in the cultivated cotton gene pool, such as delayed pigment gland morphogenesis in the seed and resistances to pests and diseases. However, it is very difficult to directly transfer favorable traits into cultivated cotton through conventional gene recombination due to the absence of pairing and crossover between chromosomes of G. australe and G. hirsutum (2n=52, AADD). To enhance the transfer of favorable genes from wild species into cultivated cotton, we developed a set of hirsutum-australe monosomic alien chromosome addition lines (MAAL) using a combination of morphological survey, microsatellite marker assisted selection, and molecular cytogenetic analysis. The amphidiploid (2n=78, AADDGG) of G. australe and G. hirsutum was consecutively backcrossed with upland cotton to develop alien addition lines of individual G. australe chromosomes in G. hirsutum. From these backcross progeny, we generated the first complete set of chromosome addition lines in cotton; twelve of 13 lines are monosomic additions, and only chromosome 7Ga is multiple addition. MAALs of 1Ga and 11Ga were the first to be isolated. The chromosome addition lines can be employed as bridges for the transfer of desired genes from G. australe into G. hirsutum, as well as for gene assignment, isolation of chromosome-specific probes, flow sorting and microdissection of chromosome, development of chromosome-specific ‘‘paints’’ for fluorochrome-labeled DNA fragments, physical mapping, and selective isolation and mapping of cDNAs for a particular G. australe chromosome.

Confirmation of Gossypium hirsutum Chromosome Substitution Lines using SSR markers S. Saha1, D. M. Stelly2, D. Raska2, A. Hulse2, O. A. Gutiérrez1,3, A. Makamov4, V. Gotmare5, F. Wang2, Sh. Manchali2, D. Deng1, J. N. Jenkins1, I. Salakhutdinov, M. Ayubov and I. Y. Abdurakhmonov4 1USDA-ARS, Crop Science Research Lab., Mississippi State, MS 39762 USA, 2Texas A&M University, College Station, TX 77843 USA, 3Current Address USDA-ARS, Subtropical Horticultural Research Station, Miami, FL, 33158 USA, 4Center of Genomic Technologies, Institute of Genetics and Plant Experimental Biology, Academy of Sciences of Uzbekistan. Yuqori Yuz, Qibray Region Tashkent District, 111226 Uzbekistan, 5Division of Crop Improvement, Central Institute for Cotton Research, Post Bag No. 2, Shankarnagar PO, Nagpur , India ABSTRACT Two major impediments in the genetic improvement of cotton are: 1) under-utilization of diverse germplasm and 2) insufficient information about genes that control important traits. The primary gene pool for cotton includes wild types of G. hirsutum L., as well as the other four 2n=52 species of Gossypium (G. barbadense, G. mustellinum, G. tomentosum and G. darwinii) all of which are readily hybridized and contain AD1 genomes (G. hirsutum L.) and thus share a common gross chromosome structure. Nonetheless, previous interspecific introgression efforts using conventional breeding methods have had very limited success, indicating deep-seated genetic conflicts in the hybrids preclude facile recovery of agronomically useful types. To help overcome these barriers, we are developing alien chromosome substitution (CS) lines from G. barbadense, G. mustellinum and G. tomentosum. The development of each CS line involves four stages: (i) create isogenic Upland chromosome-deficient stocks, by backcrossing various chromosome deficiencies (monosome or telosome) to a common line, namely ‘Texas Marker-1’ (TM-1); (ii) create a F1 substitution stock that is monosomic or monotelodisomic (i.e., partially hemizygous) by recurrent backcrossing to each isogenic cytogenetic stock as a recurrent seed parent; (iii) inbreed the backcrossed hypoaneuploid derivative to recover a euploid disomic substitution line; (iv) confirm the cytogenetic and genetic constitution of the disomic lines by cytological analysis and chromosome-specific SSR markers. We have developed several CS lines from G. barbadense, G. mustellinum and G. tomentosum respectively based on cytological analysis. These substitution lines are nearly isogenic to the common parent TM-1 for 25 chromosome pairs, as well as to each other, for 24 chromosome pairs. At the time development was initiated for some CS lines, molecular markers did not exist, and by the time several lines were released, very few accurately mapped chromosome specific SSR markers were available in the public domain. Recently in addition to the cytological analysis, we have been using SSR markers from one or more linkage maps to assess the constitution and genetic identity of the CS lines. The overall objective of this paper is to report on the genetic identity of the CS lines using SSR markers. For CS euploid lines (2n=52), we compared marker profiles of CS lines, parents and other CS germplasm. For most CS lines and most mapped markers, the observed SSR profiles were concordant with expectations. For a minority of markers and lines, however, the results were discordant; these markers, linkage groups, and CS lines are being further investigated to better define these research and breeding resources.

In Brazil, it is possible to perform in situ conservation of G. barbadense and G. mustelinum. G. barbadense plants are maintained for several domestic uses. The vast majority are isolated from cultivated upland cotton, although cotton fields are located at distances that permit gene flow in some restricted areas. G. mustelinum occurs only in the northeast region of the country, and there is no sympatry with upland cotton crops. Just few upland cotton plants derived from kernels used to feed cattle or spread via dispersal during transportation can be found near some G. mustelinum populations. Although gene flow could be a problem for in situ conservation in the places where sympatry is present, natural hybrids do not occur or occur in very low frequencies in situ in Brazil. We evaluated the presence of pollen competition as a pre-zygotic barrier in crosses between upland cotton (used exclusively as pollen donor) and G. barbadense and G. mustelinum. We found that a pollination of stigmas of G. barbadense using a mixture of 50% upland cotton and 50% G. barbadense resulted in 24.5% of hybrids on average. Similarly, stigmas of G. mustelinum pollinated with mixtures containing pollen from G. mustelinum and upland cotton in proportions of 25%:75%, 50%:50% and 75%:25% produced interspecific hybrids at frequencies of 61.4%, 23.8% and 3.6%, respectively. These low rates of hybrids demonstrate that pollen competition is present with G. mustelinum and G. barbadense and confirms this mechanism as a sexual barrier. Knowledge regarding pollen competition among cultivated and non cultivated cottons in Brasil are being used to design methods for in situ preservation of sympatric, permitting less restrictive conditions for maintaining the genetic identity of non-cultivated species.

Gossypium hirsutum L. (Upland cotton), an allotetraploid with a base chromosome number of 13 (2n = 4x = 52) and a genome size of 2500 Mbp, is the major cash crop in China. In the present study, ninety-three F2 individuals derived from the interspecific cross, G. hirsutum L. acc 08N2162 and G. tomentosum Nutt. ex Seem., were used to construct the genetic map (HT map) with simple sequence repeats (gSSR) and simple sequence repeats derived from expressed sequence tags (EST–SSR). 1045 loci were detected from 807 pairs of EST–SSR, and 755 loci from 540 pairs of gSSR. Polymorphisms from gSSR and EST-SSR was very similar, 16.0 % (540 in 3380) and 15.8% (807 in 5180), respectively. A total of 1800 loci from 1347 pairs of primers were detected, of which there were 682 co-dominant markers, 458 dominant for upland cotton and 660 for G. tomentosum. Of the 1800 loci, 1295 loci were grouped into 46 linkage groups at LOD≧4, and the map covered 3,375.3 cM with a mean density of 2.86 cM per locus. All the above linkage groups have been located on their corresponding chromosomes using the genetic map of interspecific cross between TM-1 (G. hirsutum L.) and Hai 7124 (G. barbadense L.) (HB map) as reference. The same reciprocal translocations were found between A4 and A5, A2 and A3 chromosomes on both genetic maps. There were 506 (28.1%) markers showed segregation distortion, of which 255 (14.1%) markers located and 24 SDRs totally were detected. The distribution of distorted marker loci were very uneven between At subgenome and Dt, less distorted marker loci were located into At (89) than D sub-genome (166), 7 SDRs appeared in the At and 17 SDRs appeared in the Dt. Five distorted markers located on 12 short linkage groups.

Salinity, as one of limited facts, largely affects the production of cotton. The triangle region of the Yellow River in Shandong province, as the one of the main cotton production provinces in China, occupies the 40% of the total cotton production areas and increases annually. The collection, exploration, screening and innovation of cotton germplasm were deeply carried out in our Center. The novel cotton varieties and materials such as salinity-tolerant, high fiber quality, wilt-resisted, Bt- gene varieties were bred in these years. The main varieties such as Lukengmian 33, Renhe 39, Lujinmian 36, Lukengmian 37, and et al, yield high in the triangle region of the Yellow River in Shandong province, which paved out the broad way of the super-high yield cotton breeding research.